Multi-strain synergistic and protective kitchen garbage aerobic treatment microbial inoculant

Abstract

The present application belongs to the kitchen garbage treatment technical field, disclose a kind of multi-strain synergism and protection's kitchen garbage aerobic treatment microbial inoculant, to solve the problem of single function of existing microbial inoculant, long adaptation period, insufficient degradation efficiency.The microbial inoculant includes composite microbial inoculant and diatomite, the composite microbial inoculant includes bacillus subtilis, bacillus licheniformis, bacillus velezensis and bacillus megaterium;Microbial inoculant also includes protective agent, the protective agent includes modified trehalose or trehalose.The microbial inoculant strain is weak antagonism or no antagonism, realizes kitchen garbage efficient degradation, excellent reduction rate, low live bacteria loss rate, key enzyme activity is significantly improved.It is simple in operation, low in energy consumption, suitable for kitchen garbage aerobic fermentation treatment, and has important significance for promoting urban solid waste resource.

Description

Technical Field

[0001] This invention belongs to the field of kitchen waste treatment technology, specifically relating to a multi-strain synergistic and protective aerobic microbial agent for kitchen waste treatment. Background Technology

[0002] With the acceleration of urbanization, kitchen waste has become a core component of urban solid waste. This type of waste is characterized by high moisture, high oil, and high salt content. If it enters the environment directly without effective treatment, it will cause serious pollution problems, including groundwater pollution, odor diffusion, and the growth of pathogens, directly threatening the urban ecological environment and residents' health.

[0003] Current mainstream incineration and landfill technologies face significant bottlenecks: incineration requires a large amount of auxiliary fuel to overcome the low calorific value caused by high moisture content, approximately 2100-3100 kJ / kg, resulting in poor economic efficiency and carbon footprint benefits; landfill disposal, due to its high content of biodegradable organic matter (VS / TS > 85%), is prone to leachate pollution and disorderly emission of methane greenhouse gases, posing a significant risk of secondary pollution.

[0004] Biological methods are widely recognized as the most feasible technological approach due to their high efficiency, low carbon emissions, and resource recovery potential. Their core principle is to utilize microorganisms to decompose large organic molecules in waste, such as starch (30-60%), protein (15-25%), and cellulose (5-10%), into smaller molecules, achieving waste reduction and stabilization. However, existing biological treatment technologies still have key drawbacks: Limited strain functionality: Commercially available microbial agents are mostly composed of single or limited species, making it difficult to simultaneously degrade complex components; Long adaptation period: Microbial communities require a 3-5 day adaptation period in salt and oil-inhibiting environments, resulting in low start-up efficiency; Insufficient degradation efficiency: The degradation rate of starch is generally below 70%, and the degradation cycle of cellulose exceeds 7 days, failing to meet the needs of large-scale treatment.

[0005] The highly complex composition of kitchen waste places multi-dimensional demands on the microbial community—it must possess the ability to secrete multiple enzymes, including proteases, lipases, amylases, and cellulases. Existing microbial agents lack targeted, multifunctional designs, leading to prolonged treatment cycles and significant fluctuations in waste reduction rates. Therefore, developing a highly efficient composite microbial degradation agent, through screening for dominant strains with strong environmental adaptability and multi-substrate degradation capabilities to construct a functionally complementary microbial community, has become a key pathway to overcome the bottlenecks in kitchen waste biological treatment technology and has urgent practical significance for promoting the resource utilization of urban solid waste. Summary of the Invention

[0006] The purpose of this invention is to provide a simple, low-energy-consumption, highly active, and efficient enzyme-producing aerobic microbial agent for treating kitchen waste. Addressing the shortcomings of existing biological agents, such as single-function strains, a 3-5 day adaptation period in salt and oil environments, starch degradation rates generally below 70%, and cellulose degradation cycles exceeding 7 days, this invention optimizes the agent formulation and preparation process. It eliminates the need for complex equipment, reduces energy consumption, ensures long-term bacterial activity, achieves efficient degradation of kitchen waste, improves waste reduction rates, and facilitates the resource recovery of urban solid waste through multi-enzyme secretion to adapt to the degradation of complex components.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0008] A microbial inoculant, comprising a compound microbial inoculant solution and diatomaceous earth.

[0009] Preferably, the number of effective viable bacteria in the compound microbial culture solution is 10. 9 -10 10 cfu / mL.

[0010] Preferably, the bacterial strains in the compound microbial solution include Bacillus subtilis, Bacillus licheniformis, Bacillus belesii, and Bacillus megaterium.

[0011] Preferably, the ratio of viable Bacillus subtilis to Bacillus licheniformis is 0.5-5:1.

[0012] Preferably, the ratio of viable Bacillus subtilis to Bacillus vesiculosus is 0.5-5:1.

[0013] Preferably, the ratio of viable Bacillus subtilis to Bacillus megaterium is 0.5-5:1.

[0014] Preferably, the preparation process of the compound microbial culture includes fermentation culture.

[0015] Preferably, the fermentation medium for fermentation includes LB liquid medium, supplements, and inorganic salt regulators.

[0016] Preferably, the supplement includes carbon source supplements and nutritional supplements.

[0017] Preferably, the carbon source supplement is brown sugar.

[0018] Preferably, the volume-to-mass ratio of LB liquid culture medium to brown sugar is 20 mL: 0.8-1.2 g.

[0019] Preferably, the nutritional supplement includes peptone and yeast extract.

[0020] Preferably, the volume-to-mass ratio of LB liquid culture medium to peptone is 200 mL: 0.8-1.2 g.

[0021] Preferably, the volume-to-mass ratio of LB liquid culture medium to yeast extract is 125 mL: 0.8-1.2 g.

[0022] Preferably, the inorganic salt regulator includes dipotassium hydrogen phosphate and sodium chloride.

[0023] Preferably, the volume-to-mass ratio of LB liquid culture medium to dipotassium hydrogen phosphate is 1000 mL: 0.9-1.1 g.

[0024] Preferably, the volume-to-mass ratio of LB liquid culture medium to sodium chloride is 1000 mL: 0.9-1.1 g.

[0025] Preferably, the volume-to-mass ratio of the composite microbial inoculum to diatomaceous earth is 5 mL: 0.5-2 g.

[0026] Preferably, the microbial agent includes a protectant.

[0027] Preferably, the protective agent includes modified trehalose.

[0028] Preferably, the modified trehalose is prepared by activating carboxylated trehalose with an activator and then reacting it with a modifier.

[0029] Preferably, the modifier includes 1,3-bis(2-hydroxyethoxy)propane-2-ol and methyl 3-hydroxy-2-methylpropionate.

[0030] Preferably, the mass ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to methyl 3-hydroxy-2-methylpropionate is 1-4:2.

[0031] 1,3-Bis(2-hydroxyethoxy)propane-2-ol and methyl 3-hydroxy-2-methylpropionate together form the core structure for modified trehalose, adapting it to the protection of bacteria and enzymes. Through esterification with the carboxyl groups of carboxylated trehalose, a stable network molecular backbone is formed, endowing the modified trehalose with good spatial structural stability. This, in turn, forms a physical barrier on the surface of the bacteria and around the enzyme molecules, reducing the damage of the external environment to the bacterial cell structure and enzyme protein conformation. It also enhances the flexibility of the modified trehalose molecules, making them easier to adhere to the surface of the bacterial cell membrane and the spatial structure of the enzyme molecules, thus strengthening the tightness of the protective barrier. At the same time, the presence of ester groups can reduce the exposure of amino acid residues in the enzyme's active site, reducing the probability of enzyme inactivation due to external factors, and improving the long-term stability and efficient degradation performance of the bacterial agent.

[0032] Preferably, the mass ratio of carboxylated trehalose to 1,3-bis(2-hydroxyethoxy)propane-2-ol is 1-5:1.

[0033] Preferably, the activator comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine.

[0034] Preferably, the mass ratio of carboxylated trehalose to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1-4:1.

[0035] Preferably, the mass ratio of carboxylated trehalose to 4-dimethylaminopyridine is 20-40:1.

[0036] Preferably, carboxylated trehalose is prepared by reacting trehalose with succinic anhydride.

[0037] Preferably, the mass ratio of trehalose to succinic anhydride is 5-10:1.92.

[0038] Preferably, the volume-to-mass ratio of the compound microbial inoculum to the protectant is 5 mL: 0.5-2 g.

[0039] More preferably, the modifier includes tert-butyl 3-(2-hydroxyethoxy)propionate, and the mass ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to tert-butyl 3-(2-hydroxyethoxy)propionate is 1-4:2. 1,3-bis(2-hydroxyethoxy)propane-2-ol can further crosslink to form a denser molecular network framework, enhance the structural stability of modified trehalose, and provide a stronger physical encapsulation barrier for bacteria and enzyme molecules. It also improves the compatibility of modified trehalose with the cell membrane and polar groups on the surface of enzyme proteins, effectively weakening the damage to the structural integrity of bacterial cells caused by external temperature fluctuations and humidity changes, as well as the interference with the conformation of amino acid residues in the active site of enzyme proteins, further strengthening the retention effect of modified trehalose on bacterial activity and the maintenance effect on enzyme activity.

[0040] The use of a microbial agent in the treatment of kitchen waste includes the steps of uniformly mixing compound microbial liquid with kitchen waste and carrying out aerobic fermentation and degradation of kitchen waste.

[0041] Preferably, the volume of the microbial agent is measured by the volume of the compound microbial liquid therein, and the volume-to-mass ratio of the compound microbial liquid to kitchen waste is 20-150 mL: 1 kg.

[0042] Preferably, the degradation time is 2-5 days.

[0043] This invention also provides a method for preparing activated bacterial strains, comprising:

[0044] Bacillus subtilis, Bacillus licheniformis, Bacillus belye and Bacillus megaterium were inoculated into LB solid medium and activated by culturing at 37°C for 24 hours to obtain activated strains.

[0045] Preferably, the activated bacterial strains include activated Bacillus subtilis, activated Bacillus licheniformis, activated Bacillus belye, and activated Bacillus megaterium.

[0046] This invention also provides a method for preparing primary seed culture, comprising:

[0047] The activated bacterial strain was inoculated into LB liquid medium and cultured at 36-38℃ and 80-120rpm for 1-3 days to obtain the primary seed culture.

[0048] Preferably, the primary seed culture includes primary Bacillus subtilis, primary Bacillus licheniformis, primary Bacillus belye, and primary Bacillus megaterium.

[0049] Preferably, the bacterial concentration in the primary seed culture is 10. 9 -10 10 cfu / mL.

[0050] This invention also provides a method for preparing a secondary seed culture, comprising:

[0051] The primary Bacillus subtilis, primary Bacillus licheniformis, primary Bacillus belye, and primary Bacillus megaterium were uniformly mixed and inoculated into LB liquid medium. The mixture was then cultured at 37°C and 100 rpm for 2 days to obtain the secondary seed culture.

[0052] Preferably, the mass ratio of primary Bacillus subtilis to primary Bacillus licheniformis is 0.5-5:1.

[0053] Preferably, the mass ratio of primary Bacillus subtilis to primary Bacillus vesiculosus is 0.5-5:1.

[0054] Preferably, the mass ratio of primary Bacillus subtilis to primary Bacillus megaterium is 0.5-5:1.

[0055] Preferably, the bacterial concentration in the secondary seed culture is 10. 9 -10 10 cfu / mL.

[0056] This invention also provides a method for preparing a composite microbial culture, comprising:

[0057] Brown sugar, peptone, yeast extract, dipotassium hydrogen phosphate, and sodium chloride were added to LB liquid medium and mixed evenly to obtain fermentation medium. Secondary seed culture was inoculated into fermentation medium and cultured at 36-38℃ and 80-120rpm for 40-50h to obtain compound microbial culture.

[0058] Preferably, in the fermentation medium, the volume-to-mass ratio of LB liquid medium to brown sugar is 20 mL: 0.8-1.2 g.

[0059] Preferably, in the fermentation medium, the volume-to-mass ratio of LB liquid medium to peptone is 200 mL: 0.8-1.2 g.

[0060] Preferably, in the fermentation medium, the volume-to-mass ratio of LB liquid medium to yeast extract is 125 mL: 0.8-1.2 g.

[0061] Preferably, in the fermentation medium, the volume-to-mass ratio of LB liquid medium to dipotassium hydrogen phosphate is 1000 mL: 0.9-1.1 g.

[0062] Preferably, in the fermentation medium, the volume-to-mass ratio of LB liquid medium to sodium chloride is 1000 mL: 0.9-1.1 g.

[0063] Preferably, the inoculation ratio of secondary seed culture is 3-10%.

[0064] Preferably, the number of effective viable bacteria in the compound microbial culture solution is 10. 9 -10 10 cfu / mL.

[0065] This invention also provides a method for preparing a microbial inoculant, comprising:

[0066] Diatomaceous earth is added to the compound microbial inoculum solution and dried at 30-40℃ to obtain the microbial inoculum agent.

[0067] Preferably, the volume-to-mass ratio of the composite microbial inoculum to diatomaceous earth is 5 mL: 0.5-2 g.

[0068] The present invention also provides a method for treating kitchen waste, comprising:

[0069] Kitchen waste treatment: Mix microbial agents evenly with kitchen waste, and carry out aerobic fermentation and degradation of kitchen waste.

[0070] Preferably, the volume of the microbial agent is measured by the volume of the compound microbial liquid therein, and the volume-to-mass ratio of the compound microbial liquid to kitchen waste is 20-150 mL: 1 kg.

[0071] Preferably, the degradation time is 2-5 days.

[0072] This invention also provides a method for preparing carboxylated trehalose, comprising:

[0073] Diethyl ether and acetone were mixed evenly to obtain a precipitant. Succinic anhydride was dissolved in anhydrous N,N-dimethylformamide and mixed evenly to obtain a succinic anhydride solution. Trehalose was dissolved in anhydrous N,N-dimethylformamide, and nitrogen gas was introduced. Under conditions of 75-85℃, the succinic anhydride solution and triethylamine were added, and the mixture was stirred for 10-15 hours. The reaction solution was then distilled under reduced pressure, the precipitant was added, and the mixture was centrifuged at 4000-6000 rpm for 3-10 minutes. The precipitate was dissolved in a solvent, and the dissolution process was repeated 2-5 times. The mixture was then vacuum dried at 45-55℃ for 10-15 hours to obtain carboxylated trehalose.

[0074] Preferably, the volume ratio of diethyl ether to acetone in the precipitant is 7:2-4.

[0075] Preferably, in the succinic anhydride solution, the mass-to-volume ratio of succinic anhydride to anhydrous N,N-dimethylformamide is 1.92 g: 10-30 mL.

[0076] Preferably, the mass-to-volume ratio of trehalose to anhydrous N,N-dimethylformamide is 5-10 g: 320 mL.

[0077] Preferably, the mass of the succinic anhydride solution is measured by the mass of the succinic anhydride therein, and the mass ratio of trehalose to succinic anhydride is 5-10:1.92.

[0078] Preferably, the mass-to-volume ratio of trehalose to triethylamine is 5-10 g: 2.8 mL.

[0079] Preferably, the mass-to-volume ratio of trehalose to precipitant is 5-10 g: 100 mL.

[0080] Preferably, the solvent is anhydrous N,N-dimethylformamide.

[0081] This invention also provides a method for preparing modified trehalose, comprising:

[0082] 1,3-bis(2-hydroxyethoxy)propane-2-ol and methyl 3-hydroxy-2-methylpropionate were dispersed in deionized water at 90-100℃ and stirred for 20-40 min. The mixture was then cooled to room temperature to obtain a modifier solution. Carboxylated trehalose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine were dissolved in deionized water and stirred in an ice-water bath for 20-40 min. The modifier solution was then added, and the mixture was reacted at room temperature for 2-4 days. The mixture was then dialyzed for 2-4 days and lyophilized to obtain modified trehalose.

[0083] Preferably, in the modifier solution, the mass-to-volume ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to deionized water is 1-4 g: 100 mL.

[0084] Preferably, in the modifier solution, the mass ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to methyl 3-hydroxy-2-methylpropionate is 1-4:2.

[0085] More preferably, the modifier solution includes tert-butyl 3-(2-hydroxyethoxy)propionate.

[0086] More preferably, the mass ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to tert-butyl 3-(2-hydroxyethoxy)propionate is 1-4:2.

[0087] Preferably, the mass-to-volume ratio of carboxylated trehalose to deionized water is 1-4 g: 40 mL.

[0088] Preferably, the mass ratio of carboxylated trehalose to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1-4:1.

[0089] Preferably, the mass ratio of carboxylated trehalose to 4-dimethylaminopyridine is 20-40:1.

[0090] Preferably, the mass of the modifier solution is measured by the 1,3-bis(2-hydroxyethoxy)propane-2-ol contained therein, and the mass ratio of carboxylated trehalose to 1,3-bis(2-hydroxyethoxy)propane-2-ol is 1-5:1.

[0091] Preferably, the molecular weight cutoff of the dialysis bag is 3000-4000 Da.

[0092] Preferably, the dialysate is deionized water, and the dialysate is changed 3-7 times.

[0093] This invention also provides a method for preparing a microbial inoculant, comprising:

[0094] Preparation of microbial inoculants: Add a protective agent to the compound microbial inoculant solution, mix evenly, add diatomaceous earth, and dry at 30-40℃ to obtain the microbial inoculant.

[0095] Preferably, the protective agent is modified trehalose.

[0096] Preferably, the volume-to-mass ratio of the compound microbial inoculum to the protectant is 5 mL: 0.5-2 g.

[0097] Preferably, the volume-to-mass ratio of the composite microbial inoculum to diatomaceous earth is 5 mL: 0.5-2 g.

[0098] This invention utilizes a compound strain formulation of Bacillus subtilis, Bacillus licheniformis, Bacillus belye, and Bacillus megaterium, and introduces modified trehalose as a protective agent in conjunction with diatomaceous earth as a carrier to prepare the microbial agent. Therefore, it offers the following advantages: no complex equipment is required, and the operation process is simple; the cultivation and drying temperatures are low, significantly reducing energy consumption; the viable cell loss rate is low, and the activity is well maintained; it achieves efficient degradation of kitchen waste, significantly improves the activity of key enzymes, and provides stable and excellent treatment results. Therefore, this invention is a simple, low-energy-consumption, highly active, and efficient enzyme-producing aerobic microbial agent for kitchen waste treatment. Attached Figure Description

[0099] Figure 1 This is a schematic diagram showing the test results of the degradation performance of the compound microbial inoculum on kitchen waste.

[0100] Figure 2 This is a schematic diagram showing the test results of the viable cell loss rate test for microbial inoculants.

[0101] Figure 3 This is a schematic diagram showing the results of the microbial inoculant activity test for amylase.

[0102] Figure 4 This is a schematic diagram showing the results of a microbial inoculant activity test for protease.

[0103] Figure 5 This is a schematic diagram showing the results of the microbial inoculant activity test for lipase.

[0104] Figure 6 This is a schematic diagram showing the results of the activity test of microbial inoculants on cellulase. Detailed Implementation

[0105] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0106] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0107] Example 1:

[0108] Preparation of activated bacterial strains: Bacillus subtilis, Bacillus licheniformis, Bacillus belye, and Bacillus megaterium were inoculated into LB solid medium and incubated at 37°C for 24 hours to activate the strains. The activated strains included activated Bacillus subtilis, activated Bacillus licheniformis, activated Bacillus belye, and activated Bacillus megaterium.

[0109] Preparation of primary seed culture: Activated bacterial strains were inoculated into LB liquid medium and cultured at 37℃ and 100 rpm for 2 days to obtain primary seed culture. The primary seed culture included primary Bacillus subtilis, primary Bacillus licheniformis, primary Bacillus belye, and primary Bacillus megaterium; the bacterial concentration in the primary seed culture was 10-1. 9 cfu / mL.

[0110] Preparation of secondary seed culture: Primary Bacillus subtilis, primary Bacillus licheniformis, primary Bacillus belye, and primary Bacillus megaterium were uniformly mixed and inoculated into LB liquid medium. The mixture was incubated at 37℃ and 100 rpm for 2 days to obtain the secondary seed culture. The mass ratios of primary Bacillus subtilis to primary Bacillus licheniformis, primary Bacillus subtilis to primary Bacillus belye, and primary Bacillus subtilis to primary Bacillus megaterium were all 1:1. The bacterial concentration in the secondary seed culture was 10... 9 cfu / mL.

[0111] Preparation of the compound microbial culture: Brown sugar, peptone, yeast extract, dipotassium hydrogen phosphate, and sodium chloride were added to LB liquid medium and mixed evenly to obtain the fermentation medium. The secondary seed culture was inoculated into the fermentation medium and cultured at 37℃ and 100 rpm for 48 h to obtain the compound microbial culture. In the fermentation medium, the volume-to-mass ratio of LB liquid medium to brown sugar was 20 mL:1 g, the volume-to-mass ratio of LB liquid medium to peptone was 200 mL:1 g, the volume-to-mass ratio of LB liquid medium to yeast extract was 125 mL:1 g, the volume-to-mass ratio of LB liquid medium to dipotassium hydrogen phosphate was 1000 mL:1 g, and the volume-to-mass ratio of LB liquid medium to sodium chloride was 1000 mL:1 g. The inoculation ratio of the secondary seed culture was 4%, and the effective viable count in the compound microbial culture was 102. 9 cfu / mL.

[0112] Preparation of microbial inoculant: Diatomaceous earth was added to the composite microbial inoculant solution and dried at 35℃ to obtain the microbial inoculant. The volume-to-mass ratio of the composite microbial inoculant solution to the diatomaceous earth was 5 mL: 1 g.

[0113] Kitchen waste treatment: Microbial inoculants are evenly mixed with kitchen waste for aerobic fermentation and degradation. The volume of the microbial inoculant is measured by the volume of the compound microbial liquid, and the volume-to-mass ratio of the compound microbial liquid to kitchen waste is 60 mL: 1 kg. The degradation time is 3 days.

[0114] Example 2: The only difference between this example and Example 1 is the preparation of the secondary seed culture.

[0115] Preparation of secondary seed culture: Primary Bacillus subtilis, primary Bacillus licheniformis, primary Bacillus belye, and primary Bacillus megaterium were uniformly mixed and inoculated into LB liquid medium. The mixture was incubated at 37℃ and 100 rpm for 2 days to obtain the secondary seed culture. The mass ratio of primary Bacillus subtilis to primary Bacillus licheniformis was 4:3, the mass ratio of primary Bacillus subtilis to primary Bacillus belye was 2:1, and the mass ratio of primary Bacillus subtilis to primary Bacillus megaterium was 4:1. The bacterial concentration in the secondary seed culture was 10... 9 cfu / mL.

[0116] Example 3: The only difference between this example and Example 1 is the preparation of the secondary seed culture.

[0117] Preparation of secondary seed culture: Primary Bacillus subtilis, primary Bacillus licheniformis, primary Bacillus belye, and primary Bacillus megaterium were uniformly mixed and inoculated into LB liquid medium. The mixture was incubated at 37℃ and 100 rpm for 2 days to obtain the secondary seed culture. The mass ratio of primary Bacillus subtilis to primary Bacillus licheniformis was 1:2, the mass ratio of primary Bacillus subtilis to primary Bacillus belye was 1:1, and the mass ratio of primary Bacillus subtilis to primary Bacillus megaterium was 1:1. The bacterial concentration in the secondary seed culture was 10... 9 cfu / mL.

[0118] Example 4: The only difference between this example and Example 1 is the preparation of the secondary seed culture.

[0119] Preparation of secondary seed culture: Primary Bacillus subtilis, primary Bacillus licheniformis, primary Bacillus belye, and primary Bacillus megaterium were uniformly mixed and inoculated into LB liquid medium. The mixture was incubated at 37℃ and 100 rpm for 2 days to obtain the secondary seed culture. The mass ratio of primary Bacillus subtilis to primary Bacillus licheniformis was 3:2, the mass ratio of primary Bacillus subtilis to primary Bacillus belye was 3:2, and the mass ratio of primary Bacillus subtilis to primary Bacillus megaterium was 1:1. The bacterial concentration in the secondary seed culture was 10... 9 cfu / mL.

[0120] Example 5: The only difference between this example and Example 1 is the preparation of the secondary seed culture.

[0121] Preparation of secondary seed culture: Primary Bacillus subtilis, primary Bacillus licheniformis, primary Bacillus belye, and primary Bacillus megaterium were uniformly mixed and inoculated into LB liquid medium. The mixture was incubated at 37℃ and 100 rpm for 2 days to obtain the secondary seed culture. The mass ratio of primary Bacillus subtilis to primary Bacillus licheniformis was 1:1, the mass ratio of primary Bacillus subtilis to primary Bacillus belye was 1:1, and the mass ratio of primary Bacillus subtilis to primary Bacillus megaterium was 3:1. The bacterial concentration in the secondary seed culture was 10... 9 cfu / mL.

[0122] Example 6: The only difference between this example and Example 2 is the treatment of kitchen waste.

[0123] Kitchen waste treatment: The microbial agent is evenly mixed with the kitchen waste, and aerobic fermentation and degradation of the kitchen waste are carried out. The volume of the microbial agent is measured by the volume of the compound microbial liquid, and the volume-to-mass ratio of the compound microbial liquid to the kitchen waste is 20mL:1kg. The degradation time is 3 days.

[0124] Example 7: The only difference between this example and Example 2 is the treatment of kitchen waste.

[0125] Kitchen waste treatment: Microbial inoculants are evenly mixed with kitchen waste, and aerobic fermentation and degradation of the kitchen waste are carried out. The volume of the microbial inoculant is measured by the volume of the compound microbial liquid, and the volume-to-mass ratio of the compound microbial liquid to kitchen waste is 40mL:1kg. The degradation time is 3 days.

[0126] Example 8: The only difference between this example and Example 2 is the treatment of kitchen waste.

[0127] Kitchen waste treatment: Microbial inoculants are evenly mixed with kitchen waste for aerobic fermentation and degradation. The volume of the microbial inoculant is measured by the volume of the compound microbial inoculant solution, with a volume-to-mass ratio of 80 mL to 1 kg of kitchen waste. The degradation time is 3 days.

[0128] Example 9: The only difference between this example and Example 2 is the treatment of kitchen waste.

[0129] Kitchen waste treatment: Microbial inoculants are evenly mixed with kitchen waste, and aerobic fermentation and degradation of the kitchen waste are carried out. The volume of the microbial inoculant is measured by the volume of the compound microbial liquid, and the volume-to-mass ratio of the compound microbial liquid to the kitchen waste is 100mL:1kg. The degradation time is 3 days.

[0130] Example 10: The only difference between this example and Example 2 is the treatment of kitchen waste.

[0131] Kitchen waste treatment: Microbial inoculants are evenly mixed with kitchen waste, and aerobic fermentation and degradation of the kitchen waste are carried out. The volume of the microbial inoculant is measured by the volume of the compound microbial liquid, and the volume-to-mass ratio of the compound microbial liquid to the kitchen waste is 120mL:1kg. The degradation time is 3 days.

[0132] Example 11: The only difference between this example and Example 2 is the treatment of kitchen waste.

[0133] Kitchen waste treatment: Microbial inoculants are evenly mixed with kitchen waste, and aerobic fermentation and degradation of the kitchen waste are carried out. The volume of the microbial inoculant is measured by the volume of the compound microbial liquid, and the volume-to-mass ratio of the compound microbial liquid to kitchen waste is 150mL:1kg. The degradation time is 3 days.

[0134] Example 12: The only difference between this example and Example 2 is the preparation of the microbial agent.

[0135] Preparation of carboxylated trehalose: Diethyl ether and acetone were mixed uniformly to obtain a precipitant; succinic anhydride was dissolved in anhydrous N,N-dimethylformamide and mixed uniformly to obtain a succinic anhydride solution; trehalose was dissolved in anhydrous N,N-dimethylformamide, nitrogen gas was purged, and at 80°C, the succinic anhydride solution and triethylamine were added and stirred for 12 h. The reaction solution was then distilled under reduced pressure, the precipitant was added, and the mixture was centrifuged at 5000 rpm for 5 min. The precipitate was dissolved in a solvent, and the dissolution process was repeated three times. The mixture was then vacuum dried at 50°C for 12 h to obtain carboxylated trehalose. In the precipitant, the volume ratio of diethyl ether to acetone is 7:3; in the succinic anhydride solution, the mass-to-volume ratio of succinic anhydride to anhydrous N,N-dimethylformamide is 1.92 g: 20 mL; the mass-to-volume ratio of trehalose to anhydrous N,N-dimethylformamide is 7 g: 320 mL; the mass of the succinic anhydride solution is measured by the mass of succinic anhydride therein, and the mass ratio of trehalose to succinic anhydride is 7:1.92; the mass-to-volume ratio of trehalose to triethylamine is 7 g: 2.8 mL; the mass-to-volume ratio of trehalose to the precipitant is 7 g: 100 mL; and the solvent is anhydrous N,N-dimethylformamide.

[0136] Preparation of modified trehalose: 1,3-bis(2-hydroxyethoxy)propane-2-ol and methyl 3-hydroxy-2-methylpropionate were dispersed in deionized water at 95℃ and stirred for 30 min. The mixture was then cooled to room temperature to obtain a modifier solution. Carboxylated trehalose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in deionized water and stirred in an ice-water bath for 30 min. The modifier solution was then added, and the mixture was reacted at room temperature for 3 days. The mixture was then dialyzed for 3 days and lyophilized to obtain modified trehalose. In the modifier solution, the mass-to-volume ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to deionized water is 1 g:50 mL, and the mass ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to methyl 3-hydroxy-2-methylpropionate is 1:1; the mass-to-volume ratio of carboxylated trehalose to deionized water is 1 g:20 mL, the mass ratio of carboxylated trehalose to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 2:1, and the mass ratio of carboxylated trehalose to 4-dimethylaminopyridine is 30:1; the mass of the modifier solution is measured by 1,3-bis(2-hydroxyethoxy)propane-2-ol, and the mass ratio of carboxylated trehalose to 1,3-bis(2-hydroxyethoxy)propane-2-ol is 3:1; the molecular weight cutoff of the dialysis bag is 3500 Da, the dialysate is deionized water, and the dialysate is changed 5 times.

[0137] Preparation of microbial inoculant: A protectant was added to the composite microbial inoculant solution, mixed thoroughly, and then diatomaceous earth was added. The mixture was dried at 35°C to obtain the microbial inoculant. The protectant was modified trehalose, and the volume-to-mass ratio of the composite microbial inoculant solution to the protectant was 5 mL:1 g, as was the volume-to-mass ratio of the composite microbial inoculant solution to the diatomaceous earth.

[0138] Example 13: The only difference between this example and Example 12 is the preparation of the modified trehalose.

[0139] Preparation of modified trehalose: 1,3-bis(2-hydroxyethoxy)propane-2-ol and methyl 3-hydroxy-2-methylpropionate were dispersed in deionized water at 95℃ and stirred for 30 min. The mixture was then cooled to room temperature to obtain a modifier solution. Carboxylated trehalose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in deionized water and stirred in an ice-water bath for 30 min. The modifier solution was then added, and the mixture was reacted at room temperature for 3 days. The mixture was then dialyzed for 3 days and lyophilized to obtain modified trehalose. In the modifier solution, the mass-to-volume ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to deionized water is 1 g:50 mL, and the mass ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to methyl 3-hydroxy-2-methylpropionate is 3:2; the mass-to-volume ratio of carboxylated trehalose to deionized water is 1 g:20 mL, the mass ratio of carboxylated trehalose to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 2:1, and the mass ratio of carboxylated trehalose to 4-dimethylaminopyridine is 30:1; the mass of the modifier solution is measured by the amount of 1,3-bis(2-hydroxyethoxy)propane-2-ol, and the mass ratio of carboxylated trehalose to 1,3-bis(2-hydroxyethoxy)propane-2-ol is 3:1; the molecular weight cutoff of the dialysis bag is 3500 Da, the dialysate is deionized water, and the dialysate is changed 5 times.

[0140] Example 14: The only difference between this example and Example 12 is the preparation of the modified trehalose.

[0141] Preparation of modified trehalose: 1,3-bis(2-hydroxyethoxy)propane-2-ol, methyl 3-hydroxy-2-methylpropionate and tert-butyl 3-(2-hydroxyethoxy)propionate were dispersed in deionized water at 95℃ and stirred for 30 min. The mixture was then cooled to room temperature to obtain a modifier solution. Carboxylated trehalose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in deionized water and stirred in an ice-water bath for 30 min. The modifier solution was added, and the mixture was reacted at room temperature for 3 days. The mixture was then dialyzed for 3 days and lyophilized to obtain modified trehalose. In the modifier solution, the mass-to-volume ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to deionized water is 1 g:50 mL; the mass ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to methyl 3-hydroxy-2-methylpropionate is 1:1; the mass ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to tert-butyl 3-(2-hydroxyethoxy)propionate is 1:1; the mass-to-volume ratio of carboxylated trehalose to deionized water is 1 g:20 mL; and the carboxylated trehalose... The mass ratio of trehalose to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 2:1, and the mass ratio of carboxylated trehalose to 4-dimethylaminopyridine was 30:1. The mass of the modifier solution was measured by 1,3-bis(2-hydroxyethoxy)propane-2-ol, and the mass ratio of carboxylated trehalose to 1,3-bis(2-hydroxyethoxy)propane-2-ol was 3:1. The molecular weight cutoff of the dialysis bag was 3500 Da, the dialysate was deionized water, and the dialysate was changed 5 times.

[0142] Example 15: The only difference between this example and Example 12 is the preparation of the modified trehalose.

[0143] Preparation of modified trehalose: 1,3-bis(2-hydroxyethoxy)propane-2-ol, methyl 3-hydroxy-2-methylpropionate and tert-butyl 3-(2-hydroxyethoxy)propionate were dispersed in deionized water at 95℃ and stirred for 30 min. The mixture was then cooled to room temperature to obtain a modifier solution. Carboxylated trehalose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in deionized water and stirred in an ice-water bath for 30 min. The modifier solution was added, and the mixture was reacted at room temperature for 3 days. The mixture was then dialyzed for 3 days and lyophilized to obtain modified trehalose. In the modifier solution, the mass-to-volume ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to deionized water is 1 g:50 mL; the mass ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to methyl 3-hydroxy-2-methylpropionate is 1:1; the mass ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to tert-butyl 3-(2-hydroxyethoxy)propionate is 2:3; the mass-to-volume ratio of carboxylated trehalose to deionized water is 1 g:20 mL; and the carboxylated trehalose... The mass ratio of trehalose to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 2:1, and the mass ratio of carboxylated trehalose to 4-dimethylaminopyridine was 30:1. The mass of the modifier solution was measured by 1,3-bis(2-hydroxyethoxy)propane-2-ol, and the mass ratio of carboxylated trehalose to 1,3-bis(2-hydroxyethoxy)propane-2-ol was 3:1. The molecular weight cutoff of the dialysis bag was 3500 Da, the dialysate was deionized water, and the dialysate was changed 5 times.

[0144] Comparative Example 1: The only difference between this comparative example and Example 12 is that 1,3-bis(2-hydroxyethoxy)propane-2-ol was not used in the preparation of the modified trehalose.

[0145] Comparative Example 2: The only difference between this comparative example and Example 12 is that methyl 3-hydroxy-2-methylpropionate was not used in the preparation of the modified trehalose.

[0146] Comparative Example 3: The only difference between this comparative example and Example 2 is the preparation of the microbial inoculant.

[0147] Preparation of microbial inoculant: A protectant was added to the compound microbial inoculant solution, mixed evenly, and then diatomaceous earth was added. The mixture was dried at 35℃ to obtain the microbial inoculant. The protectant was trehalose, and the volume-to-mass ratio of the compound microbial inoculant solution to the protectant was 5 mL:1 g, and the volume-to-mass ratio of the compound microbial inoculant solution to the diatomaceous earth was 5 mL:1 g.

[0148] Experimental Example 1: Degradation performance test of strain on starch, protein, oil and cellulose.

[0149] Test sample: Primary seed culture prepared in Example 1.

[0150] Test methods: Add 5g beef extract, 10g peptone, 5g sodium chloride, 10g soluble starch, and 20g agar to 1000mL of culture medium and sterilize at 115℃ for 20min to obtain starch culture medium; add 2g ammonium sulfate, 0.5g magnesium sulfate, 1g potassium dihydrogen phosphate, 20g cellulose powder, 0.2g Congo red, and 20g agar to 1000mL of culture medium and sterilize at 121℃ for 20min to obtain cellulose culture medium; add 5g beef extract and 10g peptone to 1000mL of culture medium. 5g sodium chloride and 20g agar were sterilized at 121℃ for 20min to obtain protein medium; 5g beef extract, 10g peptone, 5g sodium chloride, 10g sesame oil, 20g agar and 1mL neutral red were added to 1000mL of medium and sterilized at 121℃ for 20min to obtain oil medium; the primary seed culture was inoculated onto starch medium, cellulose medium, protein medium and oil medium respectively, and the degradation ability of the strain on starch, protein, oil and cellulose was identified by the hydrolysis zone method.

[0151] The results of the strain's degradation performance on starch, protein, oil and cellulose are shown in Table 1. √ indicates degradation ability, and × indicates no degradation ability.

[0152] Table 1. Test results of the strain's degradation performance on starch, protein, oil and cellulose.

[0153]

[0154] Bacillus subtilis exhibits excellent starch, protein, and lipid degradation capabilities. As a multifunctional core bacterium, it can simultaneously degrade the high-content organic components in three types of kitchen waste: starch, protein, and lipids. Bacillus licheniformis exhibits excellent starch and cellulose degradation capabilities. As a multifunctional bacterium, its dual degradation capabilities of starch and cellulose can effectively treat cellulose substances in kitchen waste, avoiding the overall treatment cycle extension caused by delayed cellulose degradation. Bacillus belliesi exhibits excellent protein degradation capabilities, while Bacillus megaterium exhibits excellent lipid degradation capabilities. As single-function high-efficiency bacteria, they can further enhance the degradation efficiency of compound bacterial agents on specific substrates, avoid the generation of putrid odors from high-protein substrates during treatment, and reduce the inhibitory effect of lipids on the metabolic activities of other strains.

[0155] Experimental Example 2: Test of antagonistic relationship between strains.

[0156] Test sample: Primary seed culture prepared in Example 1.

[0157] Test method: Add 0.2 mL of solution containing 10 6Add 5 mL of 0.7 wt% soft agar at 45°C to a CFU / mL bacterial suspension, and quickly pour it onto a solidified base plate. After solidification, evenly place sterile Oxford cups with an inner diameter of 6 mm on the surface. After 24 h of incubation, centrifuge at 12000 rpm and collect the supernatant. Add 100 μL of sterile filtered test bacterial fermentation supernatant to the Oxford cup. After incubation at 37°C for 18 h, measure the diameter of the inhibition zone with vernier calipers to determine the antagonistic strength. Where: inhibition zone diameter = total diameter - outer diameter of Oxford cup; antagonistic strength grading: - for none; + for weak antagonistic strength, inhibition zone < 5 mm; ++ for medium antagonistic strength, inhibition zone 5-10 mm; +++ for strong antagonistic strength, inhibition zone > 10 mm.

[0158] The results of the strain antagonism test are shown in Table 2.

[0159] Table 2 Results of strain antagonism test

[0160] strain Bacillus subtilis Bacillus licheniformis Bacillus belesiensis Bacillus megaterium Bacillus subtilis - + - - Bacillus licheniformis + - - - Bacillus belesiensis - - - - Bacillus megaterium - - - -

[0161] The weak or non-antagonistic relationship between Bacillus subtilis, Bacillus licheniformis, Bacillus belyeis, and Bacillus megaterium allows each strain to express its own metabolic functions normally in the composite system, providing a feasible guarantee for the large-scale production of composite microbial inoculum.

[0162] Experimental Example 3: Degradation performance test of compound microbial inoculum on kitchen waste.

[0163] Test sample: The composite microbial culture prepared in the example.

[0164] Test Method: 1 kg of kitchen waste was collected from the canteen and placed into a self-made kitchen waste degradation device from Hangzhou Nanda Environmental Protection Technology Co., Ltd. A compound microbial inoculant solution was evenly sprayed into the device and stirring was started. The spraying volume was 40 mL, serving as the experimental group. The control group was treated with 40 mL of deionized water at room temperature for 72 hours. The remaining mass was weighed, and the reduction rate was calculated using the formula R(%) = (M0-M1) / M0 × 100%. 有效 (%)=R 实验 -R 对照 The effective reduction rate of the compound microbial inoculum solution was calculated; where R is the reduction rate, M0 is the initial mass of kitchen waste, and M1 is the remaining mass of kitchen waste after treatment; R 有效 For the effective reduction rate, R 实验 R represents the reduction rate in the experimental group. 对照 The reduction rate is for the control group.

[0165] The test results of the degradation performance of the compound microbial inoculum on kitchen waste are as follows: Figure 1As shown, the bacterial strain combination formed by Bacillus subtilis, Bacillus licheniformis, Bacillus belye, and Bacillus megaterium can simultaneously initiate the degradation process of starch, protein, oil, and cellulose, ultimately achieving efficient weight reduction within 72 hours and significantly shortening the degradation cycle. Among Examples 1-5, Example 2 has the highest effective weight reduction rate. Under this ratio, the proportion of Bacillus subtilis and Bacillus licheniformis, as the "core multifunctional bacteria," is more balanced, ensuring the rapid degradation of starch, which accounts for 30-60% of kitchen waste, while eliminating the physical obstacles of fibrous substances to the decomposition of other substrates through the cellulose degradation ability of Bacillus licheniformis. At the same time, the dosage of Bacillus belye and Bacillus megaterium is suitable for the characteristics of waste with high protein and high oil content, avoiding the problems of protein putrefaction and oil accumulation caused by insufficient specific degrading bacteria, and ultimately maximizing the degradation efficiency. In Examples 2 and Examples 6-11, the preferred dosage of 60 mL / kg as the usage ratio of the compound microbial liquid is the most suitable, achieving better results at the lowest possible cost and having clear practical application value.

[0166] Experimental Example 4: Long-term stability test of compound microbial inoculum.

[0167] Test sample: The composite microbial culture prepared in Example 2.

[0168] Test method: The compound microbial culture solution was filled into 100mL opaque plastic bottles (80mL total) and stored at 4℃, 25℃, and 37℃ for 12 months respectively; the initial viable count was 10×10⁻⁶. 8 The number of live bacteria was measured at CFU / mL at 1, 2, 3, 6 and 12 months.

[0169] The results of the long-term stability test of the compound microbial inoculum are shown in Table 3.

[0170] Table 3. Results of long-term stability test of composite microbial inoculum

[0171]

[0172] Without the addition of a protective agent, the viable bacterial survival rate reached 71.6% after 12 months of storage at 4°C. The composite microbial solution prepared by this invention exhibits significant long-term stability. The low-temperature environment effectively inhibits the metabolic activity of the strains, reducing cell death caused by respiration and enzymatic hydrolysis, while avoiding denaturation and inactivation of biomolecules such as nucleic acids and proteins at high temperatures. After 3 months of storage at 25°C, the viable bacterial survival rate reached 73.3%. For small and medium-sized kitchen waste treatment sites without low-temperature storage conditions, this agent can meet usage needs through short-term storage at room temperature, reducing the investment cost of storage equipment. Under extreme short-term heat exposure scenarios at 37°C, such as summer storage environments and high temperatures in transport vehicles, the viable bacterial survival rate was 21.6% after 3 months of storage, proving that the agent of this invention has a certain short-term heat resistance, avoiding the defect of existing agents that are greatly inactivated when exposed to short-term high temperatures. It is also suitable for non-constant temperature logistics scenarios, eliminating the need for cold chain transportation and further reducing the application cost of the entire chain.

[0173] Experimental Example 5: Test on the live bacteria loss rate of microbial inoculants.

[0174] Test samples: Microbial agents prepared in Examples 2, 12-15, and Comparative Examples 1-3.

[0175] Test method: Add 0.1g of microbial inoculum to a sterile centrifuge tube containing 9.9mL of sterile physiological saline, vortex for 10min to completely disperse the bacterial powder, and obtain 10 -2 The bacterial suspension of a certain concentration was diluted to 10. -7 Concentration; 0.1 mL of bacterial suspension was evenly spread onto the surface of LB solid medium. The culture dish was inverted and placed in a 37°C incubator for 24 hours. The number of intact, clearly defined single colonies in each culture dish was counted, and the viable count of each bacterial powder was calculated. The culture dish was then stored at 37°C in a sealed, moisture-proof environment away from light. The viable count was measured after 60 days, and the viable count was calculated as L(%) = (N0 - N) 30 ) / N0×100%, where L is the viable cell loss rate, N0 is the initial viable cell count, and N 30 The number of viable bacteria after 30 days of storage.

[0176] The test results of the viable cell loss rate test of the microbial agent prepared in this invention are as follows: Figure 2As shown, Example 2 did not add any protective agent, and the bacterial powder lacked effective protection for the bacterial cells during normal storage, resulting in the highest live bacteria loss rate. Example 12 used a complete modification system containing 1,3-bis(2-hydroxyethoxy)propane-2-ol and methyl 3-hydroxy-2-methylpropionate to prepare modified trehalose. The modified trehalose could better encapsulate the bacterial cells, maintain the bacterial cell activity environment, and give the bacterial powder better storage stability, significantly reducing the live bacteria loss rate. Example 13 adjusted the ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to methyl 3-hydroxy-2-methylpropionate based on Example 12, making the molecular structure of the modified trehalose more suitable for the bacterial cell protection requirements, further optimizing the protective performance. Example 14 introduced tert-butyl 3-(2-hydroxyethoxy)propionate to construct a more complete bacterial cell protection system, further enhancing the protective effect on the bacterial cells, reducing the loss of live bacteria, and further reducing the live bacteria loss rate. Low; Example 15 increased the amount of tert-butyl 3-(2-hydroxyethoxy)propionate, achieving optimal synergistic effect of all modifiers, resulting in the best protective effect of modified trehalose on bacterial cells and the lowest viable cell loss rate among all samples; Comparative Example 3 used ordinary trehalose as a protective agent, which reduced viable cell loss to some extent through the basic protective effect of trehalose, but its compatibility with bacterial cells and its ability to protect bacterial cell structure were limited, resulting in a higher viable cell loss rate than Example 12; Comparative Example 1 did not introduce 1,3-bis(2-hydroxyethoxy)propane-2-ol in the preparation of modified trehalose, and Comparative Example 2 did not use methyl 3-hydroxy-2-methylpropionate in the preparation of modified trehalose, leading to insufficient molecular structural integrity of modified trehalose and a higher viable cell loss rate than Example 12; This indicates that the introduction of modified trehalose is crucial for improving the storage stability of microbial agents, and optimizing the modification system can effectively reduce viable cell loss during the storage of bacterial powder.

[0177] Experimental Example 6: Activity test of microbial inoculants on different enzymes.

[0178] Test samples: Microbial agents prepared in Examples 2, 12-15, and Comparative Examples 1-3.

[0179] Test method: Add 10g of microbial agent to 100mL of sterile physiological saline, seal and extract by shaking at 37℃ and 150rpm for 2h. Transfer the mixture to a centrifuge tube and centrifuge at 4℃ and 8000rpm for 15min. Take the supernatant and filter it through a 0.22μm microporous membrane. Measure the activities of amylase, protease, lipase and cellulase respectively.

[0180] The results of the microbial inoculant's activity test for amylase are as follows: Figure 3 As shown, the test results of the microbial inoculant for protease activity are as follows: Figure 4 As shown in the figure, the test results of the microbial inoculant for lipase activity are as follows: Figure 5 As shown, the test results of the microbial inoculant for cellulase activity are as follows: Figure 6 As shown, the microbial agent prepared in Example 2 is characterized by amylase and protease as the main enzymes, lipase as the auxiliary enzymes, and cellulase as the basic support, effectively covering the main organic components of kitchen waste, and significantly improving the activity of key enzymes. Examples 12-13 use modified trehalose, which forms a weak interaction with enzyme proteins through specific groups, which can both encapsulate enzyme molecules to reduce external interference and maintain the spatial conformation of the enzyme active center, thus significantly improving the activity of all four enzymes. Examples 14-15 introduce tert-butyl 3-(2-hydroxyethoxy)propionate, which can enhance the tightness of the modified trehalose in encapsulating enzyme molecules and reduce the exposure and loss of the enzyme active center. The activity of the four enzymes was further enhanced in Comparative Example 1, which did not introduce 1,3-bis(2-hydroxyethoxy)propane-2-ol in the preparation of modified trehalose, and Comparative Example 2 did not use methyl 3-hydroxy-2-methylpropionate, so it could not fully maintain the stability of the enzyme active site, and the overall enzyme activity was still lower than that of Example 12. Comparative Example 3 used ordinary trehalose as a protective agent. Although it could reduce the loss of enzyme activity through the basic moisturizing and molecular encapsulation effect of trehalose, it was not modified and its compatibility with enzyme protein and its ability to protect the enzyme active site were limited. Therefore, the overall enzyme activity was slightly higher than that of Example 2, but the increase was small and lower than that of Example 12.

[0181] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0182] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A microbial inoculant, characterized in that: The microbial agent comprises a compound microbial inoculum and diatomaceous earth, wherein the compound microbial inoculum contains 10 effective viable bacteria. 9 -10 10 The cfu / mL of the compound microbial solution contains Bacillus subtilis, Bacillus licheniformis, Bacillus belyssus, and Bacillus megaterium; the viable cell ratio of Bacillus subtilis to Bacillus licheniformis is 0.5-5:1, the viable cell ratio of Bacillus subtilis to Bacillus belyssus is 0.5-5:1, and the viable cell ratio of Bacillus subtilis to Bacillus megaterium is 0.5-5:

1. The microbial agent includes a protectant, which includes modified trehalose. The modified trehalose is prepared by activating carboxylated trehalose with an activator and then reacting it with the modifyer. The modifyer includes 1,3-bis(2-hydroxyethoxy)propane-2-ol and methyl 3-hydroxy-2-methylpropionate. The mass ratio of 1,3-bis(2-hydroxyethoxy)propane-2-ol to methyl 3-hydroxy-2-methylpropionate is 1-4:2, and the mass ratio of carboxylated trehalose to 1,3-bis(2-hydroxyethoxy)propane-2-ol is 1-5:

1.

2. The microbial inoculant according to claim 1, characterized in that: The preparation process of the compound microbial culture includes fermentation culture. The fermentation culture medium includes LB liquid medium, supplements and inorganic salt regulators. The supplements include carbon source supplements and nutrient supplements. The carbon source supplement is brown sugar. The volume-to-mass ratio of LB liquid medium to brown sugar is 20 mL: 0.8-1.2 g.

3. The microbial inoculant according to claim 2, characterized in that: The nutritional supplement comprises peptone and yeast extract, wherein the volume-to-mass ratio of LB liquid culture medium to peptone is 200 mL: 0.8-1.2 g, and the volume-to-mass ratio of LB liquid culture medium to yeast extract is 125 mL: 0.8-1.2 g.

4. The microbial inoculant according to claim 2, characterized in that: The inorganic salt regulator includes dipotassium hydrogen phosphate and sodium chloride. The volume-to-mass ratio of the LB liquid culture medium to dipotassium hydrogen phosphate is 1000 mL: 0.9-1.1 g, and the volume-to-mass ratio of the LB liquid culture medium to sodium chloride is 1000 mL: 0.9-1.1 g.

5. The microbial inoculant according to claim 1, characterized in that: The volume-to-mass ratio of the composite microbial inoculum to diatomaceous earth is 5 mL: 0.5-2 g.

6. The microbial inoculant according to claim 1, characterized in that: The activator comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, wherein the mass ratio of carboxylated trehalose to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1-4:1, and the mass ratio of carboxylated trehalose to 4-dimethylaminopyridine is 20-40:

1.

7. The microbial inoculant according to claim 1, characterized in that: The carboxylated trehalose is prepared by reacting trehalose with succinic anhydride, wherein the mass ratio of trehalose to succinic anhydride is 5-10:1.

92.

8. The microbial inoculant according to claim 1, characterized in that: The volume-to-mass ratio of the composite microbial inoculum to the protective agent is 5 mL: 0.5-2 g.

9. The use of the microbial agent according to any one of claims 1-8 in the treatment of kitchen waste, characterized in that: The steps of the kitchen waste treatment include uniformly mixing microbial agents with kitchen waste, and carrying out aerobic fermentation and kitchen waste degradation; the volume of the microbial agents is measured by the volume of the composite microbial liquid therein, the volume-to-mass ratio of the composite microbial liquid to the kitchen waste is 20-150mL:1kg, and the degradation time is 2-5 days.

Images