Composite bacterial agent producing high-temperature protease, high-temperature amylase and high-temperature lipase, and preparation method and application thereof

Abstract

The present invention discloses a composite microbial agent for producing high-temperature protease, high-temperature amylase and high-temperature lipase, and a preparation method and application thereof. The composite microbial agent comprises Bacillus parageous, Bacillus stearothermophilus and Thermus thermophilus. Bacillus parageous, Bacillus stearothermophilus and Thermus thermophilus are subjected to liquid fermentation respectively, and the cultured fermentation liquids are mixed to obtain a liquid composite microbial agent; the liquid composite microbial agent is evenly sprinkled on auxiliary materials composed of rice husk powder, straw powder and attapulgite, and a solid microbial agent is obtained after drying. The composite microbial agent can degrade substances such as protein, starch and fat in organic waste under high temperature conditions exceeding 65°C, and can be effectively used in high-temperature composting to increase the maximum temperature of the pile body, prolong the number of days of high-temperature composting, and significantly improve the seed germination index of the compost product.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a composite bacterial agent for producing high-temperature protease, high-temperature amylase and high-temperature lipase, and a preparation method and application thereof. Background Art

[0002] Composting is an important way to achieve harmless treatment, volume reduction and resource utilization of organic solid waste. High temperature is of great significance to the composting process. Only at high temperature can pathogens in the compost be inactivated, thus achieving the purpose of harmlessness. The high temperature stage is also a critical period for the rapid degradation of organic matter in composting, and is an important factor affecting the composting maturity cycle. On the other hand, the important driver of the composting process - "microorganisms" are sensitive to temperature. As the temperature rises, the activity of microorganisms will be greatly inhibited, which will eventually limit the rapid degradation of organic matter and affect the composting maturity cycle.

[0003] The composting efficiency can be improved and the composting cycle can be shortened by inoculating bacterial agents. However, not all bacterial agents can fully play their role in the composting process. The main reason is that most of the inoculated functional microorganisms are mesophilic bacteria and mesophilic bacteria, and their growth will be inhibited during the high temperature period of composting, resulting in insufficient decomposition and utilization of organic matter such as protein, starch, and fat in the composting environment, thus failing to achieve the expected effect. Extreme thermophiles are a type of high-temperature microorganisms with an optimal growth temperature of 60 to 80°C. Compared with mesophiles and mesophiles, they are more adaptable to the high temperature environment of composting and can quickly degrade organic matter during the high temperature period of composting, thereby shortening the composting fermentation time. Therefore, screening extreme thermophiles that are thermophilic and can proliferate using composting organic matter and compounding them into composite bacterial agents is of great significance for accelerating the composting process of organic waste and improving the quality of compost products. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a composite bacterial agent that produces high-temperature protease, high-temperature amylase and high-temperature lipase. The composite bacterial agent can degrade substances such as protein, starch and fat in organic waste under high temperature conditions exceeding 65°C, and can be effectively used in high-temperature composting.

[0005] The invention also provides a preparation method of the composite microbial agent and application of the composite microbial agent in high-temperature composting.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a composite bacterial agent for producing high-temperature protease, high-temperature amylase and high-temperature lipase, wherein the composite bacterial agent includes Parageobacillus sp., Geobacillus stearothermophilus and Thermus thermophilus.

[0007] Preferably, the Geobacillus sp. is Parageobacillus sp. NY44, the Geobacillus stearothermophilus is Geobacillus stearothermophilus CCTCC AB 2013010, and the Thermus thermophilus is Thermusthermophilus CGMCC 1.15099.

[0008] Preferably, the ratio of the viable counts of Bacillus paragea, Bacillus stearothermophilus and Thermus thermophilus is 0.5-1:1-5:1-2.

[0009] The preparation method of the composite microbial agent producing high-temperature protease, high-temperature amylase and high-temperature lipase of the present invention comprises the following steps: subjecting Bacillus paragea, Bacillus stearothermophilus and Thermus thermophilus to liquid fermentation respectively; mixing the cultured fermentation broth to obtain a liquid composite microbial agent; and evenly sprinkling the liquid composite microbial agent on auxiliary materials consisting of rice husk powder, straw powder and attapulgite, and drying to obtain a solid microbial agent.

[0010] Preferably, the preparation method is:

[0011] (1) Activation of bacterial strains: The three strains were inoculated into solid culture medium, cultured, and single colonies were selected and inoculated into liquid culture medium to obtain inoculum;

[0012] (2) Preparation of primary seed solution: inoculating the inoculum prepared in step (1) into a liquid culture medium at an inoculum volume ratio of 5-10%, and culturing to obtain a primary seed solution;

[0013] (3) Preparation of fermentation broth: The first-grade seed solution prepared in step (2) is inoculated into a fermentation tank containing a fermentation medium at an inoculum ratio of 2% to 5% by volume, and the fermentation broth of each strain is obtained by culturing;

[0014] (4) Preparation of solid microbial agent: The fermentation liquids of the bacteria prepared in step (3) are mixed to obtain a composite microbial agent; the mixed fermentation liquid is evenly spread on the auxiliary materials consisting of rice husk powder, straw powder and attapulgite, and dried to obtain a solid composite microbial agent.

[0015] The solid culture medium comprises: 10-15 g of peptone, 10-15 g of sodium chloride, 5-10 g of yeast powder, 20 g of agar, and water is added to make the volume up to 1 L; the liquid culture medium comprises: 10-15 g of peptone, 10-15 g of sodium chloride, 5-10 g of yeast powder, and water is added to make the volume up to 1 L; the fermentation culture medium comprises: 15-20 g of brown sugar, 1.0-2.0 g of ammonium sulfate, 2-3 g of peptone, 1-2 g of yeast powder, 5-10 g of sodium chloride, and water is added to make the volume up to 1 L.

[0016] In step (1), the inoculation solution is obtained by culturing until the OD600 value reaches 0.6-0.8; and the first-level seed solution is obtained by culturing until the OD600 value reaches 0.8-1.0 in step (2).

[0017] In step (3), the first-level seed solution prepared in step (2) is introduced, and the ventilation volume is 6m 3 / h, stirring speed 180-220rpm, 65-70℃ culture until the number of viable bacteria in the fermentation tank is greater than 1×10 8 cfu / mL, the fermentation broth of each strain was obtained.

[0018] The invention discloses an application of the composite bacterial agent producing high-temperature protease, high-temperature amylase and high-temperature lipase in high-temperature composting.

[0019] The application is as follows: mixing pig manure and straw, mixing the liquid composite microbial agent with the materials, and starting composting after fully mixing.

[0020] The present invention prepares a specific composite bacterial agent that can realize high temperature composting, improve the maximum temperature of the pile, extend the days of high temperature period of composting, and improve the seed germination index of the compost product. In the microbial inoculation composting experiment, the interaction between strains is very important, and there may be synergy or antagonism between bacteria and bacteria. Generally, the effect of multi-bacteria mixed inoculation may be better than the effect of single bacteria inoculation, but it is not ruled out that there is no mutual promotion between multiple strains, but antagonism is played. The present invention mixes Bacillus paragea, Bacillus stearothermophilus and Thermus thermophilus for the first time, and the three strains are used simultaneously to produce obvious unexpected synergy, while Bacillus paragea and Bacillus stearothermophilus or Thermus thermophilus combination do not have synergy. Therefore, the present invention utilizes the composite bacterial agent composed of three bacteria to effectively solve the single or two bacteria combination bacterial agent growth inhibition during composting, and the defect that the organic matter degradation effect cannot be fully exerted can effectively extend the days of high temperature period of composting, effectively improve the maturity of compost products, so that the seed germination index of compost products is improved.

[0021] Beneficial effects: (1) The present invention provides a composite bacterial agent that produces high-temperature protease, high-temperature amylase and high-temperature lipase. The bacterial agent can secrete high-temperature protease, high-temperature amylase and high-temperature lipase, and can function under conditions of ≥65°C, which is beneficial to the rapid degradation of macromolecular organic matter during the high-temperature period of composting.

[0022] (2) The microbial composite agent provided by the present invention can grow in a high-temperature composting environment of 65-70°C, and can effectively increase the maximum temperature of the compost body, so that the maximum temperature of the compost body is increased from 58°C in the control group to 75°C, which can more effectively kill pathogenic microorganisms and meet the harmlessness requirements.

[0023] (3) The microbial composite agent provided by the present invention can continue to play a role during the high temperature period of composting, and can effectively prolong the number of days in the high temperature period of composting, so that the number of days in the high temperature period of composting increases from 5 days in the control group to 18 days.

[0024] (4) The microbial agent provided by the present invention can effectively improve the maturity of the compost product, so that the seed germination index of the compost product is increased from 60.7% of the control group to 90.2%.

[0025] (5) The composite bacterial agent fermentation process of the present invention is simple, and the mixed bacterial agent can be directly used for composting, which saves energy and reduces production costs and preparation difficulty. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the colony morphology of strain Parageobacillus sp.NY44. DETAILED DESCRIPTION

[0027] The strains involved in the examples are from:

[0028] Geobacillus stearothermophilus: commercial strains may be used, including but not limited to Geobacillus stearothermophilus CCTCC AB 2013010;

[0029] Thermus thermophilus: Commercial strains may be used, including but not limited to Thermus thermophilus CGMCC 1.15099.

[0030] Parageobacillus sp.NY44 was provided by the Circular Agriculture Research Center of Jiangsu Academy of Agricultural Sciences (Screening of extremely thermophilic functional bacteria and their effect on promoting compost maturity, Journal of Agro-Environment Science, 2020, 39(7):1633-1642). The colony morphology of strain NY44 is shown in Figure 1 , white, opaque, flat, rough surface, irregular edges, diameter 1.5-2mm. Gram staining results were positive under optical microscope, rod-shaped bacteria.

[0031] The culture medium involved in the embodiment:

[0032] Solid culture medium: peptone 10g, sodium chloride 10g, yeast powder 5g, agar 20g, add water to make up to 1L;

[0033] Liquid culture medium: peptone 10g, sodium chloride 10g, yeast powder 5g, add water to make up to 1L;

[0034] Fermentation medium: 15g brown sugar, 1.0g ammonium sulfate, 3g peptone, 2g yeast powder, 5g sodium chloride, add water to make up to 1L.

[0035] Example 1

[0036] Preparation of liquid composite microbial agent containing Bacillus paragea NY44

[0037] The liquid composite microbial agent is obtained by mixing the fermentation liquid of Bacillus paragea NY44, the fermentation liquid of Bacillus stearothermophilus CCTCCAB 2013010 and the fermentation liquid of Thermus thermophilus CGMCC 1.15099. The specific preparation method is as follows:

[0038] (1) Activation of bacterial strains: The three strains were inoculated into solid culture medium and cultured at 65°C for 2 days. A single colony was picked and inoculated into liquid culture medium and cultured at 65°C and 150 rpm until the OD600 value reached 0.8 to obtain the inoculum;

[0039] (2) Preparation of primary seed solution: inoculate the inoculum prepared in step (1) into the liquid culture medium at a volume ratio of 5%, and culture at 65° C. and 150 rpm until the OD600 value reaches 1.0, which is the primary seed solution;

[0040] (3) Preparation of liquid bacterial agent: The first-grade seed solution prepared in step (2) was inoculated into a fermentation tank containing a fermentation medium at an inoculum rate of 5% by volume, and the ventilation volume was 6 m 3 / h, stirring speed 200rpm, 65℃ culture until the number of viable bacteria in the fermentation tank is greater than 1×10 8 cfu / mL (about 5×10 8 cfu / mL) to obtain fermentation broths of various strains, and mix the fermentation broths of various strains so that the ratio of the live counts of Bacillus paragea NY44, Bacillus stearothermophilus and Thermus thermophilus is 1:2:2 to obtain a liquid composite microbial agent.

[0041] Example 2

[0042] Preparation of solid composite microbial agent containing Bacillus paragea NY44

[0043] The liquid composite microbial agent is obtained by mixing the fermentation liquid of Bacillus paragea NY44, the fermentation liquid of Bacillus stearothermophilus CCTCC AB 2013010 and the fermentation liquid of Thermus thermophilus CGMCC 1.15099 with auxiliary materials. The specific preparation method is as follows:

[0044] (1) Activation of bacterial strains: The three strains were inoculated into solid culture medium and cultured at 65°C for 2 days. A single colony was picked and inoculated into liquid culture medium and cultured at 65°C and 150 rpm until the OD600 value reached 0.8 to obtain the inoculum;

[0045] (2) Preparation of primary seed solution: inoculate the inoculum prepared in step (1) into the liquid culture medium at a volume ratio of 5%, and culture at 65° C. and 150 rpm until the OD600 value reaches 1.0, which is the primary seed solution;

[0046] (3) Preparation of liquid bacterial agent: The first-grade seed solution prepared in step (2) was inoculated into a fermentation tank containing a fermentation medium at an inoculum rate of 5% by volume, and the ventilation volume was 6 m 3 / h, stirring speed 200rpm, 65℃ culture until the number of viable bacteria in the fermentation tank is greater than 1×10 8 cfu / mL (about 5×10 8 cfu / mL) to obtain fermentation broths of each strain, and mix the fermentation broths of each strain so that the ratio of the number of live bacteria of Bacillus paragea NY44, Bacillus stearothermophilus and Thermus thermophilus is 1:2:2 to obtain a liquid composite microbial agent. The liquid composite microbial agent is evenly sprinkled on an auxiliary material composed of rice husk powder, straw powder and attapulgite mixed in a mass ratio of 1:1:1, and a solid microbial agent is obtained after drying.

[0047] Example 3

[0048] Determination of high temperature enzyme activity of Bacillus paragea NY44 and composite bacterial agents

[0049] The parageobacterium NY44 stored at -80°C was activated with solid culture medium and inoculated into liquid culture medium and cultured at 65°C until OD 600 When the value reaches 1.0, the culture solution is centrifuged; the parageobacillus NY44, the fermentation broth of Bacillus stearothermophilus CCTCC AB 2013010 and the Thermus thermophilus CGMCC 1.15099 stored at -80°C are activated with solid culture medium and inoculated into liquid culture medium at the same time, and cultured at 65°C until the OD600 value reaches 1.0. The supernatants of the two cultures are used to determine the activities of high-temperature proteases, amylases and lipases, and the enzyme reaction temperature is 70-90°C. The protease activity is determined by the Folin method; the amylase activity is determined by the DNS method; and the lipase activity is determined by the alkaline titration method. The test results are shown in Table 1:

[0050] Table 1 Results of high temperature enzyme activity determination of Bacillus paragea NY44

[0051]

[0052] From the results in Table 1, it can be seen that the extracellular protease, amylase and lipase secreted by Bacillus paragea NY44 alone have certain activities in the high temperature range of 70-85°C, but the activity of lipase decreases significantly when the temperature rises. When the three specific strains of the present invention are used in combination, the extracellular protease, amylase and lipase have high activities in the high temperature range of 70-85°C, and the activity of lipase does not change substantially in the high temperature range of 70-85°C, and remains at a high level.

[0053] This indicates that the composite bacterial agent of the present invention can decompose a variety of macromolecular organic matter during the high temperature period when used for composting, thereby improving the composting efficiency and shortening the composting cycle.

[0054] Example 4

[0055] Application of microbial agents in composting

[0056] Pig manure and straw were mixed at a mass ratio of 6:1, the carbon-nitrogen ratio of the mixed material was 30:1, and the moisture content was 65%. The liquid composite microbial agent prepared in Example 1 was mixed with the material at a mass ratio of 0.5:100, and the composting was started after sufficient mixing. During the composting process, the temperature of the compost was recorded in the morning and afternoon every day. When the temperature was higher than 70°C, the compost was turned every day. As time went on, the temperature of the compost decreased. When the temperature was lower than 35°C, the composting was stopped. Samples were taken to measure various composting indicators. The measurement results are shown in Table 2. The compost exceeded 55°C for 18 days, and the highest temperature was 75°C. At the end of the composting, the seed germination index of the compost material was 90.2%.

[0057] Comparative Example 1

[0058] A single inoculum containing Bacillus paragea NY44 was cultured by the method of Example 1, and mixed with the material at a mass ratio of 0.5:100 according to the method of Example 4 for composting. Samples were taken to measure various composting indicators. The measurement results are shown in Table 2. The number of days when the compost body exceeded 55°C was 9 days, and the highest temperature was 62°C. At the end of composting, the seed germination index of the compost material was 71.7%.

[0059] Comparative Example 2

[0060] The mixed bacterial agent containing Bacillus parageobacillus NY44 and Bacillus stearothermophilus CCTCC AB2013010 (the ratio of the number of live bacteria of the two is 1:2) was cultured according to the method of Example 1, and the mixed bacterial agent was mixed with the material at a mass ratio of 0.5:100 according to the method of Example 4 for composting. Sampling was performed to measure various composting indicators, and the measurement results are shown in Table 2. The number of days when the compost body exceeded 55° C. was 8 days, and the highest temperature was 60° C.; at the end of composting, the seed germination index of the compost material was 70.1%.

[0061] Comparative Example 3

[0062] The mixed bacterial agent containing Bacillus paragea NY44 and Thermus thermophilus CGMCC 1.15099 (the ratio of the number of live bacteria of the two is 1:2) was cultured according to the method of Example 1, and mixed with the material at a mass ratio of 0.5:100 according to the method of Example 4 for composting. Sampling was performed to measure various composting indicators. The measurement results are shown in Table 2. The number of days when the compost body exceeded 55°C was 10 days, and the highest temperature was 65°C; at the end of composting, the seed germination index of the compost material was 73.5%.

[0063] Comparative Example 4

[0064] The mixed bacterial agent containing Geobacillus stearothermophilus CCTCC AB 2013010 and Thermus thermophilus CGMCC 1.15099 (the ratio of the number of live bacteria of the two is 1:1) was cultured by the method of Example 1, and mixed with the material at a mass ratio of 0.5:100 according to the method of Example 4 for composting. Sampling was performed to measure various composting indicators, and the measurement results are shown in Table 2. The number of days when the compost body exceeded 55° C. was 9 days, and the highest temperature was 63° C.; at the end of composting, the seed germination index of the compost material was 72.3%.

[0065] Comparative Example 5

[0066] Pig manure and straw were mixed in a mass ratio of 6:1, the carbon-nitrogen ratio of the mixed material was 30:1, and the moisture content was 65%. The materials were fully mixed and then composting began. During the composting process, the temperature of the pile was recorded in the morning and afternoon every day. When the temperature was higher than 70°C, the pile was turned every day. As time went on, the temperature of the pile dropped. When the temperature was lower than 35°C, the turning of the pile was stopped. Samples were taken to measure various composting indicators. The results are shown in Table 2. The pile exceeded 55°C for 5 days and the highest temperature was 58°C. At the end of composting, the seed germination index of the compost material was 60.7%.

[0067] The composting index test results of Example 4 and Comparative Examples 1-5 are shown in Table 2.

[0068] Table 2 Composting index test results of each embodiment and comparative example

[0069]

[0070] As can be seen from the results of Table 2, the composting treatment inoculated with the composite microbial agent of the present invention can reach a higher composting temperature, and the high temperature period lasts longer. At the end of the composting, the seed germination index of the composting treatment inoculated with the agent is higher than that of the control, indicating that the inoculation of extreme thermophilic microbial agents is beneficial to accelerate the maturity of the compost and improve the quality of the compost. The present invention uses Bacillus parageolyticus, Bacillus stearothermophilus and Thermus thermophilus for the first time. The simultaneous use of the three strains will produce a synergistic effect, while the combination of Bacillus parageolyticus and Bacillus stearothermophilus reduces the composting temperature, while the high temperature duration is shortened, and the combination of Bacillus parageolyticus and Thermus thermophilus has basically no synergistic effect; and the use effect of Bacillus stearothermophilus CCTCC AB2013010 and Thermus thermophilus CGMCC 1.15099 single bacteria is not as good as Bacillus parageolyticus NY44. The composite agent of the present invention using three bacteria at the same time can effectively extend the days of the high temperature period of the compost, effectively improve the maturity of the compost product, and improve the seed germination index of the compost product.

[0071] Example 5

[0072] Application of microbial agents in composting

[0073] Pig manure and straw were mixed in a mass ratio of 6:1, the carbon-nitrogen ratio of the mixed material was 30:1, and the moisture content was 65%. According to the preparation method of Example 1, a liquid composite microbial agent containing Bacillus paragea NY44, Bacillus stearothermophilus CCTCC AB 2013010 and Thermus thermophilus CGMCC 1.15099 with a live bacterial count ratio of 0.8:2.5:1.5 was prepared and mixed with the material in a mass ratio of 0.5:100, and the composting was started after sufficient mixing. During the composting process, the temperature of the compost was recorded in the morning and afternoon every day. When the temperature was higher than 70°C, the compost was turned every day. As time went on, the temperature of the compost decreased. When the temperature was lower than 35°C, the composting was stopped. Samples were taken to measure various composting indicators. The measurement results are shown in Table 2. The compost exceeded 55°C for 17 days and the highest temperature was 74°C. At the end of the composting, the seed germination index of the compost material was 88.4%.

[0074] Example 6

[0075] Application of microbial agents in composting

[0076] Pig manure and straw were mixed in a mass ratio of 6:1, the carbon-nitrogen ratio of the mixed material was 30:1, and the moisture content was 65%. According to the preparation method of Example 1, a liquid composite microbial agent containing Bacillus paragea NY44, Bacillus stearothermophilus CCTCC AB 2013010 and Thermus thermophilus CGMCC 1.15099 live bacteria in a ratio of 1:5:2 was prepared and mixed with the material in a mass ratio of 0.5:100, and the composting was started after sufficient mixing. During the composting process, the temperature of the compost was recorded in the morning and afternoon every day. When the temperature was higher than 70°C, the compost was turned every day. As time went on, the temperature of the compost decreased. When the temperature was lower than 35°C, the composting was stopped. Samples were taken to measure various composting indicators. The measurement results are shown in Table 2. The compost exceeded 55°C for 16 days and the highest temperature was 74°C. At the end of the composting, the seed germination index of the compost material was 86.7%.

Claims

1. A composite bacterial agent for producing high-temperature protease, high-temperature amylase and high-temperature lipase, characterized in that: The composite bacterial agent is composed of Bacillus paragea ( Parageobacillus sp.), Geobacillus stearothermophilus ( Geobacillus stearothermophilus ) and Thermus thermophilus ( Thermus thermophilus ) composition; the parageobacterium is Parageobacillus sp.NY44, the thermophilic Geobacillus is Geobacillus stearothermophilus CCTCC AB 2013010; the thermophilic bacterium is Thermus thermophilus CGMCC 1.15099; the ratio of the viable counts of Bacillus paragea, Bacillus stearothermophilus and Thermus thermophilus is 0.5-1:1-5:1-2.

2. A method for preparing the composite bacterial agent producing high-temperature protease, high-temperature amylase and high-temperature lipase according to claim 1, characterized in that: The bacillus paragea, the bacillus stearothermophilus and thermus thermophilus are subjected to liquid fermentation respectively; the cultured fermentation liquids are mixed to obtain a liquid composite microbial agent; the liquid composite microbial agent is evenly sprinkled on auxiliary materials consisting of rice husk powder, straw powder and attapulgite, and then dried to obtain a solid microbial agent; The specific preparation method is: (1) Activation of bacterial strains: The three strains were inoculated into solid culture medium, cultured, and single colonies were selected and inoculated into liquid culture medium to obtain inoculum; (2) Preparation of primary seed solution: inoculating the inoculum prepared in step (1) into a liquid culture medium at an inoculum volume ratio of 5% to 10%, and culturing to obtain a primary seed solution; (3) Preparation of fermentation broth: The first-grade seed solution prepared in step (2) is inoculated into a fermentation tank containing a fermentation medium at an inoculum ratio of 2% to 5% by volume, and the fermentation broth of each strain is obtained by culturing; (4) Preparation of microbial agent: the fermentation broths of the strains prepared in step (3) are mixed to obtain a liquid composite microbial agent; the liquid composite microbial agent is evenly sprinkled on the auxiliary materials consisting of rice husk powder, straw powder and attapulgite, and then dried to obtain a solid composite microbial agent.

3. The preparation method according to claim 2, characterized in that: The solid culture medium comprises: 10-15 g of peptone, 10-15 g of sodium chloride, 5-10 g of yeast powder, 20 g of agar, and water is added to make the volume up to 1 L; the liquid culture medium comprises: 10-15 g of peptone, 10-15 g of sodium chloride, 5-10 g of yeast powder, and water is added to make the volume up to 1 L; the fermentation culture medium comprises: 15-20 g of brown sugar, 1.0-2.0 g of ammonium sulfate, 2-3 g of peptone, 1-2 g of yeast powder, 5-10 g of sodium chloride, and water is added to make the volume up to 1 L.

4. The preparation method according to claim 2, characterized in that: In step (1), the culture is carried out until the OD600 value reaches 0.6-0.8 to obtain the inoculum solution; in step (2), the culture is carried out until the OD600 value reaches 0.8-1.0 to obtain the primary seed solution.

5. The preparation method according to claim 2, characterized in that: In step (3), the first-level seed solution prepared in step (2) is introduced, and the ventilation volume is 6m 3 / h, stirring speed 180-220rpm, 65-70℃ culture until the number of viable bacteria in the fermentation tank is greater than 1×10 8 cfu / mL, the fermentation broth of each strain was obtained.

6. Use of the composite bacterial agent producing high-temperature protease, high-temperature amylase and high-temperature lipase according to claim 1 in high-temperature composting.

7. The use according to claim 6, characterized in that: The application is: mixing pig manure and straw to obtain a mixed material, mixing the composite bacterial agent with the material, and starting composting after fully mixing.

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