Solid culture medium for fermenting abamectin by utilizing agricultural wastes and application of solid culture medium

Abstract

The invention discloses a solid culture medium for fermenting abamectin by utilizing agricultural wastes and application of the solid culture medium, and belongs to the technical field of microbial fermentation. In order to solve the problems of high consumption of water resources and organic reagents, high energy consumption, high cost, unfriendliness to the environment and the like in production of abamectin through liquid fermentation and the problem of low utilization rate of agricultural wastes, the method comprises the following steps: screening optimal agricultural wastes as a solid fermentation culture medium of streptomyces avermitilis; and through optimization of a streptomyces avermitilis solid fermentation culture medium and fermentation process parameters, a green streptomyces avermitilis solid fermentation strategy is developed, so that the fermentation cost is reduced, the energy consumption of equipment, the waste of water resources and the generation of wastewater and waste residues are reduced, and the low-cost and environment-friendly application of the abamectin is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to a solid medium for fermenting avermectin by using agricultural waste and its application. Background Art

[0002] China produces approximately 4 billion tons of agricultural waste every year, ranking among the countries with the largest output of agricultural waste in the world, and the output of agricultural waste is increasing at an annual rate of 5% - 10%. Landfilling and incineration of solid waste will cause pollution and are not conducive to achieving carbon neutrality. Therefore, it is of great significance to improve the recycling and utilization rate of solid waste. Solid fermentation is one of the effective solutions to improve the utilization rate of solid waste and convert it into value-added products. This process neither requires a large amount of aeration and stirring nor consumes too much water, and the fermented substrate can also be recycled as biological fertilizer and antibacterial feed. Compared with liquid fermentation, it is more economical and environmentally friendly. Currently, solid fermentation has been applied to the production of a variety of different target products, including succinic acid, galactosidase, paclitaxel, tylosin, etc.

[0003] Avermectin is currently the only biopesticide with an annual output value exceeding 3 billion. The main contributors to its derivative ivermectin, Satoshi Ōmura and William C. Campbell, even won the 2015 Nobel Prize in Physiology or Medicine. Avermectin is thus considered one of the greatest discoveries for humanity after penicillin. Currently, avermectin is mainly obtained through liquid fermentation of Streptomyces avermitilis. The fermentation broth is extracted twice with an ethanol solution with a volume nearly 4 times that of the broth, and then the avermectin in the extract is adsorbed onto a column using macroporous resin. After elution and concentration with acetone, an ointment-like product rich in avermectin is obtained. Finally, the ointment is refined with ethanol to obtain avermectin. This process consumes a large amount of water resources and organic reagents, has high energy consumption, high costs and is not environmentally friendly. Compared with liquid fermentation, in the solid fermentation process, there is no need for continuous stirring of the fermenter, and the fermented solid substrate can directly be used as antibacterial and insecticidal feed and fertilizer in agriculture and animal husbandry, which not only reduces power consumption but also saves water resources and organic reagents. At the same time, it is more environmentally friendly than liquid fermentation. Therefore, exploring a low-energy solid fermentation production method for avermectin using cheap raw materials has important practical significance. On the one hand, it can save energy, reduce consumption and lower costs; on the other hand, the fermented solid substrate can be directly used as antibacterial and insecticidal feed or antibacterial fertilizer without generating too much waste residue. Summary of the Invention

[0004] To address the problems of high consumption of water resources and organic reagents, high energy consumption, high cost, and environmental unfriendliness in the production of avermectin by liquid fermentation, and to solve the problem of low utilization rate of agricultural waste, the present invention screens the optimal agricultural waste as the solid fermentation medium for Streptomyces avermitilis, and through the optimization of the solid fermentation medium of Streptomyces avermitilis and fermentation process parameters, develops a green solid fermentation strategy for Streptomyces avermitilis to reduce fermentation costs, reduce equipment energy consumption, waste of water resources, and generation of wastewater and waste residues, thereby realizing the application of avermectin at a lower cost and in a green and environmentally friendly manner.

[0005] To solve the above technical problems and achieve the corresponding technical effects, the present invention provides the following technical solutions:

[0006] The first object of the present invention is to provide a solid medium for fermenting avermectin using agricultural waste, and the solid medium is composed of wheat bran, corncob, earthworm manure, bagasse, cane molasses, ammonium sulfate, and CoCl2.

[0007] In an embodiment of the present invention, wheat bran and corncob in the solid medium serve as carbon sources, and the mass ratio of wheat bran to corncob is 2:1. Earthworm manure serves as a nitrogen source, and the mass ratio of carbon source to nitrogen source is 3:1. The addition amounts of bagasse, cane molasses, and ammonium sulfate are 8.62%, 3%, and 10.07% of the total mass of carbon and nitrogen sources, respectively. The final concentration of CoCl2 is 0.1 mM based on the total mass of carbon and nitrogen sources. The initial water content of the solid medium is 78.5% based on the total mass of carbon and nitrogen sources.

[0008] The second object of the present invention is to provide a preparation method for the above solid medium, and the preparation method includes the following steps:

[0009] Corncob, wheat bran, earthworm manure, and bagasse need to be broken, dried, and sieved respectively before use, then stored in a cold room, and dried to a constant weight before use. Weigh each component according to the composition of the solid medium, and then add water according to the initial water content and mix evenly.

[0010] In an embodiment of the present invention, the corncob, wheat bran, earthworm manure, and bagasse pass through a 1-3 mm filter screen.

[0011] The third object of the present invention is to provide a method for fermenting and producing avermectin using the above solid medium. The method inoculates the seed liquid of Streptomyces avermitilis into the solid medium at an inoculation amount of 25% (v / w), and ferments at 28°C for 12-16 days. The inoculation amount is calculated based on the total mass of carbon and nitrogen sources.

[0012] In an embodiment of the present invention, the Streptomyces avermitilis is NEAU12.

[0013] In one embodiment of the present invention, the method for preparing the seed liquid is to inoculate an agar block with Streptomyces avermitilis spores into a seed medium and ferment and culture it at 28°C and 250 rpm for 40 h.

[0014] In one embodiment of the present invention, the composition of the seed medium is as follows: corn starch 30 g / L, soybean cake powder 8 g / L, peanut cake powder 10 g / L, yeast powder 4 g / L, CoCl2·6H2O 30 mg / L, α-amylase 28 mg / L, and the balance is water.

[0015] In one embodiment of the present invention, the method is single-batch fermentation or continuous repeated batch fermentation.

[0016] In one embodiment of the present invention, the method of continuous repeated batch fermentation is to inoculate the seed liquid of Streptomyces avermitilis into a solid medium at an inoculation amount of 25% (v / w), and perform the first batch of fermentation at 28°C to obtain a fermentation culture; transfer 15% by weight of the fermentation culture to the second batch of solid medium and perform the second batch of fermentation at 28°C; subsequent batches are fermented using the same method until the last batch of fermentation is completed.

[0017] Advantages of the present invention:

[0018] 1. The present invention provides a solid fermentation medium and a solid fermentation method capable of successfully producing avermectin, and through the optimization of the medium and culture conditions, the yield of avermectin B 1a reaches a maximum of 3.83 mg / gds.

[0019] 2. In order to further determine the advantages of solid fermentation using agricultural waste, the costs of solid fermentation and liquid fermentation of avermectin were preliminarily compared in the experiment. Calculated according to the market price (https: / / www.1688.com, December 19, 2024), as shown in Table 1, the total raw material costs of liquid fermentation and solid fermentation are estimated to be $0.119 per liter and $0.103 per kilogram, respectively. In our study, the highest yields of avermectin B 1a by liquid fermentation and solid fermentation were 3.70 g / L and 3.83 mg / gds, respectively. On this premise, 1 kilogram of avermectin B 1aThe raw material production cost through solid-state fermentation ($26.222) is 18.24% lower than that through liquid fermentation ($32.071) (Table 1). Industrial models for the annual production of 10 tons of avermectin by SSF and SmF were established and simulated using SuperPro Designer V10.7 software. By comprehensively calculating the costs of raw materials, equipment, and utilities, it was found that the total cost of solid-state fermentation is approximately 8.38% lower than that of liquid fermentation (Table 2). Although the time required for solid-state fermentation to reach the same production level is 1.4 times that of liquid fermentation, the efficiency of liquid fermentation is achieved with an increase in energy consumption due to the need for rapid stirring and efficient mass transfer. In contrast, solid-state fermentation provides a low-cost, energy-saving, and environmentally friendly alternative. The solid-state fermentation of avermectin reduces environmental pollution through the reuse of agricultural waste while reducing costs. Moreover, the solid waste generated by solid-state fermentation can be directly used as biofertilizer and antibacterial feed, without generating a large amount of wastewater and waste residue. Therefore, the solid-state fermentation of Streptomyces avermitilis is a more economical and environmentally friendly production method.

[0020] Table 1 Preliminary comparison of the medium costs for solid-state fermentation and liquid fermentation of avermectin

[0021]

[0022]

[0023] Note: a The production in solid-state fermentation is based on the amount per liter of liquid medium; b The production in solid-state fermentation is based on the amount per kilogram of solid substrate.

[0024] Table 2 Comparison of the total costs for solid-state fermentation and liquid fermentation of avermectin

[0025]

[0026] Note: a The avermectin B of SSF and SmF 1a The production is analyzed at 3.83 mg / gds and 3.70 g / L respectively. Description of the Drawings

[0027] Figure 1 It is a result graph showing the effect of different carbon sources in the solid-state fermentation medium on the production of avermectin B 1a ;

[0028] Figure 2 It is a result graph showing the effect of different nitrogen sources in the solid-state fermentation medium on the production of avermectin B 1a ;

[0029] Figure 3Results graph of the effect of the mass ratio of wheat bran to corncob in the mixture of wheat bran and corncob as the carbon source in the solid fermentation medium on the yield of avermectin B 1a ;

[0030] Figure 4 Results graph of the effect of different carbon / nitrogen source ratios in the solid fermentation medium on the yield of avermectin B 1a ;

[0031] Figure 5 Results graph of the effect of different aeration and water-permeable materials in the solid fermentation medium on the yield of avermectin B 1a ; Among them, Figure 5 a in 1a is the results graph of the effect of different addition amounts of rice husk as the aeration and water-permeable material in the solid fermentation medium on the yield of avermectin B Figure 5 b in 1a is the results graph of the effect of different addition amounts of wheat straw as the aeration and water-permeable material in the solid fermentation medium on the yield of avermectin B Figure 5 c in 1a is the results graph of the effect of different addition amounts of bagasse as the aeration and water-permeable material in the solid fermentation medium on the yield of avermectin B 1a ; Control is the yield of avermectin B when the addition amount of the aeration and water-permeable material is the same as that of the basic medium

[0032] Figure 6 Results graph of the effect of the content of the auxiliary carbon source in the solid fermentation medium on the yield of avermectin B 1a ; Control is the yield of avermectin B when using the basic medium 1a ;

[0033] Figure 7 Results graph of the effect of the content of the auxiliary nitrogen source (NH4)SO4 in the solid fermentation medium on the yield of avermectin B 1a ;

[0034] Figure 8 Results graph of the effect of different trace elements in the solid fermentation medium on the yield of avermectin B 1a ; Control is the yield of avermectin B when using the basic medium 1a ;

[0035] Figure 9 Results graph of the effect of different water contents in the solid fermentation medium on the yield of avermectin B 1a ;

[0036] Figure 10 Results graph of the effect of different inoculation amounts in the solid fermentation medium on the yield of avermectin B 1a ;

[0037] Figure 11 is the contour map and 3D response surface map of the interaction between the independent variable and avermectin B 1a production; wherein, Figure 11 a in is the result map of the influence of the initial water content of the solid medium and the bagasse content on the avermectin B 1a production, Figure 11 b in is the result map of the influence of the initial water content of the solid medium and the ammonium sulfate content on the avermectin B 1a production, Figure 11 c in is the result map of the influence of the bagasse content and the ammonium sulfate content of the solid medium on the avermectin B 1a production;

[0038] Figure 12 is the result map of the avermectin B 1a production obtained by solid fermentation using a 250 mL shake flask under the optimal fermentation conditions with the optimal solid fermentation medium obtained in Example 1;

[0039] Figure 13 is the result map of the avermectin B 1a production obtained by solid fermentation using a 5 L flask under the optimal fermentation conditions with the optimal solid fermentation medium obtained in Example 1;

[0040] Figure 14 is the result map of the avermectin B 1a production obtained by solid fermentation using a 5.6 L tray under the optimal fermentation conditions with the optimal solid fermentation medium obtained in Example 1;

[0041] Figure 15 is the result map of the avermectin B 1a production obtained by repeated batch solid fermentation using a 5 L flask under the optimal fermentation conditions with the optimal solid fermentation medium obtained in Example 1;

[0042] Figure 16 is the result map of the avermectin B 1a production obtained by repeated batch solid fermentation using a 5.6 L tray under the optimal fermentation conditions with the optimal solid fermentation medium obtained in Example 1. Detailed implementation manners

[0043] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific implementation manners and the accompanying drawings of the specification. It should be noted that the embodiments mentioned below are only applicable to explaining the present invention, but not for limiting the scope of the present invention. The embodiments mentioned below are only a part of the embodiments of the present invention rather than all the embodiments. In the art, if other technical personnel do not make creative efforts, the embodiments obtained by them are protected by the present invention.

[0044] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, culture media and instruments used are all conventional materials, reagents, culture media and instruments in this field and can be obtained by those skilled in the art through commercial channels without special instructions.

[0045] The sources of the strains used in the present invention are as follows:

[0046] The Streptomyces avermitilis used in the present invention is Streptomyces avermitilis NEAU12, which was randomly mutagenized from Streptomyces avermitilis S0. The strain NEAU12 was disclosed in Jin, P., Li, S., Zhang, Y., Chu, L., He, H., Dong, Z., & Xiang, W. (2020). Mining and fine-tuning sugar uptake system for titer improvement of milbemycins in Streptomyces bingchenggensis. Synthetic and systems biotechnology, 5(3), 214–221. https: / / doi.org / 10.1016 / j.synbio.2020.07.001.

[0047] The culture media related to the present invention are as follows:

[0048] The composition of the solid fermentation basal medium is as follows: 5 g of corncob, 2.5 g of earthworm manure, 0.225 g of rice husk, 0.3 g of cane molasses, and 0.225 g of ammonium sulfate.

[0049] The detection methods related to the present invention are as follows:

[0050] Detection method of avermectin: To determine the yield of avermectin, 0.3 g of solid fermentation culture was mixed with 1 mL of methanol and ultrasonically treated for 40 min to extract the total avermectin and extracellular avermectin in the fermentation broth. Subsequently, the ultrasonically treated sample was centrifuged at a high speed of 12000 rpm for 15 min, and the upper avermectin extract was aspirated and filtered through a 0.22 μM organic filter membrane for liquid phase detection. The detection instrument was Shimadzu HPLC (Shimadzu LC), the chromatographic column was a C18 column (Zorbax, 4.6 mm × 250 mm, 5 μm), the detection wavelength was 246 nm, the injection volume was 20 μL, the mobile phase was a methanol solution with a volume fraction of 90%, the flow rate was 1.0 mL / min, and the column temperature was 35°C.

[0051] Example 1: Optimization of solid fermentation medium and fermentation conditions for the production of avermectin by Streptomyces avermitilis

[0052] This embodiment provides a method for solid-state fermentation of avermectin using Streptomyces avermitilis, and provides the optimization process of the solid-state fermentation medium and the optimization process of the fermentation conditions used in this method.

[0053] (1) A method for solid-state fermentation of avermectin using Streptomyces avermitilis, which comprises the following steps:

[0054] 1. Preparation of seed liquid

[0055] 1) Preparation of seed medium:

[0056] The composition of the seed medium is as follows: corn starch 30 g / L, soybean cake powder 8 g / L, peanut cake powder 10 g / L, yeast powder 4 g / L, CoCl2·6H2O 30 mg / L, α-amylase 28 mg / L, and the balance is water.

[0057] The preparation method of the seed medium is as follows: calculate the material consumption by volume, add corn starch and α-amylase to 200 mL / L of water, heat to 50 - 70 °C, stir continuously until clear and transparent, then add other raw materials, mix well and make up the volume, dispense into 250 mL Erlenmeyer flasks, with the liquid volume of 25 mL, sterilize by moist heat at 114 - 116 °C for 30 min, and reserve for use.

[0058] 2) Seed culture:

[0059] Inoculate the agar block with Streptomyces avermitilis spores into the sterilized seed medium cooled to 28 °C, and ferment and culture at 28 °C and 250 rpm for 40 h.

[0060] 2. Solid-state fermentation

[0061] 1) Preparation of solid-state fermentation medium:

[0062] The composition of the solid-state fermentation medium is as follows: carbon source (wheat bran / liquor residue / rice bran / corn cob / rice husk / corn straw), nitrogen source (soybean meal / rapeseed cake / chicken manure / mushroom residue / earthworm manure), aeration and water-permeable material (rice husk / wheat straw / sugarcane bagasse), auxiliary carbon source (cane molasses), auxiliary nitrogen source (ammonium sulfate).

[0063] The preparation method of the solid-state fermentation medium is as follows: Corn cob, rice bran, wheat bran, liquor residue, rice husk, corn straw, mushroom residue, soybean meal, earthworm manure, chicken manure and rapeseed cake need to be broken and dried before use, and then sieved through a sieve with a diameter of 1 mm for use. Sugarcane bagasse, rice husk and wheat straw used as aeration and water-permeable materials need to be broken and dried first, and then sieved through a sieve, and the base materials with a diameter of 1 - 3 mm are selected for subsequent experiments. The ground residues are stored in a cold room (4 °C) and dried to constant weight at 60 °C before use.

[0064] 2) Solid state fermentation culture:

[0065] The seed liquid prepared in step 1 was inoculated into a 250 mL Erlenmeyer flask containing a solid fermentation medium with an initial water content of 50% (calculated based on the total mass of the carbon and nitrogen sources) that had been sterilized and cooled to 28°C at an inoculation amount of 15% (v / w) (the inoculation amount was calculated based on the total mass of the carbon and nitrogen sources). The substrate was loaded at 7.5 g according to the total amount of carbon and nitrogen sources and cultured at 28°C for 10 days.

[0066] (II) Optimization of the solid state fermentation medium

[0067] 1. Optimization of the carbon source

[0068] The seed liquid of Streptomyces avermitilis was prepared according to the method in (I) above, and the seed liquid was inoculated into the solid fermentation medium for solid state fermentation culture using the same method and conditions. The only difference was to change the composition of the solid fermentation medium: the carbon sources were respectively selected as corncob, rice bran, wheat bran, distiller's grains residue, rice husk, and corn straw with the same dosage (5 g), the nitrogen source was selected as earthworm manure (2.5 g), the aeration and water-permeable material was selected as rice husk (0.225 g), the auxiliary carbon source was cane molasses (0.3 g), and the auxiliary nitrogen source was ammonium sulfate (0.225 g).

[0069] After the solid state fermentation was completed, the yield of avermectin B 1a was measured. As Figure 1 could be seen, when corncob was selected as the carbon source, the corresponding yield of avermectin B 1a was the highest, followed by wheat bran. Therefore, corncob was selected as the optimal carbon source.

[0070] 2. Optimization of the nitrogen source

[0071] The seed liquid of Streptomyces avermitilis was prepared according to the method in (I) above, and the seed liquid was inoculated into the solid fermentation medium for solid state fermentation culture using the same method and conditions. The only difference was to change the composition of the solid fermentation medium: the carbon source was selected as corncob (5 g), the nitrogen sources were respectively selected as soybean meal, rapeseed cake, chicken manure, mushroom residue, and earthworm manure with the same dosage (2.5 g), the aeration and water-permeable material was selected as rice husk (0.225 g), the auxiliary carbon source was cane molasses (0.3 g), and the auxiliary nitrogen source was ammonium sulfate (0.225 g).

[0072] After the solid state fermentation was completed, the yield of avermectin B 1a was measured. As Figure 2 could be seen, when earthworm manure was selected as the nitrogen source, the corresponding yield of avermectin B 1a was the highest. Therefore, earthworm manure was selected as the optimal nitrogen source.

[0073] 3. Optimization of the mixed carbon source

[0074] Prepare the seed liquid of Streptomyces avermitilis according to the method in (1) above, and inoculate the seed liquid into the solid fermentation medium for solid fermentation culture using the same method and conditions. The only difference is to change the composition of the solid fermentation medium: the carbon source is 5 g of wheat bran and corncob in total, and the mixing ratio is (3:1, 2:1, 1:1, 1:2, 1:3); the nitrogen source is earthworm manure (2.5 g); the aeration and water-permeable material is rice husk (0.225 g); the auxiliary carbon source is cane molasses (0.3 g); the auxiliary nitrogen source is ammonium sulfate (0.225 g).

[0075] After the solid fermentation is completed, measure the yield of avermectin B 1a It can be seen from Figure 3 that when the mass ratio of wheat bran to corncob is 2:1, the corresponding yield of avermectin B 1a is the highest. Therefore, select wheat bran and corncob with a mass ratio of 2:1 as the optimal mixed carbon source.

[0076] 4. Optimization of carbon-nitrogen ratio

[0077] Prepare the seed liquid of Streptomyces avermitilis according to the method in (1) above, and inoculate the seed liquid into the solid fermentation medium for solid fermentation culture using the same method and conditions. The only difference is to change the composition and ratio of the solid fermentation medium: the carbon source is corncob, the nitrogen source is earthworm manure, and the mass ratios of corncob to earthworm manure are selected as 5:1, 3:1, 2:1, 1:1, and 1:2 respectively (the total amount of carbon source and nitrogen source is 7.5 g). The medium also contains 0.225 g of rice husk, 0.3 g of cane molasses, and 0.225 g of ammonium sulfate.

[0078] After the solid fermentation is completed, measure the yield of avermectin B 1a It can be seen from Figure 4 that when the mass ratio of corncob to earthworm manure is 3:1, the corresponding yield of avermectin B 1a is the highest. Therefore, select 3:1 as the optimal carbon-nitrogen ratio.

[0079] 5. Optimization of aeration and water-permeable material

[0080] Prepare the seed liquid of Streptomyces avermitilis according to the method in (1) above, and inoculate the seed liquid into the solid fermentation medium for solid fermentation culture using the same method and conditions. The only difference is to change the composition of the solid fermentation medium: 5 g of corncob, 2.5 g of earthworm manure, and the aeration and water-permeable materials are respectively selected as bagasse, wheat straw or rice husk with 2%, 4%, 6%, 8%, and 10% of the total carbon and nitrogen sources, 0.3 g of cane molasses, and 0.225 g of ammonium sulfate. The Control group means that the addition amount of the aeration and water-permeable material is the same as that of the basic medium.

[0081] After the solid fermentation is completed, measure the yield of avermectin B 1aThe yield of Figure 5 As can be seen from Figure 5 , when bagasse is selected as the aeration and water infiltration material and its content is limited to 6%, the corresponding avermectin B 1a has the highest yield. Therefore, bagasse is selected as the optimal aeration and water infiltration material, and its optimal content is limited to 6%.

[0082] 6. Optimization of the content of auxiliary carbon source

[0083] Prepare the seed liquid of Streptomyces avermitilis according to the method in (1) above, and inoculate the seed liquid into the solid fermentation medium for solid fermentation culture under the same method and conditions. The difference is only to change the components of the solid fermentation medium: 5 g of corncob, 2.5 g of earthworm manure, 0.225 g of rice husk, and the contents of cane molasses are respectively selected as 0%, 3%, 6%, 9% and 12% of the total carbon and nitrogen sources, and 0.225 g of ammonium sulfate. Use the solid fermentation basal medium as the Control group.

[0084] After the solid fermentation is completed, measure the yield of avermectin B 1a The yield of Figure 6 As can be seen from Figure 6 , when the content of cane molasses is 3%, the corresponding avermectin B 1a has the highest yield. Therefore, 3% is selected as the optimal content of cane molasses.

[0085] 7. Optimization of the content of auxiliary nitrogen source

[0086] Prepare the seed liquid of Streptomyces avermitilis according to the method in (1) above, and inoculate the seed liquid into the solid fermentation medium for solid fermentation culture under the same method and conditions. The difference is only to change the components of the solid fermentation medium: 5 g of corncob, 2.5 g of earthworm manure, 0.225 g of rice husk, 0.3 g of cane molasses, and the contents of ammonium sulfate are respectively selected as 0%, 3%, 6%, 9% and 12% of the total carbon and nitrogen sources.

[0087] After the solid fermentation is completed, measure the yield of avermectin B 1a The yield of Figure 7 As can be seen from Figure 7 , when the content of ammonium sulfate is 6%, the corresponding avermectin B 1a has the highest yield. Therefore, 6% is selected as the optimal content of ammonium sulfate.

[0088] 8. Optimization of trace elements

[0089] Prepare the seed liquid of Streptomyces avermitilis according to the method in (1) above, and inoculate the seed liquid into the solid fermentation medium for solid fermentation culture under the same method and conditions. The only difference is to change the composition of the solid fermentation medium: 5 g of corncob, 2.5 g of earthworm manure, 0.225 g of rice husk, 0.3 g of cane molasses, 0.225 g of ammonium sulfate, and select trace elements of MnSO4, CoCl2, MgSO4, Na2MoO4, FeSO4 and CaCl2 with a final concentration of 0.1 mM respectively. The final concentration is calculated based on the total mass of the carbon and nitrogen sources.

[0090] After the solid fermentation is completed, measure the yield of avermectin B 1a From Figure 8 it can be seen that when CoCl2 is selected as the trace element, the corresponding yield of avermectin B 1a is the highest. Therefore, CoCl2 is selected as the best trace element.

[0091] 9. Optimization of water content

[0092] Prepare the seed liquid of Streptomyces avermitilis according to the method in (1) above, and inoculate the seed liquid into the solid fermentation medium for solid fermentation culture under the same method and conditions. The solid fermentation medium is selected as the above basic medium, and the initial water content of the solid fermentation medium (calculated based on the total weight of the carbon and nitrogen sources) is respectively selected as 30%, 50%, 70% and 90%.

[0093] After the solid fermentation is completed, measure the yield of avermectin B 1a From Figure 9 it can be seen that when the initial water content of the solid fermentation medium is 70%, the corresponding yield of avermectin B 1a is the highest. Therefore, 70% is selected as the best initial water content of the solid fermentation medium.

[0094] (3) Optimization of solid fermentation conditions

[0095] Optimization of inoculum size

[0096] Prepare the seed liquid of Streptomyces avermitilis according to the method in (1) above, and inoculate the seed liquid into the solid fermentation medium for solid fermentation culture under the same method and conditions. The solid fermentation medium is selected as the above basic medium, and the inoculum size of the seed liquid is respectively set as 5%, 15%, 25%, 35% and 50% (v / w) (the inoculum size is calculated based on the total mass of the carbon and nitrogen sources).

[0097] After the solid fermentation is completed, measure the yield of avermectin B 1a From Figure 10 it can be seen that when the inoculum size of the seed liquid is 25%, the corresponding yield of avermectin B 1a is the highest. Therefore, 25% is selected as the best inoculum size of the seed liquid.

[0098] (4) Optimization of solid fermentation method based on central composite experimental design

[0099] The present invention uses the Plackett - Burman design (PBD) method to screen the key factors for solid - state fermentation to produce avermectin. Based on single - factor analysis, six factors including C / N ratio, content of auxiliary carbon source, content of auxiliary nitrogen source, content of aeration and water - permeable material, initial water content of the medium, and inoculum amount were investigated in the PBD experiment, and the levels of each factor were determined according to the single - factor test. Based on the PBD results, the central composite design (CCD) was used to further optimize the composition of the medium for solid - state fermentation to produce avermectin. Each key factor was evaluated at five different levels. Analysis of variance (ANOVA) was used to test statistical significance. Further experiments verified the optimal culture conditions for solid - state fermentation to produce avermectin. Both PBD and CCD were designed using Design Expert (Version12).

[0100] This continuous optimization strategy was adopted to increase the yield of avermectin in solid - state fermentation. Preliminary screening using PBD identified the key factors affecting fermentation efficiency (see Tables 3 and 4). Experimental data revealed significant differences in the yield of avermectin B 1a under different culture conditions, highlighting the necessity of systematic optimization (see Table 4).

[0101] Based on the results of PBD, a regression model (Eq.1) was established to quantify the relationship between culture conditions and yield:

[0102] Y = 0.5968 + 0.0117X1 - 0.0094X2 + 0.1379X3 + 0.1352X4 + 0.0274X5 + 0.1732X6 (Eq.1)

[0103] where Y is the predicted response (yield of avermectin B 1a ), X1 is the C / N ratio, X2 is the content of cane molasses, X3 is the content of bagasse, X4 is the content of ammonium sulfate, X5 is the inoculum amount, and X6 is the water content. Statistical analysis showed that the p - value of this model was less than 0.0001, and the lack - of - fit F - value was 2.43. In addition, the R 2 , adjusted R 2 and predicted R 2 of this model were 90.75%, 86.12% and 73.67% respectively, demonstrating the excellent stability of this model and its applicability in navigating the design space. This model pointed out three important factors affecting the yield of avermectin: water content, bagasse content, and ammonium sulfate content (see Table 5).

[0104] To determine the optimal levels of these key factors, CCD (see Table 6) was adopted. Based on the experimental data, the quadratic model is as follows in the regression equation (Eq. 2):

[0105] Y = 0.9318 + 0.1463X6 + 0.1444X3 + 0.0803X4 - 0.0281X6X3 + 0.0056X6X4 - 0.0014X3X4 - 0.1114X6 2 - 0.1133X3 2 - 0.0589X4 2 (Eq. 2)

[0106] where Y is the predicted response (avermectin B 1a yield), X3 is the bagasse content, X4 is the ammonium sulfate content, and X6 is the moisture content. The p - value of this model is less than 0.0001, and the lack - of - fit F - value is 4.23. The R 2 , adjusted R 2 and predicted R 2 are 98.27%, 96.32% and 80.67% respectively, indicating that this model is qualified (see Table 7). This model predicts that the maximum predicted value of avermectin B 1a yield may reach 0.99 mg / gds under the following conditions (see Figure 11 ): The carbon source is a mixture of wheat bran and corncob with a mass ratio of 2:1, the nitrogen source is earthworm manure, and the mass ratio of carbon to nitrogen source is 3:1; The aeration and water - permeable material is bagasse, and the addition amount is 8.62% of the total mass of the carbon and nitrogen sources; The auxiliary carbon source is molasses, and the addition amount is 3% of the total mass of the carbon and nitrogen sources; The auxiliary nitrogen source is ammonium sulfate, and the addition amount is 10.07% of the total mass of the carbon and nitrogen sources; The final concentration of trace element CoCl2 is 0.1 mM, the inoculation amount is 25% (v / w t ) and the initial water content is 78.5%. The final concentration of trace element CoCl2, the inoculation amount and the initial water content are all calculated based on the total mass of the carbon and nitrogen sources. Under these conditions, the actual yield of avermectin B 1a reaches 1.22 mg / gds on the 10th day, which is about 3 times the yield under the fermentation conditions of using the basal medium with a water content of 50% and an inoculation amount of 15%.

[0107] Table 3 Levels of the test factors in the Plackett - Burman design experiment

[0108]

[0109] Table 4 Plackett - Burman design matrix and the corresponding yields of avermectin B 1a

[0110] ​

[0111] ANOVA Variance Analysis of the Selection Factor Model in Table 5

[0112]

[0113]

[0114] Central Composite Design for Medium Optimization in Table 6

[0115]

[0116] ANOVA Variance Analysis of the Quadratic Model in Table 7

[0117]

[0118]

[0119] Example 2: Solid State Fermentation Experiment in 250 mL Erlenmeyer Flasks

[0120] Using the optimal solid medium obtained in Example 1, with an initial water content of 78.5%, inoculate 25% of the seed solution, and keep other fermentation conditions the same as in (1) of Example 1. Conduct a solid state fermentation experiment in 250 mL Erlenmeyer flasks (loading amount: 7.5 g of carbon source + nitrogen source). During the experiment, collect solid fermentation samples every two days for the detection of the yield of avermectin B 1a Through the detection of the yield of avermectin B 1a It is found that after 14 days of solid state fermentation, the highest yield of avermectin B 1a reaches 1.89 mg / gds (see Figure 12 ).

[0121] Example 3: Solid State Fermentation Experiment in 5 L Flasks

[0122] According to the optimal solid medium obtained in Example 1, prepare 150 g (calculated based on 150 g of carbon and nitrogen sources) of solid medium, load it into a 5 L flask, with an initial water content of 78.5%, inoculate 25% of the seed solution, and keep other fermentation conditions the same as in (1) of Example 1. Conduct a solid state fermentation experiment. During the experiment, collect solid fermentation samples every two days for the detection of the yield of avermectin B 1a Through the detection of the yield of avermectin B 1a It is found that in the 5 L flask, after 14 days of solid state fermentation, the highest yield of avermectin B 1a reaches 2.47 mg / gds (see Figure 13 ).

[0123] Example 4: Solid State Fermentation Experiment in 5.6 L Trays

[0124] The optimal solid medium obtained according to Example 1 was used to prepare 150 g (calculated based on 150 g of carbon and nitrogen sources) of solid medium, which was placed in a 5.6 L tray. Under the condition of an initial water content of 78.5%, 25% of the seed liquid was inoculated, and other fermentation conditions were the same as those in (1) of Example 1. A solid fermentation experiment was carried out. During the experiment, solid fermentation samples were collected every two days for the detection of the yield of avermectin B 1a The yield of avermectin B was detected. By detecting the yield of avermectin B 1a , it was found that in the 5.6 L tray, after 14 days of solid fermentation, the highest yield of avermectin B 1a reached 3.64 mg / gds, which was 47.4% higher than the yield in the 5 L flask (see Figure 14 ).

[0125] Example 5: Repeated batch fermentation experiment in a 5 L flask

[0126] The fermentation method of the first batch of repeated batch fermentation in a 5 L flask was the same as that in Example 3. After one batch of fermentation, approximately 15% by weight of the fermentation culture was transferred to another flask. The specific method was as follows: The weight of the empty flask was recorded before fermentation, and the total weight of the flask and the fermentation culture was weighed after fermentation to calculate the weight of the fermentation culture. Then, a 0.5 mm top layer was removed with a spatula, and 15% by weight of the solid fermentation culture was weighed from the remaining culture with a long-handled sterilized spoon and transferred to a new flask containing the same mass of medium. Each batch of fermentation lasted for 14 days, and the entire process of repeated batch solid fermentation was repeated for 84 days. It was shaken or rotated three times a day to improve the aeration of the medium. The production parameters of each cycle are shown in Table 8. The results showed that for the repeated batch fermentation in a 5 L flask, the lag phase of the subsequent batches was shorter than that of the first batch, the high productivity was maintained for up to four cycles, and the maximum yield in the repeated batch culture was 8% higher than that in the batch culture (see Figure 15 ).

[0127] Example 6: Repeated batch fermentation experiment in a 5.6 L tray

[0128] The first batch fermentation method for repeated batch fermentation in a 5.6 L tray is the same as that in Example 4. After one batch of fermentation, approximately 15% by weight of the fermentation culture is transferred to another tray. The specific method is as follows: Record the weight of the empty tray before fermentation. After fermentation is completed, weigh the total weight of the tray and the fermentation culture to calculate the weight of the fermentation culture. Then, scrape off the top layer of 0.5 mm, and weigh 15% by weight of the solid fermentation culture from the remaining culture using a long-handled sterilized spoon and transfer it to a new tray containing the same mass of medium. Each batch of fermentation lasts for 14 days, and the entire process of repeated batch solid fermentation is repeated for 84 days. Shake or rotate three times a day to improve the aeration of the medium. The production parameters for each cycle are shown in Table 8. The results show that for repeated batch fermentation in a 5.6 L tray, the lag phase of subsequent batches is shorter than that of the first batch, high productivity is maintained for up to four cycles, and the maximum yield in repeated batch culture is 5% higher than that in batch culture. In shallow tray repeated batch culture, the highest yield of avermectin B 1a is 3.83 mg / gds, which is also the highest yield reported for solid fermentation of avermectin. (See Figure 16 ).

[0129] Table 8 Parameters for the production of avermectin B by repeated batch solid fermentation in a 5 L flask and a 5.6 L tray 1a

[0130]

[0131] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.​

Claims

1. A solid medium for fermenting avermectin using agricultural waste, characterized in that, The solid medium is composed of wheat bran, corncob, earthworm manure, bagasse, cane molasses, ammonium sulfate and CoCl2.

2. The solid culture medium according to claim 1, wherein In the solid medium, wheat bran and corncob serve as carbon sources, and the mass ratio of wheat bran to corncob is 2:

1. Earthworm manure serves as a nitrogen source, and the mass ratio of carbon source to nitrogen source is 3:

1. The addition amounts of bagasse, cane molasses and ammonium sulfate are 8.62%, 3% and 10.07% of the total mass of carbon and nitrogen sources respectively. The final concentration of CoCl2 is 0.1 mM based on the total mass of carbon and nitrogen sources. The initial water content of the solid medium is 78.5% based on the total mass of carbon and nitrogen sources.

3. The preparation method of the solid culture medium according to claim 1 or 2, characterized in that, It includes the following steps: Before use, corncob, wheat bran, earthworm manure and bagasse need to be broken, dried and sieved respectively, then stored in a cold room and dried to constant weight before use. Weigh each component according to the composition of the solid medium, and then add water according to the initial water content and mix evenly.

4. The preparation method according to claim 3, characterized in that, The corncob, wheat bran, earthworm manure and bagasse pass through a 1 - 3 mm sieve.

5. A method for fermenting and producing avermectin using the solid culture medium described in claim 1 or 2, characterized in that, Inoculate the seed liquid of Streptomyces avermitilis into the solid medium at an inoculation amount of 25% (v / w), and ferment at 28 °C for 12 - 16 d. The inoculation amount is calculated based on the total mass of carbon and nitrogen sources.

6. The method according to claim 5, characterized in that, The Streptomyces avermitilis is NEAU12.

7. The method according to claim 5, characterized in that, The preparation method of the seed liquid is to inoculate an agar block with Streptomyces avermitilis spores into the seed medium and ferment and culture at 28 °C and 250 rpm for 40 h.

8. The method according to claim 7, wherein The composition of the seed medium is as follows: corn starch 30 g / L, soybean cake powder 8 g / L, peanut cake powder 10 g / L, yeast powder 4 g / L, CoCl2·6H2O 30 mg / L, α - amylase 28 mg / L, and the balance is water.

9. The method according to claim 5, wherein The method is single - batch fermentation or continuous repeated batch fermentation.

10. The method according to claim 9, wherein, The method of continuous repeated batch fermentation is to inoculate the seed liquid of Streptomyces avermitilis into the solid medium at an inoculation amount of 25% (v / w), and carry out the first - batch fermentation at 28 °C to obtain the fermentation culture. Transfer 15% by weight of the fermentation culture to the second - batch solid medium and carry out the second - batch fermentation at 28 °C. The subsequent batches are fermented in the same way until the last batch of fermentation is completed.

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