Solid medium for fermentation of avermectin using agricultural waste and application thereof

Abstract

The application discloses a solid culture medium for fermenting avermectin by using agricultural wastes and an application thereof, and belongs to the technical field of microbial fermentation. In order to solve problems of large water resource and organic reagent consumption, high energy consumption, high cost, environmental unfriendliness and the like in the production of avermectin by using liquid fermentation, and to solve the problem of low utilization rate of agricultural wastes, the optimal agricultural wastes are screened as the solid fermentation culture medium of Streptomyces avermitilis, and the green solid fermentation strategy of Streptomyces avermitilis is developed by optimizing the solid fermentation culture medium of Streptomyces avermitilis and fermentation process parameters, so as to reduce the fermentation cost, reduce the waste of equipment energy consumption, water resource and the generation of waste water and waste residue, and to realize the application of avermectin at a lower cost and in a green and environmental-friendly manner.

Description

TECHNICAL FIELD

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

[0002] China produces about 40 billion tons of agricultural waste per year, which is one of the largest countries in the world in terms of agricultural waste production, and the agricultural waste is increasing at an average annual rate of 5% to 10%. Landfilling and incineration of solid waste can cause pollution and is 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 does not require a large amount of aeration and stirring, nor does it consume excessive water. The substrate after fermentation can also be recycled as biofertilizer and antibacterial feed. Compared with liquid fermentation, it is more economical and environmentally friendly. Solid fermentation has been applied to the production of a variety of different target products, including succinic acid, galactosidase, paclitaxel and tylosin.

[0003] Avermectin is currently the only biological pesticide with an annual output value of more than 3 billion. The main contributors of its derivative ivermectin, Satoshi Omura and William Campbell, even won the Nobel Prize in Physiology or Medicine in 2015. Avermectin is therefore considered one of the greatest discoveries for mankind after penicillin. Currently, avermectin is mainly obtained by liquid fermentation of Streptomyces avermitilis in production. The fermentation broth is extracted twice with nearly 4 times the volume of ethanol solution, and then the avermectin in the extracted solution is adsorbed onto a column using a macroporous resin. After elution with acetone and concentration, an oil paste product rich in avermectin is obtained, and finally the oil paste is refined with ethanol to obtain avermectin. This process consumes a lot of water resources and organic reagents, has high energy consumption, high cost and is not environmentally friendly. Compared with liquid fermentation, the fermentation tank does not need to be stirred all the time in the solid fermentation process, and the solid substrate after fermentation can be directly used as antibacterial and insect-resistant 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, compared with liquid fermentation, it is more environmentally friendly. Therefore, it is of great practical significance to explore a low-energy solid fermentation production method for avermectin using cheap raw materials. On the one hand, it can save energy, reduce consumption and reduce cost; on the other hand, the solid substrate after fermentation can be directly used as antibacterial and insect-resistant feed or antibacterial fertilizer, without generating too much waste residue. SUMMARY

[0004] In order to solve the problems of large water resource and organic reagent consumption, high energy consumption, high cost and environmental unfriendliness in the production of abamectin by liquid fermentation, and to solve the problem of low utilization rate of agricultural waste, the optimal agricultural waste is screened as the solid fermentation medium of Streptomyces avermitilis, and through optimization of the solid fermentation medium of Streptomyces avermitilis and fermentation process parameters, a green solid fermentation strategy of Streptomyces avermitilis is developed, so as to reduce the fermentation cost, reduce the waste of equipment energy consumption, water resource and the generation of waste water and waste residue, thereby realizing the application of abamectin with lower cost and green environmental protection.

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

[0006] The first object of the present application is to provide a solid medium for fermenting abamectin by using agricultural waste, which is composed of wheat bran, corn cob, earthworm manure, sugarcane residue, sugarcane molasses, ammonium sulfate and CoCl2.

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

[0008] The second object of the present application is to provide a preparation method of the above-mentioned solid medium, which comprises the following steps:

[0009] The corn cob, the wheat bran, the earthworm manure and the sugarcane residue need to be respectively crushed, dried and sieved before use, and then are stored in a cold room and dried to a constant weight before use; each component is weighed according to the composition of the solid medium, and then water is added according to the initial water content and mixed uniformly.

[0010] In an embodiment of the present application, the corn cob, the wheat bran, the earthworm manure and the sugarcane residue are sieved through a 1-3 mm filter screen.

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

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

[0013] In an embodiment of the present application, the seed liquid is prepared by inoculating agar blocks with spores of Streptomyces avermitilis into seed culture medium and incubating at 28℃, 250rpm for 40h.

[0014] In an embodiment of the present application, the composition of the seed culture medium is as follows: corn starch 30g / L, soybean meal 8g / L, peanut meal 10g / L, yeast powder 4g / L, CoCl2·6H2O 30mg / L, alpha amylase 28mg / L, and the rest is water.

[0015] In an embodiment of the present application, the method is single batch fermentation or continuous repeated batch fermentation.

[0016] In an embodiment of the present application, the method of continuous repeated batch fermentation is as follows: inoculate Streptomyces avermitilis seed liquid into solid culture medium at an inoculation amount of 25%(v / w), and perform the first batch fermentation at 28℃ to obtain fermentation culture; transfer 15% by weight of the fermentation culture into the second batch of solid culture medium, and perform the second batch fermentation at 28℃; and subsequently use the same method until the last batch of fermentation is completed.

[0017] Advantages of the present application:

[0018] 1. The present application provides a solid fermentation culture medium and a solid fermentation method that can successfully produce avermectin, and through optimization of the culture medium and culture conditions, the yield of avermectin B 1a can reach up to 3.83mg / gds.

[0019] 2. In order to further determine the advantages of solid fermentation using agricultural waste, the cost of avermectin solid fermentation and liquid fermentation was preliminarily compared. According to the market price (https: / / www.1688.com, December 19, 2024), the total cost of raw materials for liquid fermentation and solid fermentation is estimated to be $0.119 per liter and $0.103 per kilogram, respectively, as shown in Table 1. In our study, the highest yield of avermectin B 1a by liquid fermentation and solid fermentation was 3.70g / L and 3.83mg / gds, respectively, and under this premise, the cost of 1kg of avermectin B 1aThe raw material cost for solid state fermentation (26.222 USD) was 18.24% lower than that for liquid fermentation (32.071 USD) (Table 1). The industrial models for SSF and SmF producing 10 tons of avermectin per year were established and simulated using SuperPro Designer V10.7 software, and the total cost of raw materials, equipment and utilities was calculated, and it was found that the total cost of solid fermentation was about 8.38% lower than that of liquid fermentation (Table 2). Although the time required for solid fermentation to achieve the same yield was 1.4 times that of liquid fermentation, the efficiency of liquid fermentation was achieved at the cost of increased energy consumption, as rapid stirring and effective mass transfer were required. In contrast, solid fermentation provides a low-cost, energy-saving and environmentally friendly alternative. Solid state fermentation of avermectin reduces environmental pollution by reusing agricultural waste while reducing costs. And the solid waste produced by solid state fermentation can also be directly used as bio-fertilizer and antibacterial feed, without generating a large amount of wastewater and slag. Therefore, solid state fermentation of Streptomyces avermitilis is a more economical and environmentally friendly production method.

[0020] Table 1 Preliminary comparison of the cost of avermectin solid fermentation and liquid fermentation medium

[0021]

[0022]

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

[0024] Table 2 Comparison of the total cost of avermectin solid fermentation and liquid fermentation

[0025]

[0026] Note: a Avermectin B1a in SSF and SmF 1a The yield was analyzed as 3.83 mg / g ds and 3.70 g / L, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Graph of the results of the effect of different carbon sources in the solid fermentation medium on the yield of avermectin B1a; 1a

[0028] Figure 2 Graph of the results of the effect of different nitrogen sources in the solid fermentation medium on the yield of avermectin B1a; 1a

[0029] Figure 3 ​​Effect of mass ratio of wheat bran to corn cob in the mixture of wheat bran and corn cob as carbon source in solid fermentation medium on the yield of avermectin B 1a Result graph of the effect of different carbon / nitrogen source ratios in solid fermentation medium on the yield of avermectin B

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

[0031] Figure 5 Effect of different aeration water-permeable materials in solid fermentation medium on the yield of avermectin B 1a Result graph of the effect of different aeration water-permeable materials in solid fermentation medium on the yield of avermectin B Figure 5 Effect of different amounts of rice husk as aeration water-permeable material in solid fermentation medium on the yield of avermectin B 1a Result graph of the effect of different amounts of rice husk as aeration water-permeable material in solid fermentation medium on the yield of avermectin B Figure 5 Effect of different amounts of wheat straw as aeration water-permeable material in solid fermentation medium on the yield of avermectin B 1a Result graph of the effect of different amounts of wheat straw as aeration water-permeable material in solid fermentation medium on the yield of avermectin B Figure 5 Effect of different amounts of sugarcane residue as aeration water-permeable material in solid fermentation medium on the yield of avermectin B 1a Result graph of the effect of different amounts of sugarcane residue as aeration water-permeable material in solid fermentation medium on the yield of avermectin B; Control is the yield of avermectin B when the amount of aeration water-permeable material added is the same as the basic medium 1a Yield of avermectin B

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

[0033] Figure 7 Effect of content of auxiliary nitrogen source (NH4)2SO4 in solid fermentation medium on the yield of avermectin B 1a Result graph of the effect of content of auxiliary nitrogen source (NH4)2SO4 in solid fermentation medium on the yield of avermectin B

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

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

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

[0037] Figure 11 a is the initial water content and bagasse content of the solid medium on the production of abamectin B 1a contour plot and 3D response surface plot of the interaction of yield; wherein, Figure 11 b is the initial water content and ammonium sulfate content of the solid medium on the production of abamectin B 1a result plot of the interaction of yield, Figure 11 b is the initial water content and ammonium sulfate content of the solid medium on the production of abamectin B 1a result plot of the interaction of yield, Figure 11 c is the bagasse content and ammonium sulfate content of the solid medium on the production of abamectin B 1a result plot of the interaction of yield;

[0038] Figure 12 is a result plot of the yield of abamectin B obtained by solid fermentation using the optimal solid fermentation medium obtained in Example 1 under the optimal fermentation conditions using 250 mL shake flasks; 1a result plot of the yield of abamectin B;

[0039] Figure 13 is a result plot of the yield of abamectin B obtained by solid fermentation using the optimal solid fermentation medium obtained in Example 1 under the optimal fermentation conditions using 5 L flasks; 1a result plot of the yield of abamectin B;

[0040] Figure 14 is a result plot of the yield of abamectin B obtained by solid fermentation using the optimal solid fermentation medium obtained in Example 1 under the optimal fermentation conditions using 5.6 L trays; 1a result plot of the yield of abamectin B;

[0041] Figure 15 is a result plot of the yield of abamectin B obtained by repeated batch solid fermentation using the optimal solid fermentation medium obtained in Example 1 under the optimal fermentation conditions using 5 L flasks; 1a result plot of the yield of abamectin B;

[0042] Figure 16 is a result plot of the yield of abamectin B obtained by repeated batch solid fermentation using the optimal solid fermentation medium obtained in Example 1 under the optimal fermentation conditions using 5.6 L trays. 1a result plot of the yield of abamectin B. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the present application more clear and understandable, the present application will be further described in detail below in combination with specific embodiments and the drawings of the specification. It should be noted that the following mentioned embodiments are only applicable to explain the present application, but not to limit the scope of the present application. The following mentioned embodiments are only a part of the embodiments of the present application, but not all the embodiments. Other skilled persons in the art, without making creative efforts, can obtain the embodiments which are protected by the present application.

[0044] The experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art, unless otherwise specified, which can be obtained by commercial channels.

[0045] The strains used in the present application are as follows:

[0046] The Streptomyces avermitilis used in the present application is Streptomyces avermitilis NEAU12, which is obtained by random mutagenesis of Streptomyces avermitilis S0, and the strain NEAU12 is 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 medium involved in the present application is as follows:

[0048] The composition of the solid fermentation basic medium is as follows: corn cob 5 g, earthworm manure 2.5 g, rice husk 0.225 g, sugarcane molasses 0.3 g, ammonium sulfate 0.225 g.

[0049] The detection method involved in the present application is as follows:

[0050] The detection method of avermectin: in order to determine the production of avermectin, 0.3 g of solid fermentation culture was mixed with 1 mL of methanol, and ultrasonic treatment was carried out for 40 min for extracting total avermectin in the fermentation broth and extracellular avermectin. Then the ultrasonic treated sample was centrifuged at high speed of 12000 rpm for 15 min, and the upper layer of the avermectin extract was passed through a 0.22 μM organic filter membrane, and then liquid phase detection was carried out, the detection instrument was Shimadzu LC, the chromatographic column was C18 column (Zorbax, 4.6 mm x 250 mm, 5 μm), the detection wavelength was 246 nm, the injection volume was 20 μL, the mobile phase was 90% (v / v) methanol solution, the flow rate was 1.0 mL / min, and the column temperature was 35℃.

[0051] Example 1: Optimization of solid fermentation medium and fermentation conditions for Streptomyces avermitilis to produce avermectin

[0052] The present embodiment provides a method for producing avermectin by solid fermentation using Streptomyces avermitilis, and provides an optimization process of a solid fermentation medium used in the method and an optimization process of fermentation conditions.

[0053] (I) A method for producing avermectin by solid fermentation using Streptomyces avermitilis, comprising 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 meal 8 g / L, peanut cake powder 10 g / L, yeast powder 4 g / L, CoCl2·6H2O 30 mg / L, alpha amylase 28 mg / L, and the balance is water.

[0057] The preparation method of the seed medium is as follows: according to the volume, the amount of corn starch and alpha amylase is added to 200 mL / L water, heated to 50-70℃, stirred continuously until clear and transparent, then other raw materials are added, mixed, constant volume, divided into 250 mL triangular bottles, liquid volume is 25 mL, 114-116℃ wet heat sterilization for 30 min, standby.

[0058] 2) Seed culture:

[0059] The agar block with Streptomyces avermitilis spores is inoculated into the sterilized and cooled to 28℃ seed culture medium, and fermented and cultured at 28℃, 250 rpm for 40 h.

[0060] 2. Solid fermentation

[0061] 1) Preparation of solid fermentation medium:

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

[0063] The preparation method of the solid fermentation medium is as follows: corn cob, rice bran, wheat bran, white wine residue, rice hull, corn straw, mushroom residue, soybean meal, earthworm manure, chicken manure and rapeseed cake need to be crushed, dried before use, then sieved with a filter screen with a diameter of 1 mm. The cane residue, rice hull and wheat straw used as water-permeable and air-permeable material need to be crushed, dried, then sieved with a filter screen, and the base material with a diameter of 1-3 mm is selected for subsequent experiments. The ground residues are stored in a cold room (4℃), and dried to constant weight at 60℃ before use.

[0064] 2) Solid-state fermentation culture:

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

[0066] (II) Optimization of solid-state fermentation media

[0067] 1. Optimization of carbon sources

[0068] The seed culture of Streptomyces avermitilis was prepared according to the method described in (I) above, and the seed culture was inoculated into a solid fermentation medium for solid fermentation culture under the same method and conditions. The only difference was that the composition of the solid fermentation medium was changed: the carbon source was selected in the same amount (5g) of corn cob, rice bran, wheat bran, liquor residue, rice husk and corn stalk, the nitrogen source was earthworm castings (2.5g), the aeration and water permeability material was rice husk (0.225g), the auxiliary carbon source was sugarcane molasses (0.3g) and the auxiliary nitrogen source was ammonium sulfate (0.225g).

[0069] Avermectin B was determined after solid-state fermentation. 1a The output of, by Figure 1 It can be seen that when corn cob is chosen as the carbon source, the corresponding abamectin B... 1a Corn cob yields the highest amount of carbon, followed by wheat bran. Therefore, corn cob is chosen as the optimal carbon source.

[0070] 2. Optimization of nitrogen source

[0071] The seed culture of *Streptomyces avermitilis* was prepared according to the method described in (I) above, and the seed culture was inoculated into a solid fermentation medium for solid fermentation culture under the same method and conditions. The only difference was that the composition of the solid fermentation medium was changed: the carbon source was corn cob (5g), the nitrogen sources were soybean meal, rapeseed cake, chicken manure, mushroom residue and earthworm castings in the same amount (2.5g), the aeration and water permeability material was rice husk (0.225g), the auxiliary carbon source was sugarcane molasses (0.3g), and the auxiliary nitrogen source was ammonium sulfate (0.225g).

[0072] Avermectin B was determined after solid-state fermentation. 1a The output of, by Figure 2 It can be seen that when earthworm castings are chosen as the nitrogen source, the corresponding avermectin B... 1a Earthworm castings have the highest yield. Therefore, earthworm castings are chosen as the optimal nitrogen source.

[0073] 3. Optimization of mixed carbon sources

[0074] Seed liquid of Streptomyces avermitilis was prepared according to the method in (1) above, and the seed liquid was inoculated into solid fermentation medium to carry out solid fermentation culture by using the same method and conditions, the only difference being that the composition of the solid fermentation medium was changed: the carbon source was selected to be wheat bran and corn cob in a total amount of 5 g, and the mixing ratio was (3:1, 2:1, 1:1, 1:2, 1:3), the nitrogen source was selected to be earthworm manure (2.5 g), the water-permeable and air-permeable material was selected to be rice husk (0.225 g), the auxiliary carbon source was sugarcane molasses (0.3 g), and the auxiliary nitrogen source was ammonium sulfate (0.225 g).

[0075] The yield of avermectin B 1a was determined after the solid fermentation, and it was found that Figure 3 when the mass ratio of wheat bran and corn cob was 2:1, the corresponding yield of avermectin B 1a was the highest. Therefore, wheat bran and corn cob with a mass ratio of 2:1 were selected as the optimal mixed carbon source.

[0076] 4. Optimization of carbon-nitrogen ratio

[0077] Seed liquid of Streptomyces avermitilis was prepared according to the method in (1) above, and the seed liquid was inoculated into solid fermentation medium to carry out solid fermentation culture by using the same method and conditions, the only difference being that the composition and ratio of the solid fermentation medium were changed: the carbon source was selected to be corn cob, the nitrogen source was selected to be earthworm manure, and the mass ratio of corn cob and earthworm manure was selected to be 5:1, 3:1, 2:1, 1:1 and 1:2 (the total amount of carbon source and nitrogen source was 7.5 g), the medium also contained rice husk 0.225 g, sugarcane molasses 0.3 g, and ammonium sulfate 0.225 g.

[0078] The yield of avermectin B 1a was determined after the solid fermentation, and it was found that Figure 4 when the mass ratio of corn cob and earthworm manure was 3:1, the corresponding yield of avermectin B 1a was the highest. Therefore, 3:1 was selected as the optimal carbon-nitrogen ratio.

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

[0080] Seed liquid of Streptomyces avermitilis was prepared according to the method in (1) above, and the seed liquid was inoculated into solid fermentation medium to carry out solid fermentation culture by using the same method and conditions, the only difference being that the composition of the solid fermentation medium was changed: corn cob 5 g, earthworm manure 2.5 g, and the water-permeable and air-permeable material was selected to be sugarcane residue, wheat straw or rice husk in an amount of 2%, 4%, 6%, 8% and 10% of the total amount of carbon and nitrogen source, sugarcane molasses 0.3 g, and ammonium sulfate 0.225 g. The Control group indicates that the amount of water-permeable and air-permeable material added is the same as the basic medium.

[0081] The yield of avermectin B 1aThe output of, by Figure 5 It can be seen that when sugarcane bagasse is selected as the aeration and permeability material, and its content is limited to 6%, the corresponding abamectin B... 1a Sugarcane bagasse was chosen as the optimal aeration and permeability material due to its high yield. Its optimal content was set at 6%.

[0082] 6. Optimization of auxiliary carbon source content

[0083] Seed culture of *Streptomyces avermitilis* was prepared according to the method described in (I) above, and the seed culture was inoculated into a solid-state fermentation medium for solid-state fermentation under the same method and conditions, the only difference being the change in the composition of the solid-state fermentation medium: 5g corn cob, 2.5g earthworm castings, 0.225g rice husk, and sugarcane molasses content of 0%, 3%, 6%, 9%, and 12% of the total carbon and nitrogen sources, respectively, and 0.225g ammonium sulfate. The solid-state fermentation basal medium was used as the Control group.

[0084] Avermectin B was determined after solid-state fermentation. 1a The output of, by Figure 6 It can be seen that when the sugarcane molasses content is 3%, the corresponding abamectin B... 1a The highest yield is achieved through this process. Therefore, 3% is chosen as the optimal content for sugarcane molasses.

[0085] 7. Optimization of auxiliary nitrogen source content

[0086] The seed culture of *Streptomyces avermitilis* was prepared according to the method described in (I) above, and the seed culture was inoculated into a solid fermentation medium for solid fermentation culture under the same method and conditions. The only difference was that the composition of the solid fermentation medium was changed: 5g of corn cob, 2.5g of earthworm castings, 0.225g of rice husk, 0.3g of sugarcane molasses, and the ammonium sulfate content was selected as 0%, 3%, 6%, 9% and 12% of the total carbon and nitrogen sources, respectively.

[0087] Avermectin B was determined after solid-state fermentation. 1a The output of, by Figure 7 It can be seen that when the ammonium sulfate content is 6%, the corresponding avermectin B... 1a The highest yield is achieved. Therefore, 6% is chosen as the optimal content for ammonium sulfate.

[0088] 8. Optimization of trace elements

[0089] Seed liquid of Streptomyces avermitilis was prepared according to the method in (I) above, and inoculated into solid fermentation medium to carry out solid fermentation culture by the same method and condition, the only difference is that the composition of the solid fermentation medium was changed: corn cob 5 g, earthworm excrement 2.5 g, rice husk 0.225 g, cane molasses 0.3 g, ammonium sulfate 0.225 g, and trace elements were selected as MnSO4, CoCl2, MgSO4, Na2MoO4, FeSO4 and CaCl2 with a final concentration of 0.1 mM, respectively, and the final concentration was calculated based on the total mass of carbon and nitrogen source.

[0090] The yield of avermectin B 1a was determined after the end of solid fermentation, and it can be seen from Figure 8 that when CoCl2 was selected as the trace element, the corresponding yield of avermectin B 1a was the highest. Therefore, CoCl2 was selected as the best trace element.

[0091] 9. Optimization of water content

[0092] Seed liquid of Streptomyces avermitilis was prepared according to the method in (I) above, and inoculated into solid fermentation medium to carry out solid fermentation culture by the same method and condition, the only difference is that the composition of the solid fermentation medium was changed: corn cob 5 g, earthworm excrement 2.5 g, rice husk 0.225 g, cane molasses 0.3 g, ammonium sulfate 0.225 g, and trace elements were selected as MnSO4, CoCl2, MgSO4, Na2MoO4, FeSO4 and CaCl2 with a final concentration of 0.1 mM, respectively, and the final concentration was calculated based on the total mass of carbon and nitrogen source.

[0093] The yield of avermectin B 1a was determined after the end of solid fermentation, and it can be seen from Figure 9 that when the initial water content of the solid fermentation medium was 70%, the corresponding yield of avermectin B 1a was the highest. Therefore, 70% was selected as the best initial water content of the solid fermentation medium.

[0094] (III) Optimization of solid fermentation conditions

[0095] Optimization of inoculation amount

[0096] Seed liquid of Streptomyces avermitilis was prepared according to the method in (I) above, and inoculated into solid fermentation medium to carry out solid fermentation culture by the same method and condition, the only difference is that the composition of the solid fermentation medium was changed: corn cob 5 g, earthworm excrement 2.5 g, rice husk 0.225 g, cane molasses 0.3 g, ammonium sulfate 0.225 g, and trace elements were selected as MnSO4, CoCl2, MgSO4, Na2MoO4, FeSO4 and CaCl2 with a final concentration of 0.1 mM, respectively, and the final concentration was calculated based on the total mass of carbon and nitrogen source.

[0097] The yield of avermectin B 1a was determined after the end of solid fermentation, and it can be seen from Figure 10 that when the inoculation amount of the seed liquid was 25%, the corresponding yield of avermectin B 1a was the highest. Therefore, 25% was selected as the best inoculation amount of the seed liquid.

[0098] (iv) Optimization of solid-state fermentation process based on central composite design

[0099] The present invention employs Plackett-Burman design (PBD) method to screen the key factors for the production of avermectin by solid-state fermentation. Based on single factor analysis, six factors, C / N ratio, auxiliary carbon source content, auxiliary nitrogen source content, aeration and water permeable material content, initial water content of medium and inoculum size, were investigated in PBD experiment, and the levels of each factor were determined according to single factor experiment. Based on the results of PBD, central composite design (CCD) was further employed to optimize the composition of medium for the production of avermectin by solid-state fermentation. Each key factor was evaluated at five different levels. Analysis of variance (ANOVA) was used to test the statistical significance. Further experiment was performed to verify the optimal culture conditions for the production of avermectin by solid-state fermentation. Both PBD and CCD were designed by Design Expert (Version 12).

[0100] This sequential optimization strategy was employed to improve the production of avermectin by solid-state fermentation. The preliminary screening using PBD identified the key factors affecting the fermentation efficiency (see Table 3 and Table 4). The experimental data revealed significant differences in the production of avermectin B 1a under different culture conditions, emphasizing the necessity of system 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 production:

[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 (production of avermectin B 1a ), X1is C / N ratio, X2is sugarcane molasses content, X3is sugarcane bagasse content, X4is ammonium sulfate content, X5is inoculum size, and X6is water content. Statistical analysis showed that the model p-value 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 the model were 90.75%, 86.12%, and 73.67%, respectively, demonstrating the excellent stability of the model and its applicability in navigating the design space. The model pointed out three important factors affecting the production of avermectin: water content, sugarcane bagasse content, and ammonium sulfate content (see Table 5).

[0104] To determine the optimal levels of these key factors, a CCD was used (see Table 6). Based on the experimental data, the quadratic model is shown in the following 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 X3 is the sugarcane 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 F-value of the fit is 4.23. The R-value of this model is... 2 Adjust R 2 and prediction R 2 The accuracy rates were 98.27%, 96.32%, and 80.67%, respectively, indicating that the model is qualified (see Table 7). This model predicts the effectiveness of avermectin B under the following conditions. 1a The maximum predicted yield could reach 0.99 mg / gds (see...). Figure 11 The carbon source was a mixture of wheat bran and corn cob at a mass ratio of 2:1; the nitrogen source was earthworm castings, with a carbon-to-nitrogen ratio of 3:1; the aeration and drainage material was sugarcane bagasse, added at 8.62% of the total carbon and nitrogen source mass; the auxiliary carbon source was sugarcane molasses, added at 3% of the total carbon and nitrogen source mass; the auxiliary nitrogen source was ammonium sulfate, added at 10.07% of the total carbon and nitrogen source mass; the final concentration of trace element CoCl2 was 0.1 mM, and the inoculum size was 25% (v / w). t The initial moisture content was 78.5%, and the final concentration of trace element CoCl2, inoculum size, and initial moisture content were all calculated based on the total mass of the carbon and nitrogen sources. Under these conditions, avermectin B... 1a The actual yield reached 1.22 mg / gds on day 10, which was about three times the yield under fermentation conditions with a basic culture medium, a water content of 50%, and an inoculum size of 15%.

[0107] Table 3. Levels of test factors in Plackett-Burman design experiments

[0108]

[0109] Table 4. Plackett-Burman Design Matrix and Avermectin B 1a corresponding output

[0110]

[0111] Table 5 ANOVA analysis of the selected factor model

[0112]

[0113]

[0114] Table 6 Central composite design for medium optimization

[0115]

[0116] Table 7 ANOVA analysis of the quadratic model

[0117]

[0118]

[0119] Example 2: 250 mL flask solid fermentation experiment

[0120] Using the optimal solid medium obtained in Example 1, 25% of seed broth was inoculated into 250 mL flasks (loading capacity: carbon source + nitrogen source 7.5 g) under the condition of initial water content of 78.5%, and other fermentation conditions were the same as in Example 1 (I), and solid fermentation experiment was carried out. During the experiment, solid fermentation samples were collected every two days for detection of the yield of avermectin B 1a . Through detection of the yield of avermectin B 1a , it was found that after 14 days of solid fermentation, the highest yield of avermectin B 1a was 1.89 mg / g ds (see Figure 12 ).

[0121] Example 3: 5 L flask solid fermentation experiment

[0122] According to the optimal solid medium obtained in Example 1, 150 g (calculated according to carbon and nitrogen source 150 g) of solid medium was prepared and loaded into a 5 L flask, 25% of seed broth was inoculated under the condition of initial water content of 78.5%, and other fermentation conditions were the same as in Example 1 (I), and solid fermentation experiment was carried out. During the experiment, solid fermentation samples were collected every two days for detection of the yield of avermectin B 1a . Through detection of the yield of avermectin B 1a , it was found that after 14 days of solid fermentation in a 5 L flask, the highest yield of avermectin B 1a was 2.47 mg / g ds (see Figure 13 ).

[0123] Example 4: 5.6 L tray solid fermentation experiment

[0124] Using the optimal solid medium obtained in Example 1, 150 g (calculated based on 150 g of carbon and nitrogen sources) of solid medium was prepared and placed in a 5.6 L tray, inoculated with 25% seed broth under the condition of initial moisture content of 78.5%, and other fermentation conditions were the same as in Example 1 (I). During the experiment, solid fermentation samples were collected every two days for detection of the production of avermectin B 1a . By detecting the production of avermectin B 1a , it was found that after 14 days of solid fermentation in a 5.6 L tray, the maximum production of avermectin B 1a was 3.64 mg / g ds, which was 47.4% higher than the production in a 5 L flask (see Figure 14 ).

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

[0126] The first batch of fermentation in a 5 L flask was the same as in Example 3. After one batch of fermentation, about 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 was completed to calculate the weight of the fermentation culture, and then a scraper was used to remove the top layer of 0.5 mm, and a long-handled sterilized spoon was used to weigh 15% by weight of the solid fermentation culture from the remaining culture and transfer it to a new flask containing the same amount of medium. Each batch was continuously fermented for 14 days, and the whole process of repetitive batch solid fermentation lasted for 84 days. It was shaken or rotated three times a day to improve the ventilation of the medium. The production parameters of each cycle are shown in Table 8. The results showed that for the repetitive batch fermentation in a 5 L flask, the lag phase of the subsequent batches was shorter than the first batch, the high production rate was maintained for up to four cycles, and the maximum production in the repetitive batch culture was 8% higher than that in the batch culture (see Figure 15 ).

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

[0128] 5.6L tray repeated fed-batch fermentation The first batch of the 5.6L tray repeated fed-batch fermentation was identical to that of Example 4. After one batch of fermentation, about 15% of the weight of the fermentation culture was transferred to another tray. The specific method was as follows: the weight of the empty tray was recorded before fermentation, and the total weight of the tray and fermentation culture was weighed after fermentation was completed to calculate the weight of the fermentation culture, then the top 0.5 mm was removed with a spatula, and 15% of the weight of the solid fermentation culture was weighed with a long-handled sterilized spoon from the remaining culture and transferred to a new tray containing the same weight of medium. Each batch was continuously fermented for 14 days, and the whole process of repeated fed-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 5.6L tray repeated fed-batch fermentation, the lag phase of the subsequent batches was shorter than the first batch, the high production rate was maintained for up to four cycles, and the maximum yield in repeated fed-batch culture was 5% higher than that in fed-batch culture. In the shallow tray repeated fed-batch culture, the highest yield of avermectin B 1a was 3.83 mg / gds, which was also the highest yield reported for avermectin solid fermentation. Figure 16

[0129] Table 8 Parameters of 5.6L flask and 5.6L tray repeated fed-batch solid fermentation for producing avermectin B 1a

[0130]

[0131] Although the present application has been disclosed in its preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore, the protection scope of the present application should be defined by the claims.​​

Claims

1. A solid culture medium for fermenting avermectin using agricultural waste, characterized in that, The solid culture medium is composed of wheat bran, corn cob, earthworm castings, sugarcane bagasse, sugarcane molasses, ammonium sulfate, and CoCl2. Wheat bran and corn cob serve as carbon sources in a 2:1 mass ratio, while earthworm castings serve as a nitrogen source in a 3:1 mass ratio. The amounts of sugarcane bagasse, sugarcane molasses, and ammonium sulfate added are 8.62%, 3%, and 10.07% of the total mass of the carbon and nitrogen sources, respectively. The final concentration of CoCl2 is 0.1 mM based on the total mass of the carbon and nitrogen sources. The initial moisture content of the solid culture medium is 78.5% based on the total mass of the carbon and nitrogen sources.

2. The method for preparing the solid culture medium according to claim 1, characterized in that, Includes the following steps: Corn cobs, wheat bran, earthworm castings, and sugarcane bagasse need to be crushed, dried, and sieved separately before use, and then stored in a cold room and dried to constant weight before use; weigh each component according to the composition of the solid culture medium, and then add water according to the initial moisture content and mix evenly.

3. The preparation method according to claim 2, characterized in that, The corn cobs, wheat bran, earthworm castings, and sugarcane bagasse are filtered through a 1-3 mm screen.

4. A method for producing avermectin by fermentation using the solid culture medium of claim 1, characterized in that, The seed culture of Streptomyces avermitilis was inoculated into solid culture medium at an inoculation rate of 25% (v / w) and fermented at 28°C for 12-16 days. The inoculation amount was calculated based on the total mass of carbon and nitrogen sources.

5. The method according to claim 4, characterized in that, The Streptomyces avermitilis is NEAU12.

6. The method according to claim 4, characterized in that, The seed culture is prepared by inoculating an agar block containing Streptomyces avermitilis spores into a seed culture medium and fermenting it at 28°C and 250 rpm for 40 h.

7. The method according to claim 6, characterized in that, The seed culture medium consists of the following components: corn starch 30 g / L, soybean meal 8 g / L, peanut meal 10 g / L, yeast powder 4 g / L, and CoCl2·6H2O 30 mg / L. α Amylase 28 mg / L, balance water.

8. The method according to claim 4, characterized in that, The method is either single-batch fermentation or continuous repeated batch fermentation.

9. The method according to claim 8, characterized in that, The method of continuous repeated batch fermentation involves inoculating a seed culture of *Streptomyces avermitilis* into a solid culture medium at an inoculation rate of 25% (v / w), and carrying out the first batch fermentation at 28°C to obtain a fermentation culture; then transferring 15% by weight of the fermentation culture to a second batch of solid culture medium and carrying out the second batch fermentation at 28°C; the same method is used thereafter until the last batch of fermentation is completed.

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