Microbial agent and application thereof in field of rapid rotting of rotten vegetable leaves

Abstract

The invention relates to the technical field of resource utilization of rotten vegetable waste, in particular to a microbial agent and application thereof in the field of rapid rotting of rotten vegetable waste. The microbial agent comprises bacillus pumilus and bacillus subtilis, bacillus subtilis is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.33122; the bacillus pumilus is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No. 33121. The invention also provides an application of the microbial agent in the field of rapid rotting of rotten vegetable leaves. The treatment method for rapid rotting of the rotten vegetable leaves is widened, the types of microorganisms capable of being used for rapid rotting of the rotten vegetable leaves are enriched, the rotting rate of the rotten vegetable leaves can be effectively increased when the microbial agent is used for rapid rotting of the rotten vegetable leaves, and meanwhile the yield of next-stubble crops can be effectively increased.

Description

Technical Field

[0001] The present application relates to the technical field of resource utilization of waste vegetable, and in particular to a microbial agent and its application in the field of quick-rot waste vegetable. Background Art

[0002] Vegetable waste, also known as vegetable scraps, is generated in every process from harvesting to transporting and putting vegetables on the shelves. This includes the inedible parts of ripe fresh vegetables removed when harvesting, as well as roots left in the soil (source waste). The process of transporting vegetables from the field to the market can damage some vegetables, requiring the removal of damaged parts. The vegetables undergo some simple processing before being put on the shelves to enhance their appearance, requiring the removal of parts during the processing. Studies have shown that vegetable scraps account for over 30% of total vegetable production. With the booming vegetable planting industry, the amount of scraps produced is increasing day by day.

[0003] At present, the main methods for handling waste vegetables include direct disposal, landfill, simple composting, chemical treatment, biological treatment, etc. Directly discarding waste vegetables not only takes up a large amount of land resources, but also breeds a large number of harmful microorganisms due to the rotting and deterioration of the waste vegetables, emits a foul odor, pollutes the surrounding environment, and has a negative impact on the soil, water and air. Although landfilling can alleviate environmental pressure to a certain extent, the waste vegetables degrade slowly and are prone to produce leachate, which pollutes the soil and groundwater. The simple composting treatment method has the problems of long decomposition cycle and low efficiency, which is difficult to meet the needs of large-scale waste vegetable treatment. Although chemical treatment can speed up the decomposition process, it may introduce chemical residues, affecting soil quality and subsequent crop growth.

[0004] Biological treatment primarily utilizes microbial agents, which can rapidly decompose organic matter in wastewater through the metabolic activities of specific microorganisms. This significantly shortens the composting cycle, improves treatment efficiency, and meets the urgent needs of large-scale wastewater treatment. However, the application of traditional microbial agents in wastewater composting also has shortcomings such as poor adaptability and limited decomposition capacity.

[0005] Based on the above, in order to further improve the application of microbial agents in the field of waste vegetable composting, finding more effective microorganisms for waste vegetable composting has become an urgent problem to be solved. Summary of the Invention

[0006] This application provides a microbial agent and its application in the field of rapid spoilage of waste vegetables. This application expands the treatment methods for rapid spoilage of waste vegetables and enriches the types of microorganisms that can be used for rapid spoilage of waste vegetables. Using the microbial agent provided in this application to decompose waste vegetables can effectively increase the decomposition rate of waste vegetables and effectively increase the yield of the next crop.

[0007] In a first aspect, the present application provides a Bacillus subtilis, which adopts the following technical solution:

[0008] The Bacillus subtilis provided in this application was deposited in the General Microbiology Center of the China Culture Collection Administration on December 18, 2024, with the deposit number CGMCC No.33122.

[0009] The above-mentioned Bacillus subtilis was isolated from a rotten baby cabbage sample.

[0010] Multiple reference strain sequences were obtained from the NCBI (GenBank) database, and the isolated strains and reference strains were analyzed using the software BioEdit and MEGA11 to construct a phylogenetic tree of the isolated strains and the reference strains, thereby confirming that the strain lineage of the isolated strain was Bacillus subtilis.

[0011] The above-mentioned Bacillus subtilis is used in combination with Bacillus pumilus with a preservation number of CGMCC No. 33121 for decomposing tail vegetables, which can effectively improve the decomposition rate of tail vegetables.

[0012] In a second aspect, the present application provides a culture comprising the above-mentioned Bacillus subtilis.

[0013] In this application, culture includes fermentation broth and fermentation supernatant.

[0014] In a third aspect, the present application provides a fermentation broth comprising the aforementioned Bacillus subtilis.

[0015] In a fourth aspect, the present application provides a bacterial suspension comprising the aforementioned Bacillus subtilis.

[0016] In a fifth aspect, the present application provides a microbial agent, which adopts the following technical solution:

[0017] A microbial agent comprises Bacillus pumilus and Bacillus subtilis.

[0018] Among them, Bacillus subtilis was deposited in the General Microbiology Center of China Culture Collection Administration on December 18, 2024, with the deposit number CGMCC No.33122.

[0019] Among them, Bacillus pumilus was deposited in the General Microbiology Center of China Culture Collection Administration on December 18, 2024, with the deposit number CGMCC No.33121.

[0020] The Bacillus pumilus strain was isolated from a sample of rotten baby cabbage. Its colonies are off-white with smooth, neat edges. The bacteria are short, rod-shaped, and have a relatively simple individual cell morphology, lacking filamentous hyphae.

[0021] Multiple reference strain sequences were obtained from the NCBI (GenBank) database, and the isolated strains and reference strains were analyzed using BioEdit and MEGA11 software to construct a phylogenetic tree of the isolated strains and the reference strains, thereby confirming that the strain lineage of the isolated strain was Bacillus pumilus.

[0022] The use of the Bacillus pumilus for decomposing the tail vegetable can effectively improve the decomposition rate of the tail vegetable. At the same time, the combined use of the Bacillus pumilus and the Bacillus subtilis for decomposing the tail vegetable can further effectively improve the decomposition rate of the tail vegetable.

[0023] Optionally, the effective viable count of the Bacillus subtilis in the microbial agent is (6-7)×10 9 CFU / g.

[0024] Optionally, the effective viable count of the Bacillus subtilis in the microbial agent is 6.5×10 9 CFU / g.

[0025] Optionally, the effective viable count of the Bacillus pumilus in the microbial agent is (5-6)×10 9 CFU / g.

[0026] Optionally, the effective viable count of the Bacillus pumilus in the microbial agent is 5.4×10 9 CFU / g.

[0027] In a sixth aspect, the present application provides an application of the above-mentioned Bacillus subtilis, culture, fermentation broth, bacterial suspension, and microbial agent in the field of quick-rot of vegetable waste.

[0028] Optionally, the Bacillus subtilis is used in combination with the Bacillus pumilus, and the decomposition rate of the vegetable tail can be increased by more than 13%.

[0029] In summary, this application includes at least one of the following beneficial technical effects:

[0030] 1. The present application provides a microbial agent comprising Bacillus pumilus and Bacillus subtilis.

[0031] Among them, Bacillus subtilis was deposited in the General Microbiology Center of China Culture Collection Administration on December 18, 2024, with the deposit number CGMCC No.33122.

[0032] Among them, Bacillus pumilus was deposited in the General Microbiology Center of China Culture Collection Administration on December 18, 2024, with the deposit number CGMCC No.33121.

[0033] 2. Using microbial agents composed of Bacillus pumilus and Bacillus subtilis for the decomposition of tail vegetables can further effectively improve the decomposition rate of tail vegetables and increase the content of soil organic matter, nutrients and beneficial microorganisms, improve the soil aggregate structure, and help increase the yield of the next crop.

[0034] 3. This application expands the treatment methods for the rapid decay of waste vegetables and enriches the types of microorganisms that can be used for the decomposition of waste vegetables. The use of the microbial agents provided in this application for the decomposition of waste vegetables can effectively increase the decomposition rate of waste vegetables and effectively increase the yield of the next crop. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This refers to the growth state of Bacillus pumilus cultured on beef extract peptone medium for 48 hours in this application.

[0036] Figure 2 This refers to the growth state of Bacillus subtilis cultured on beef extract peptone medium for 48 hours in this application.

[0037] Figure 3 This is a comparison chart of the results of the blank group and the experimental group in the verification of the degradation effect of pepper (Figure A is the control group - pure water; Figure B is the experimental group - microbial agent).

[0038] Figure 4 This is a comparison chart of the results of the blank group and the experimental group in the verification of the degradation effect of tomatoes (Figure A is the control group - pure water; Figure B is the experimental group - microbial agent).

[0039] Figure 5 This is a comparison chart of the results of the blank group and the experimental group after 10 days of cultivation in the verification of the degradation effect of baby cabbage (Figure A is the control group - pure water; Figure B is the experimental group - microbial agent).

[0040] Figure 6 This is a comparison chart of the results of the blank group and the experimental group after 15 days of cultivation in the verification of the degradation effect of baby cabbage (Figure A is the control group - pure water; Figure B is the experimental group - microbial agent).

[0041] Figure 7 These are the test results of the next-crop lettuce yield of different groups in the open-field vegetable waste microbial decomposition and return to the field experiment. DETAILED DESCRIPTION

[0042] Before describing the embodiments of the present application in detail, it should be understood that the terminology used herein is only for the purpose of describing particular embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the term belongs.

[0043] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this application, unless otherwise specified, "plurality" means two or more.

[0044] The endpoints of the ranges and any values disclosed in this application are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0045] In this application, the term "comprise" or "include" is an open expression, that is, it includes the content specified in this application, but does not exclude other aspects of the content.

[0046] The present application provides a microbial agent comprising Bacillus subtilis with a deposit number of CGMCC No. 33122 and Bacillus pumilus with a deposit number of CGMCC No. 33121. The microbial agent provided by the present application is used in the field of quick-rotten waste vegetables, which can effectively increase the decomposition rate of waste vegetables. Wherein, waste vegetables can be discarded branches and rotten leaves of all vegetables known in the art. For example, vegetables include peppers, tomatoes, baby cabbage, broccoli, etc.

[0047] To make the purpose, technical solutions, and advantages of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without making any creative work shall fall within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be understood as limiting this application.

[0048] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0049] The reagents, solvents and other experimental materials used in the following examples can all be obtained commercially.

[0050] In the following examples, the formula of the LB solid medium involved is as follows: based on a volume of 1 L, it includes 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar; the formula of the LB liquid medium involved is as follows: based on a volume of 1 L, it includes 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl.

[0051] The present application is further described in detail below with reference to the examples, drawings and test results.

[0052] Example

[0053] Example 1 - Screening of strains

[0054] This embodiment provides a screening process for new bacterial strains. Specifically, it includes the following steps:

[0055] (1) Sample collection: The samples were rotten baby cabbages (rotten baby cabbages obtained by placing the remaining baby cabbages at room temperature for 7 days after being picked from a plot of baby cabbages grown for many years).

[0056] (2) Preliminary isolation of target bacteria: Weigh 5 g of the sample collected above and add it to a pre-sterilized flask containing 500 ml of physiological saline under aseptic conditions. After vibrating thoroughly at room temperature for 30 minutes, dilute it to 10 -5 , select a dilution concentration of 10 -3 , 10 -4 , 10 -5 Dilutions of 0.1-0.2 ml were drawn respectively and spread on cellulose Congo red medium plates (formula: sodium nitrate 1 g / L; disodium hydrogen phosphate 1.2 g / L; potassium dihydrogen phosphate 0.9 g / L; magnesium sulfate 0.5 g / L; potassium chloride 0.5 g / L; yeast extract powder 0.5 g / L; acid hydrolyzed casein 0.5 g / L; Congo red 0.2 g / L; cellulose powder 5 g / L; agar 15 g / L; pH value 7.0±0.1 at 25°C). After the liquid was completely drawn out, the coated cellulose Congo red medium plates were placed upside down and cultured at 28°C for 48 h.

[0057] After culturing, two strains produced clear zones. The colonies that produced clear zones were streaked onto LB solid medium plates for isolation and purification (cultured according to the above culture conditions) 2-3 times until a single colony was obtained, indicating that the isolation and purification was complete. The colonies were stored for future use.

[0058] (3) Preliminary identification: The isolated and purified strains (i.e., isolated strains) were examined and observed under a microscope. Based on the microscopic examination results, it was preliminarily determined that both strains were of the genus Bacillus.

[0059] Example 2

[0060] This example provides the properties and sequencing results of a single strain of the isolated strain obtained in Example 1. The details are as follows:

[0061] (1) Properties of single strains

[0062] (1) Morphological characteristics

[0063] Isolated strain: The colony is off-white with smooth and neat edges. Figure 1 shown. Figure 1 To isolate the growth state of the strain, culture it on beef extract peptone medium for 48 hours. The formula of beef extract peptone medium is as follows: per 1L of distilled water, it contains 3g beef extract, 10g peptone, 5g sodium chloride, 15-20g agar, pH is 7.0, and the culture condition is 30℃.

[0064] (2) Culture characteristics

[0065] The optimal growth conditions for the strain are: pH 6.6-7.0, temperature 4-30℃.

[0066] (3) Functional characteristics

[0067] The above-mentioned isolated strains are used for decomposing tail vegetables, which can effectively improve the decomposition rate of tail vegetables.

[0068] (2) Sequencing

[0069] The isolated strain was sent to a third-party company for sequencing, and its 16s rRNA was obtained. Sequences of multiple reference strains were obtained from the NCBI (GenBank) database. The 16s rRNA of the isolated strain and the reference strains were analyzed using BioEdit and MEGA11 software to construct a phylogenetic tree of the isolated strain and the reference strains. This confirmed that the isolated strain was Bacillus pumilus.

[0070] Compared with related technologies, the strain isolated in this application has the following effects: through experimental results, the isolated strain was used to decompose peppers, tomatoes, and baby cabbages, and the decomposition rates of the tail cabbage reached 85.83%, 87.66%, and 85.83%, respectively. This shows that the Bacillus pumilus obtained in this application can effectively improve the decomposition rate of tail cabbage.

[0071] Bacillus pumilus was deposited in the General Microbiology Center of China Culture Collection Administration on December 18, 2024, with the deposit number CGMCC No. 33121, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0072] Example 3

[0073] This embodiment provides a fermentation broth of Bacillus pumilus.

[0074] The preparation method of the above-mentioned fermentation liquid is specifically as follows:

[0075] The isolated strain obtained in Example 1 was activated and transferred to LB liquid culture medium with a pH of 7. The culture was shaken in a shaker at 34° C. and 150 rpm for 24 h to obtain a fermentation broth of Bacillus pumilus.

[0076] Example 4

[0077] This embodiment provides a fermentation supernatant of Bacillus pumilus.

[0078] The preparation method of the above fermentation supernatant is as follows:

[0079] The isolated strain obtained in Example 1 was activated and transferred to LB liquid culture medium with a pH of 7. The culture was shaken in a shaker at 34° C. and 150 rpm for 24 h to obtain a fermentation broth of Bacillus pumilus. The fermentation broth was centrifuged and the supernatant was retained as the fermentation supernatant of Bacillus pumilus.

[0080] Example 5

[0081] This embodiment provides a bacterial suspension of Bacillus pumilus.

[0082] The preparation method of the above bacterial suspension is as follows:

[0083] The isolated strain obtained in Example 1 was activated and transferred to LB liquid culture medium with a pH of 7. The culture was shaken in a shaker at 34° C. and 150 rpm for 24 h to obtain a fermentation broth of Bacillus pumilus. The fermentation broth was centrifuged to retain the precipitate, which was resuspended in sterile physiological saline to obtain a bacterial suspension of Bacillus pumilus.

[0084] Example 6

[0085] This example provides the properties and sequencing results of another isolated strain obtained in Example 1. The details are as follows:

[0086] (1) Properties of single strains

[0087] (1) Morphological characteristics

[0088] Isolated strain: The colony is white, round, opaque, flat and dry. Figure 2 shown. Figure 2To isolate the growth state of the strain, culture it on beef extract peptone medium for 48 hours. The formula of beef extract peptone medium is as follows: per 1L of distilled water, it contains 3g beef extract, 10g peptone, 5g sodium chloride, and 15-20g agar, with a pH of 7.4-7.6 and the culture condition is 30℃.

[0089] (2) Culture characteristics

[0090] The optimal growth conditions for the strain are: pH = 7.0-7.2, temperature 34-36°C.

[0091] (3) Functional characteristics

[0092] When the above-mentioned isolated strains and the bacillus pumilus deposit numbered CGMCC No.33121 are used in combination, the tail vegetable decomposition rate of bacillus pumilus can be effectively improved.It can be seen from this that although the above-mentioned isolated strains are used alone and almost have no influence on the tail vegetable decomposition rate, it can promote the tail vegetable decomposition rate improving bacillus pumilus.Therefore, the application provides a microbial inoculum comprising this isolated strain and the bacillus pumilus deposit numbered CGMCC No.33121.Compared with prior art, this microbial inoculum can effectively improve the tail vegetable decomposition rate.

[0093] (2) Sequencing

[0094] The isolated strain was sent to a third-party company for sequencing, and its 16s rRNA was obtained. Sequences of multiple reference strains were obtained from the NCBI (GenBank) database. The 16s rRNA of the isolated strain and the reference strains were analyzed using BioEdit and MEGA11 software to construct a phylogenetic tree of the isolated strain and the reference strains. This confirmed that the isolated strain was Bacillus subtilis.

[0095] Compared with the use of Bacillus pumilus with the deposit number CGMCC No. 33121 alone, the strain isolated and obtained in this application has the following effect: through the test results, the microbial inoculum composed of the above-mentioned isolated strain and Bacillus pumilus with the deposit number CGMCC No. 33121 is used for the decomposition of peppers, tomatoes and baby cabbages, and the decomposition rates of the tail cabbage can reach 89.74%, 89.10% and 91.72% respectively. It can be seen that the microbial inoculum comprising Bacillus subtilis and Bacillus pumilus provided by this application can effectively improve the decomposition rate of the tail cabbage.

[0096] Bacillus subtilis was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on December 18, 2024, with the deposit number CGMCC No. 33122, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0097] Example 7

[0098] This embodiment provides a fermentation broth of Bacillus subtilis.

[0099] The preparation method of the above-mentioned fermentation liquid is specifically as follows:

[0100] The isolated strain obtained in Example 1 was activated and transferred to LB liquid culture medium with a pH of 7.2. The culture was shaken in a shaker at 36° C. and 150 rpm for 24 h to obtain a fermentation broth of Bacillus subtilis.

[0101] Example 8

[0102] This embodiment provides a fermentation supernatant of Bacillus subtilis.

[0103] The preparation method of the above fermentation supernatant is as follows:

[0104] The isolated strain obtained in Example 1 was activated and transferred to LB liquid culture medium with a pH of 7.2. The culture was shaken in a shaker at 36° C. and 150 rpm for 24 h to obtain a fermentation broth of Bacillus subtilis. The fermentation broth was centrifuged and the supernatant was retained as the fermentation supernatant of Bacillus subtilis.

[0105] Example 9

[0106] This embodiment provides a bacterial suspension of Bacillus subtilis.

[0107] The preparation method of the above bacterial suspension is as follows:

[0108] The isolated strain obtained in Example 1 was activated and transferred to LB liquid culture medium with a pH of 7.2. The culture was shaken in a shaker at 36° C. and 150 rpm for 24 h to obtain a fermentation broth of Bacillus subtilis. The fermentation broth was centrifuged to retain the precipitate, which was resuspended in sterile physiological saline to obtain a bacterial suspension of Bacillus subtilis.

[0109] Example 10

[0110] This embodiment provides a microbial agent.

[0111] The preparation method of the above-mentioned microbial agent is as follows:

[0112] The bacterial suspension of Bacillus pumilus with the deposit number CGMCC No. 33121 obtained in Example 5 (OD600 = 1) and the bacterial suspension of Bacillus subtilis with the deposit number CGMCC No. 33122 obtained in Example 9 (OD600 = 1) were mixed in a volume ratio of 1:1 to prepare a microbial agent. The effective bacterial content of Bacillus pumilus in the microbial agent was 5.4×10 9 CFU / g, the effective bacterial content of Bacillus subtilis is 6.5×10 9 CFU / g.

[0113] Performance testing

[0114] (1) Verification of the degradation effect on pepper

[0115] (1) The sample preparation process is as follows: peppers that have been chopped into 1-2 cm are divided into 6 groups, each group is 50 g and placed in plastic cups. Different test objects are inoculated at a 10% (V / V) inoculation rate and placed in an incubator at 35°C for static cultivation. After 15 days, samples are taken to determine the pepper decomposition rate.

[0116] The subjects to be tested in each group are as follows:

[0117] The microbial agent provided in Example 10 (the effective bacterial content of Bacillus pumilus was 5.4×10 9 CFU / g, the effective bacterial content of Bacillus subtilis is 6.5×10 9 / g) is the experimental group;

[0118] Inoculate the Bacillus pumilus provided in Example 5 (effective bacterial content of 5.4×10 9 CFU / g) was control group 1;

[0119] Inoculate the Bacillus subtilis provided in Example 9 (effective bacterial content of 6.5×10 9 CFU / g) was control group 2;

[0120] Inoculate the relevant technology of Streptomyces albidoflavus CGMCC No.12136 (effective bacterial content of 5.8×10 10 CFU / g) was control group three;

[0121] Inoculate the Bacillus tequilensis of CGMCC No. 22956 in the related technology (with an effective bacterial content of 6.2×10 10 CFU / g) was the control group IV;

[0122] Purified water was inoculated as the blank group.

[0123] (2) The calculation method of the decomposition rate of tail vegetables is as follows:

[0124] Decomposition rate of tail vegetables = (initial dry mass of tail vegetables (g) - dry mass of tail vegetables at the end of cultivation (g)) / initial dry mass of tail vegetables (g) × 100%.

[0125] (3) Test results

[0126] The test results are shown in Table 1. Among them, the results of the test group and the blank group are compared. Figure 3 shown.

[0127] Table 1 Degradation effect on pepper

[0128]

[0129] As shown in Table 1, when the Bacillus pumilus obtained in this application is used to decompose peppers, the decomposition rate of chili peppers can reach 85.83%, indicating that the Bacillus pumilus provided by this application can effectively improve the decomposition rate of chili peppers.

[0130] Combined with Table 1 and Figure 3 It can be seen that when the microbial agent obtained in this application is used for pepper decomposition, the decomposition rate of pepper can reach 89.74%, which is significantly improved by 13.71% compared with the relevant technology.

[0131] (2) Verification of degradation effect on tomatoes

[0132] (1) The test sample preparation process is as follows: Tomatoes that have been chopped into 1-2 cm are divided into 6 groups, each group of 50 g and placed in plastic cups. Different test objects are inoculated at a 10% (V / V) inoculation rate and placed in an incubator at 35°C for static cultivation. After 30 days, samples are taken to determine the tomato decomposition rate.

[0133] The subjects to be tested in each group are as follows:

[0134] The microbial agent provided in Example 10 (the effective bacterial content of Bacillus pumilus was 5.4×10 9 CFU / g, the effective bacterial content of Bacillus subtilis is 6.5×10 9 / g) is the experimental group;

[0135] Inoculate the Bacillus pumilus provided in Example 5 (effective bacterial content of 5.4×10 9 CFU / g) was control group 1;

[0136] Inoculate the Bacillus subtilis provided in Example 9 (effective bacterial content of 6.5×10 9 CFU / g) was control group 2;

[0137] Inoculate the relevant technology of Streptomyces albidoflavus CGMCC No.12136 (effective bacterial content of 5.8×10 10 CFU / g) was control group three;

[0138] Inoculate the Bacillus tequilensis of CGMCC No. 22956 in the related technology (with an effective bacterial content of 6.2×10 10 CFU / g) was the control group IV;

[0139] Purified water was inoculated as the blank group.

[0140] (2) The calculation method of the decomposition rate of tail vegetables is as follows:

[0141] Decomposition rate of tail vegetables = (initial dry mass of tail vegetables (g) - dry mass of tail vegetables at the end of cultivation (g)) / initial dry mass of tail vegetables (g) × 100%.

[0142] (3) Test results

[0143] The test results are shown in Table 2. Among them, the results of the test group and the blank group are compared. Figure 4 shown.

[0144] Table 2 Degradation effect on tomatoes

[0145]

[0146] As shown in Table 2, when the Bacillus pumilus obtained in this application is used to decompose tomatoes, the decomposition rate of tomato can reach 87.66%, indicating that the Bacillus pumilus provided by this application can effectively improve the decomposition rate of tomato.

[0147] Combined with Table 2 and Figure 4 It can be seen that when the microbial agent obtained in the present application is used for tomato decomposition, the decomposition rate of tomato can reach 89.10%, which is significantly increased by 16.59% compared with the related technology.

[0148] (3) Verification of the degradation effect on baby cabbage

[0149] (1) The preparation process of the test samples is as follows: the baby cabbage that has been chopped into 1-2 cm is divided into 6 groups, each group is 50 g and placed in plastic cups respectively. Different test objects are inoculated at a rate of 10% (V / V) and placed in an incubator at 35°C for static culture. After 10 days and 15 days, samples are taken to determine the decomposition rate of the baby cabbage.

[0150] The subjects to be tested in each group are as follows:

[0151] The microbial agent provided in Example 10 (the effective bacterial content of Bacillus pumilus was 5.4×10 9 CFU / g, the effective bacterial content of Bacillus subtilis is 6.5×10 9 / g) is the experimental group;

[0152] Inoculate the Bacillus pumilus provided in Example 5 (effective bacterial content of 5.4×10 9 CFU / g) was control group 1;

[0153] Inoculate the Bacillus subtilis provided in Example 9 (effective bacterial content of 6.5×10 9 CFU / g) was control group 2;

[0154] Inoculate the relevant technology of Streptomyces albidoflavus CGMCC No.12136 (effective bacterial content of 5.8×10 10 CFU / g) was control group three;

[0155] Inoculate the Bacillus tequilensis of CGMCC No. 22956 in the related technology (with an effective bacterial content of 6.2×10 10 CFU / g) was the control group IV;

[0156] Purified water was inoculated as the blank group.

[0157] (2) The calculation method of the decomposition rate of tail vegetables is as follows:

[0158] Decomposition rate of tail vegetables = (initial dry mass of tail vegetables (g) - dry mass of tail vegetables at the end of cultivation (g)) / initial dry mass of tail vegetables (g) × 100%.

[0159] (3) Test results

[0160] The test results are shown in Table 3 and Table 4. Among them, the results of the test group and the blank group are compared. Figure 5 and Figure 6 shown.

[0161] Table 3 Degradation effect on baby cabbage (I)

[0162]

[0163]

[0164] Table 4 Degradation effect on baby cabbage (II)

[0165]

[0166] As shown in Tables 3 and 4, when the Bacillus pumilus obtained in this application is used to decompose baby cabbage, the decomposition rate of baby cabbage after 10 days of cultivation can reach 86.90%, and the decomposition rate of baby cabbage after 15 days of cultivation can reach 85.83%, indicating that the Bacillus pumilus provided by this application can effectively improve the decomposition rate of baby cabbage.

[0167] Combined with Table 3-4 and Figure 5-6 It can be seen that when the microbial agent obtained in this application is used for tomato decomposition, the decomposition rate of tomato tail can reach 88.05% after 10 days of cultivation, and the decomposition rate of tomato tail can reach 91.72% after 15 days of cultivation. Compared with the relevant technology, the decomposition rate after 10 days of cultivation increased by 23.96%, and the decomposition rate after 15 days of cultivation increased by 16.21%.

[0168] (IV) Experiment on microbial decomposition and return of vegetable waste to the field

[0169] (1) Experimental design

[0170] The experiment was conducted on July 22, 2023 at Yongchang Experimental Station (101°04′ east longitude, 37°47′ north latitude) to conduct microbial decomposition and return of open-field vegetable tailings to the field.

[0171] The test material was broccoli, and its physical and chemical properties are shown in Table 5.

[0172] Table 5 Physical and chemical properties of broccoli leaves

[0173] Group N (mg / kg) P (mg / kg) K (mg / kg) Moisture content (%) Broccoli (dried) 3.44 0.33 3.61 - Broccoli (fresh) 1.25 0.12 1.31 63.75％

[0174] The experiment was conducted using fresh tail vegetables. The experiment was designed with 3 groups, 3 parallels in each group, and 9 treatments in total. Each treatment area was 18m 2 The categories are as follows:

[0175] A. Control (NC): Conventional fertilization was performed without the return of broccoli. Specifically, 0.5 t / mu of organic fertilizer (500 kg / mu of commercial organic fertilizer (30% organic matter, 4% total nutrients) and 30 kg / mu of 21-6-13 compound fertilizer) were evenly spread on the soil surface. A rotary tiller was used to work the organic fertilizer into the soil, tilling it to a depth of approximately 20 cm. The next crop, lettuce, was planted on July 24.

[0176] B. Control Group (CK): Conventional fertilization was performed alongside the return of broccoli to the field. Specifically, the broccoli was crushed to 3-5 cm and evenly spread on the soil surface. 0.5 t / mu of organic fertilizer (500 kg / mu of commercial organic fertilizer (30% organic matter, 4% total nutrients) and 30 kg / mu of 21-6-13 compound fertilizer) were evenly spread over the crushed broccoli (2240 kg / mu). The broccoli and organic fertilizer were then rotary-tilled into the field, tilling the broccoli to approximately 20 cm. The next crop, lettuce, was planted on July 24.

[0177] C. Experimental Group (RM): Conventional fertilization (10% reduction in fertilizer application) was performed, while broccoli was returned to the field. The microbial agent provided in Example 10 (2 billion / mL) was applied at a dosage of 0.5 kg / mu. Specifically, the broccoli was crushed to 3-5 cm, and the crushed broccoli (2240 kg / mu) was evenly spread on the soil surface. 0.5 t / mu of organic fertilizer (500 kg / mu of commercial organic fertilizer; 27 kg / mu of triple compound fertilizer (21-6-13)) and 0.5 kg / mu of microbial agent were mixed and evenly applied to the surface of the crushed broccoli. The broccoli and microbial agent were then rotated into the field using a rotary tiller, and the straw was rotary tilled to approximately 20 cm. The next crop, lettuce, was planted on July 24.

[0178] (2) Detection of decomposition rate of broccoli

[0179] To determine the impact of returning broccoli tails to the field using open-field microbial decomposition technology, the decomposition rate was determined using a nylon bag method. Four nylon bags, each filled with 40g of straw, were placed in each treatment. The bags were retrieved 5, 10, 15, and 20 days after returning to the field. The tails were washed and dried, and the decomposition rate was measured.

[0180] The calculation method of the decomposition rate of tail vegetables is as follows:

[0181] Decomposition rate of tail vegetables = (initial dry mass of tail vegetables (g) - dry mass of tail vegetables at the end of cultivation (g)) / initial dry mass of tail vegetables (g) × 100%.

[0182] Decomposition rate of tail vegetables = (dry mass of tail vegetables at the beginning (g) - dry mass of tail vegetables at the end (g)) / decomposition time (d).

[0183] The test results are shown in Table 6.

[0184] Table 6 Decomposition rate of broccoli

[0185]

[0186]

[0187] As shown in Table 6, the decomposition rate of broccoli tail was determined by nylon mesh bag method in the control group and the test group. The results showed that the decomposition rate of broccoli tail was the highest in the treatment group, and the decomposition rate reached 98.98% after 20 days of treatment. It can be seen that the microbial agent provided by this application is used for the microbial decomposition and field return technology of open-field vegetable tail, which can effectively improve the decomposition rate of tail.

[0188] (3) Yield measurement of the next crop, lettuce

[0189] After the broccoli was returned to the field, the yield of the next crop of lettuce was measured in September 2023. The experiment consisted of 3 groups, 3 replicates in each group, and 9 treatments in total. Each treatment was divided into 1m 2 Calculate the weight of lettuce leaves, roots, clean vegetables and 1m 2 The number of lettuce leaves can be used to estimate the weight of tail leaves, clean leaves and total weight in each treatment.

[0190] The test results are shown in Table 7 and Figure 7 shown.

[0191] Table 7 Lettuce yield under different treatments

[0192]

[0193] As shown in Table 7, the yield index was used to measure the growth of lettuce, the next crop after returning broccoli tails to the field. The results showed that: comparing the results of the blank group, control group, and experimental group, the net lettuce yield of the experimental group was significantly higher than that of the other treatments, reaching 7260.30 kg / mu. The blank group had the lowest lettuce yield, which was 5779.56 kg / mu. The results show that the microbial agent provided by this application can be used for the microbial decomposition and return of open-field vegetable tails to the field, which can significantly increase the yield of the next crop.

[0194] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A Bacillus subtilis, characterized in that The Bacillus subtilis ( Bacillus subtilis ) was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 33122.

2. A culture, characterized in that The culture comprises the Bacillus subtilis according to claim 1.

3. A fermentation broth, characterized in that: The fermentation broth comprises the Bacillus subtilis according to claim 1.

4. A bacterial suspension, characterized in that The bacterial suspension comprises the Bacillus subtilis according to claim 1.

5. A microbial agent, characterized in that: The microbial agent includes Bacillus pumilus and the Bacillus subtilis according to claim 1; The Bacillus pumilus ( Bacillus pumilus ) was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 33121.

6. The microbial agent according to claim 5, characterized in that The effective viable bacteria count of the Bacillus subtilis in the microbial agent is (6-7)×10 9 CFU / g; Optionally, the effective viable count of the Bacillus subtilis in the microbial agent is 6.5×10 9 CFU / g.

7. The microbial agent according to claim 5, characterized in that The effective viable count of the Bacillus pumilus in the microbial agent is (5-6)×10 9 CFU / g; Optionally, the effective viable count of the Bacillus pumilus in the microbial agent is 5.4×10 9 CFU / g.

8. Use of the Bacillus subtilis according to claim 1, the culture according to claim 2, the fermentation liquid according to claim 3, the bacterial suspension according to claim 4, and the microbial agent according to claim 5 in the field of quick-rotten tail vegetables.

9. The use according to claim 8, characterized in that When the Bacillus subtilis and the Bacillus pumilus are used in combination, the decomposition rate of the tail vegetable can be increased by more than 13%.

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