A strain with both cellulose degradation and deodorization functions

Abstract

This invention discloses a strain with both cellulose degradation and deodorization functions. The invention provides *Proteus alimentorum*, strain number YCY1-1, registered with the China General Microbiological Culture Collection Center (CGMCC) under number CGMCC No. 30359. The strain YCY1-1 provided by this invention can promote the decomposition of agricultural and forestry waste and remove the odor produced during the decomposition process. It has good application prospects in the development of microbial agents for the resource utilization of agricultural and forestry waste and specialized microbial fertilizers for the odor produced during decomposition.

Description

Technical Field

[0001] This invention relates to the field of agricultural production technology, and to a strain that has both cellulose degradation and deodorization functions, specifically to a strain YCY1-1 that has both cellulose degradation and deodorization functions, and removes the odor produced by the decomposition of vegetable waste. Background Technology

[0002] my country is the world's largest vegetable producer, and vegetables are the country's second largest crop after grains. In recent years, the vegetable industry has developed rapidly, with planting area and total output continuously increasing, accounting for more than 50% of the world's total output. While providing essential agricultural products for people's "vegetable basket," vegetable production also generates a large amount of waste. Furthermore, with the continuous increase in vegetable production and the rising demands for vegetable quality, the amount of waste is also increasing dramatically. Statistics show that the rate of waste generated during the production process of various vegetables ranges from 16.5% to 63.5%, averaging approximately 33.6%. Due to limitations in economic and technological levels, as well as the high water content of vegetable waste, a large amount of vegetable waste is improperly handled and indiscriminately dumped, leading to rotting, foul odors, mosquito and fly breeding, and the spread of pathogens, producing significant amounts of stench. Common agricultural and forestry wastes include crop straw, livestock and poultry manure, vegetable waste, and forest and fruit tree branches. Vegetable waste, in particular, contains a large amount of water, dietary fiber, enzymes, and vitamins, and is also a rich source of protein, soluble sugars, and minerals. Currently, the main methods of utilizing vegetable waste in my country are feed production, direct return to the field, and composting. Direct return to the field refers to the process of directly tilling agricultural waste into the soil, allowing its own nutrients to nourish crops. While this method can process a certain amount of vegetable waste in a short period, the natural degradation of vegetable waste has a long processing cycle and low efficiency.

[0003] Vegetable waste is mainly composed of cellulose, hemicellulose, and lignin. Cellulose, hemicellulose, and lignin are tightly linked by numerous covalent and non-covalent bonds to form lignocellulose, creating a three-dimensional structure that is insoluble in water and organic solvents and difficult to degrade at room temperature. This is the most critical reason for the low utilization rate of vegetable waste. Microorganisms secrete various enzymes to break down the chemical bonds between the components of vegetable waste, thereby disrupting its structure and degrading it. Therefore, the degradation process of vegetable waste is primarily driven by various enzymes. The enzyme systems that degrade vegetable waste are mainly divided into those that degrade cellulose, those that degrade hemicellulose, and those that degrade lignin. The addition of decomposing bacteria can accelerate the degradation of vegetable waste, increase soil organic matter, enzyme activity, and nutrients, improve soil structure, reduce agricultural waste pollution, and increase crop yields.

[0004] In cold, high-altitude regions, leafy vegetables, if not processed promptly after harvest, will cause bacteria and other microorganisms to decompose their organic matter, resulting in unpleasant odors. Malodorous gases are a serious type of environmental pollutant, affecting not only people's perception and environmental experience but also directly harming human health. Malodorous gases can strongly irritate the respiratory, nervous, circulatory, and endocrine systems. Biological deodorization uses the physiological metabolic activities of microorganisms to decompose the source of odors. It not only masks odors on the surface but also quickly and safely eliminates them at their source, inhibiting the growth of harmful bacteria. It is also relatively inexpensive and suitable for various environments. Typical substances in malodorous gases are ammonia and hydrogen sulfide. Many microorganisms in nature can degrade odor-causing substances by producing amino oxidases and enzymes that decompose sulfides. Furthermore, many microorganisms can produce antibacterial substances that inhibit the growth and reproduction of harmful bacteria such as pathogenic Escherichia coli and Salmonella. Therefore, isolating and screening deodorizing strains can effectively control odors and improve the problem of odors generated during the untimely disposal and decomposition of vegetable waste.

[0005] Therefore, screening strains that can both degrade agricultural and forestry waste / vegetable waste and remove odors and applying them to the autumn vegetable waste return treatment is particularly important for the resource utilization of vegetable waste. Summary of the Invention

[0006] The purpose of this invention is to provide a strain that combines cellulose degradation and deodorization functions.

[0007] Firstly, this invention claims protection for a Proteus bacillus.

[0008] The Proteus strain claimed in this invention is *Proteus alimentorum*, strain number YCY1-1, and its registration number at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) is CGMCC No. 30359. This strain simultaneously possesses the abilities of cellulose degradation, potassium solubilization, siderophore production, and IAA production, and also has deodorizing capabilities.

[0009] Secondly, the present invention claims protection for a culture of Proteus alimentorum YCY1-1 described in the first aspect above. The culture is the substance obtained by culturing Proteus alimentorum YCY1-1 described in the first aspect above in a bacterial culture medium (all substances within the culture container).

[0010] The substances in the above-mentioned culture include Proteus alimentorum YCY1-1 (the bacterial cell itself) and its metabolites.

[0011] The term "metabolite" refers to the primary and / or secondary metabolites produced during microbial metabolism. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the external environment and, through catabolism and anabolism, generate substances and energy to sustain life activities. The products of primary metabolism are called primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolism refers to the process by which microorganisms, at a certain growth stage, use primary metabolites as precursors to synthesize substances that have no clearly defined function for their life activities. The products of secondary metabolism are called secondary metabolites, and are mostly compounds with relatively complex molecular structures. Based on their functions, they can be classified into types such as antibiotics, hormones, alkaloids, and toxins.

[0012] In the above-mentioned cultures, the bacterial culture medium can be a solid culture medium or a liquid culture medium.

[0013] The term "culture" refers to any liquid or solid culture medium that has grown a microbial community after artificial inoculation and cultivation. It is the product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0014] Thirdly, this invention claims protection for a microbial agent.

[0015] The bacterial agent claimed in this invention contains Proteus alimentorum YCY1-1 as described in the first aspect above, metabolites of Proteus alimentorum YCY1-1, and / or the culture as described in the second aspect above.

[0016] The microbial agent is a microbial agent that promotes the decomposition of agricultural and forestry waste and / or vegetable waste and / or removes the odor produced by the decomposition of agricultural and forestry waste and / or vegetable waste.

[0017] In the aforementioned microbial agent, the active ingredient may be Proteus alimentorum YCY1-1, metabolites of Proteus alimentorum YCY1-1, and / or a culture of Proteus alimentorum YCY1-1. The active ingredient may also contain other biological and / or non-biological components. Other active ingredients of the microbial agent can be determined by those skilled in the art based on the decomposition or deodorization capabilities of vegetable waste.

[0018] In the aforementioned microbial agent, in addition to the active ingredient, a carrier is also included. The carrier can be a commonly used and biologically inert carrier in the pesticide field. The carrier can be a solid or liquid carrier; the solid carrier can be a mineral material, plant material, or polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0019] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0020] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the bacterial agent.

[0021] Fourthly, the present invention claims protection for any of the following applications of Proteus alimentorum YCY1-1 or its metabolites as described in the first aspect above, or the cultures described in the second aspect above, or the bacterial agents described in the third aspect above:

[0022] (A1) Potassium dissolution;

[0023] (A2) Prepare potassium-solubilizing products;

[0024] (A3) Iron-producing carrier;

[0025] (A4) Products for preparing iron-producing carriers;

[0026] (A5) Produces IAA;

[0027] (A6) Prepare products that produce IAA;

[0028] (A7) Degradation of cellulose;

[0029] (A8) Prepare products for degrading cellulose;

[0030] (A9) Prepare cellulase or products with cellulase activity;

[0031] (A10) Prepare filter paper enzymes or products with filter paper enzyme activity;

[0032] (A11) Removes ammonia and / or hydrogen sulfide;

[0033] (A12) Prepare products for removing ammonia and / or hydrogen sulfide.

[0034] Fifthly, the present invention claims protection for any of the following applications of Proteus alimentorum YCY1-1 or its metabolites as described in the first aspect above, or the cultures described in the second aspect above, or the bacterial agents described in the third aspect above:

[0035] (B1) Promotes the decomposition of agricultural and forestry waste;

[0036] (B2) Prepare products that promote the decomposition of agricultural and forestry waste;

[0037] (B3) Promotes the decomposition of vegetable waste;

[0038] (B4) Prepare products that promote the decomposition of vegetable waste;

[0039] (B5) Remove the odor produced by the decomposition of agricultural and forestry waste and / or vegetable waste;

[0040] (B6) Prepare products that remove the odors produced by the decomposition of agricultural and forestry waste and / or vegetable waste.

[0041] In the above aspects, the product may specifically be a microbial inoculant or microbial fertilizer. For example, a microbial inoculant or microbial fertilizer that promotes the decomposition and / or removes the odor of agricultural and forestry waste and / or vegetable waste during resource utilization.

[0042] Sixthly, the present invention claims a method for promoting the decomposition of agricultural and forestry waste and / or vegetable waste.

[0043] The method for promoting the decomposition of agricultural and forestry waste and / or vegetable waste claimed in this invention may include the following steps: treating the agricultural and forestry waste and / or vegetable waste to be decomposed with Proteus alimentorum YCY1-1 as described in the first aspect or its metabolites, or the culture as described in the second aspect or the microbial agent as described in the third aspect, thereby promoting the decomposition of agricultural and forestry waste and / or vegetable waste.

[0044] In the method, when the material to be decomposed is agricultural and forestry waste (such as sawdust), the method further includes the steps of adjusting the moisture content of the material to be decomposed to 40%-65% (e.g., 50%) and adding 0.5kg-2.0kg of urea per cubic meter of the material to be decomposed to adjust the carbon-nitrogen ratio to 25:1-35:1 (e.g., 30:1).

[0045] In the method, when the material to be decomposed is agricultural and forestry waste (such as sawdust), the decomposition time can be 60 days. When the material to be decomposed is vegetable waste, the decomposition time can be 15 days.

[0046] Seventhly, the present invention claims a method for removing odors produced by the decomposition of agricultural and forestry waste and / or vegetable waste.

[0047] The method for removing odors generated from the decomposition of agricultural and forestry waste and / or vegetable waste claimed in this invention may include the following steps: treating the agricultural and forestry waste and / or vegetable waste to be decomposed with Proteus alimentorum YCY1-1 as described in the first aspect or its metabolites, or the culture as described in the second aspect or the microbial agent as described in the third aspect, thereby removing the odors generated from the decomposition of agricultural and forestry waste and / or vegetable waste.

[0048] Eighthly, the present invention claims protection for the application of Proteus alimentorum YCY1-1 or its metabolites, or the culture, or the agent, described in the first aspect above, or the method described in the third aspect above, in the treatment of vegetable waste (such as vegetable tails) by returning it to the field.

[0049] In all of the above aspects, the vegetable may be any of the following:

[0050] (C1) Baby cabbage;

[0051] (C2) Leafy vegetables;

[0052] (C3) Chili peppers;

[0053] (C4) Fruits and vegetables.

[0054] Of the above aspects, the agricultural and forestry waste is wood chips.

[0055] The decomposition and deodorizing bacteria experiments on vegetable waste provided by this invention show that the decomposition rate of the baby bok choy inoculated with strain YCY1-1 is significantly better than that of the control group without strain YCY1-1. The decomposition rates of agricultural and forestry waste (wood chips) and chili peppers inoculated with strain YCY1-1 are also higher than those of the control group without strain YCY1-1. Deodorizing experiments show that the removal rates of ammonia and hydrogen sulfide in the baby bok choy inoculated with strain YCY1-1 are significantly higher than those in the control group without strain YCY1-1. Therefore, strain YCY1-1 provided by this invention has potential application prospects in promoting the decomposition of baby bok choy and removing the odor generated during the decomposition process. It also shows good application prospects in developing microbial agents for returning vegetable waste to the field and special microbial fertilizers for removing odors generated during the decomposition of vegetable waste.

[0056] Preservation Instructions

[0057] Category naming: Proteus alimentorum;

[0058] Biological material from ginseng: YCY1-1;

[0059] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0060] The abbreviation for the depository institution is CGMCC.

[0061] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0062] Deposit date: April 16, 2024;

[0063] Registration number at the Preservation Center: CGMCC No. 30359. Attached Figure Description

[0064] Figure 1 This is a colony morphology diagram of Proteus alimentorum YCY1-1 on a plate.

[0065] Figure 2 This is a Gram staining result of Proteus alimentorum YCY1-1.

[0066] Figure 3 This image shows the initial screening results for Proteus alimentorum YCY1-1 cellulose.

[0067] Figure 4 This is a diagram showing the potassium-solubilizing capacity of Proteus alimentorum YCY1-1.

[0068] Figure 5 A graph showing the siderophore production capacity of Proteus alimentorum YCY1-1.

[0069] Figure 6 This is a diagram illustrating the experiment on the decomposition of baby bok choy promoted by Proteus alimentorum YCY1-1.

[0070] Figure 7 This is a diagram of an experiment on the decomposition of sawdust by Proteus alimentorum YCY1-1.

[0071] Figure 8 This is a diagram of an experiment on the promotion of pepper decomposition by Proteus alimentorum YCY1-1. Detailed Implementation

[0072] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0073] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0074] Example 1: Isolation and Identification of Strain YCY1-1

[0075] I. Isolation of bacterial strains

[0076] The strain YCY1-1 used was an odor-removing strain that was previously isolated from rubber in the laboratory.

[0077] Weigh 10g of Thai raw rubber sample and add it to 90ml of sterile water containing glass beads. Shake thoroughly at 150r / min for 30min to obtain the bacterial suspension of the rubber sample mother liquor. Dilute the above bacterial suspension with sterile water to a concentration of 10:1. -1 10 -2 10 -3 10 -4 and 10 -5 Gradient, set to 10 -3 10 -4 10 -5Three consecutive 100 μL dilutions were spread onto prepared beef extract peptone medium (formulation: 3 g beef extract, 10 g peptone, 5 g sodium chloride, 15-20 g agar, 1000 mL distilled water, pH adjusted to 7.0) plates, with three replicates for each gradient. Sterile distilled water was used as a blank control. All plates were incubated at 30°C. After 3 days, single colonies of bacteria with different colors and morphologies were streaked onto plates, purified, and transferred to glycerol tubes for storage. Further deodorization tests were conducted to screen the strains, and strain YCY1-1, with better deodorization performance, was obtained.

[0078] II. Bacterial Genetic Identification and Phylogeny

[0079] DNA was extracted from the isolated strains using a bacterial genomic DNA extraction kit (TIANGEN). PCR amplification primers were the universal 16S rDNA primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′), with DNA fragments approximately 1000 bp in size, synthesized by Shanghai Sangon Biotech Co., Ltd. The PCR amplification reaction mixture consisted of 3 μL of DNA template, and 10 μmol / L each of the 27F and 1492R primers. -1 1 μL of 2×mixTaq enzyme (GenStar) and 7.5 μL of ddH2O were added to prepare a 25 μL system. PCR amplification reaction program: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 55℃ annealing for 1 min, 72℃ extension for 1.5 min, 30 cycles; final extension at 72℃ for 10 min, and storage at 4℃.

[0080] The 16S rDNA gene of the bacteria was amplified, and the sequencing results were uploaded to the NCBI database (https: / / www.ncbi.nlm.nih.gov / nuccore / / ) for online comparison. After comparison with the NCBI database, strain YCY1-1 was identified as *Proteus alimentorum*, with a maximum similarity of 99.85%. The 16S rDNA sequence of strain YCY1-1 is shown in SEQ ID No. 1.

[0081] III. Biological Characteristics Analysis of Strain YCY1-1

[0082] 1. Morphological observation and Gram staining

[0083] Strawberry strain YCY1-1 was streaked onto beef extract peptone medium using an inoculation loop and incubated upside down at 30°C for 24 hours. The basic morphology of the colonies was observed. A sterile glass slide was prepared in a clean bench. A drop of sterile water was placed on the slide, and a small amount of bacterial cells was dipped in and spread into a uniform thin layer using an inoculation loop. With the specimen side up, the slide was held by one end and carefully heated slightly over an alcohol lamp to evaporate the water. After cooling, staining began. Add 1-2 drops of ammonium oxalate crystal violet to the smear film to cover the smear with the staining solution. Stain for about 1 minute. Tilt the slide and rinse under a small stream of tap water until the rinsing water is colorless. Take 300 μL of iodine solution and apply it to the smear film to cover the smear with the staining solution. Stain for about 1 minute. Tilt the slide and rinse under a small stream of tap water until the rinsing water is colorless. Tilt the slide and add 95% ethanol to decolorize until the ethanol no longer appears purple. Rinse with water after about 20-30 seconds. Add 1-2 drops of safranin stain to the smear film to cover the smear with the staining solution. Stain for about 1 minute. Tilt the slide and rinse under a small stream of tap water until the rinsing water is colorless. Absorb the water droplets with absorbent paper and observe the specimen under a microscope after it dries.

[0084] The results showed that strain YCY1-1 formed round colonies with irregular edges, semi-transparent surfaces, and a pale yellow upper surface on beef extract peptone medium. Figure 1 Under a microscope, the colonies appear red, indicating they are Gram-negative bacteria. Figure 2 ).

[0085] 2. Determination of physiological and biochemical characteristics of strain YCY1-1

[0086] (1) Inoculate strain YCY1-1 into tryptone soybean agar medium (TSA, formula: 15g tryptone, 5g soybean peptone, 5g sodium chloride, 15g agar powder, 1000mL distilled water, pH=7.3) and culture for no more than 16h to avoid the formation of spores.

[0087] (2) Place the turbidimeter on a stable surface, calibrate and adjust the turbidimeter reading to 100, and use an Inoculatorz swab to collect a colony with a diameter of approximately 3 mm from the prepared plate and inoculate it into the IF-A inoculation solution. During the inoculation process, gently smear the swab on the wall of the inoculation bottle, shake the inoculation tube several times to ensure even inoculation of the strain, repeat several times, and adjust the turbidity of the inoculation solution to 90-98%.

[0088] (3) Pour the bacterial suspension containing the colonies into a sterile V-shaped sample loading trough. Using an 8-channel pipette, carefully add 100 μL of the bacterial suspension to each well in sequence. Cover the microplate with the cap, being careful not to touch the top and bottom surfaces to avoid inaccurate measurements.

[0089] (4) Place the Biolog GENEIII 96-well plate with the added samples directly into the OmniLog incubator and incubate at 33℃ for 24-48 hours. Then, read the data and perform identification. The results are shown in Table 1.

[0090] Table 1. Results of physiological and biochemical characteristics determination of strain YCY1-1

[0091]

[0092]

[0093]

[0094]

[0095] Note: + indicates positive; - indicates negative; w indicates weak positive.

[0096] Based on the above identification, strain YCY1-1 was identified as *Proteus alimentorum*, which was deposited on April 16, 2024, at the China General Microbiological Culture Collection Center (CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 30359. Hereinafter referred to as strain YCY1-1.

[0097] Example 2: Determination of cellulose degradation ability of strain YCY1-1

[0098] I. Initial screening of the cellulose degradation ability of strain YCY1-1

[0099] The ability of strain YCY1-1 to degrade cellulose was determined using the Congo red staining method. The differential culture medium used was formulated as follows: sodium carboxymethyl cellulose 5.5g, ammonium nitrate 0.6g, magnesium sulfate 0.2g, potassium hydrogen phosphate 0.34g, yeast extract 0.5g, NaCl 0.5g, agar 15g, distilled water 1000mL, pH 7.2-7.4. The specific procedures are as follows:

[0100] After activating strain YCY1-1, single colonies were picked and inoculated onto differential culture medium in triplicate, and incubated at 30℃ for 6 days. The plates were stained with 1.0 g / L Congo red solution for 30 min to prepare sodium carboxymethyl cellulose-Congo red solid medium. The medium was then destained twice with 1.0 mol / L sodium chloride solution for 30 min each time. The cellulose degradation ability of the strain was determined by the size of the clear zone (Dc value). Dc = diameter of the hydrolysis zone (D, cm) / colony diameter (d, cm).

[0101] Cellulose degradation ability of strain YCY1-1 Figure 3 As shown in the figure. The results showed that strain YCY1-1 had cellulose degradation ability, and the Dc value was 3.74±1.16cm.

[0102] II. Determination of cellulase activity and filter paper enzyme activity of strain YCY1-1

[0103] Culture media and reagents used:

[0104] Liquid fermentation enzyme production medium: 10.0g sodium carboxymethyl cellulose; 10.0g peptone; 5.0g yeast extract; 5.0g sodium chloride; 1.0g potassium hydrogen phosphate; 0.5g magnesium sulfate; 1000mL distilled water, pH 7.0. The medium is sterilized at 121℃ for 20min before use.

[0105] DNS reagent: Dissolve 182g of potassium sodium tartrate in 500mL of distilled water and heat in a constant temperature water bath at 50℃. Add 0.63g of 3,5-dinitrosalicylic acid, 2.1g of NaOH, and 0.5g of phenol to the hot solution and stir until dissolved. After cooling, dilute to 1000mL with distilled water and store in a brown bottle. Let stand at room temperature in the dark for 7 days before use.

[0106] Citric acid-sodium citrate buffer: Weigh 21.01g of citric acid, add distilled water and stir well, then bring the volume to 1000mL to prepare a 0.1mol / L citric acid solution; weigh 29.41g of sodium citrate, add distilled water and stir well, then bring the volume to 1000mL to prepare a 0.1mol / L sodium citrate solution; mix 9.2mL of 0.1mol / L citric acid solution with 10.8mL of 0.1mol / L sodium citrate buffer to obtain a citric acid-sodium citrate buffer with a pH of 4.8.

[0107] 1% CMC-Na solution: Weigh 10g of CMC-Na, add 990mL of citrate-sodium citrate buffer, and dissolve completely to obtain a 1% CMC-Na solution.

[0108] (1) Preparation of crude enzyme solution

[0109] The *Proteus alimentorum* YCY1-1 strain of the present invention was inoculated into liquid fermentation enzyme-producing medium (inoculation amount: 5%, volume percentage), and cultured at 37°C with shaking at 120 rpm for 4 days. The cultured fermentation broth substrate was then transferred to a centrifuge tube, balanced, and centrifuged at 4500 rpm for 10 min. The supernatant was collected to obtain the crude enzyme solution.

[0110] (2) Plotting the standard curve

[0111] Weigh 5g of glucose using a balance, heat and dry until the weight no longer changes, accurately weigh 1g of it into 50mL of deionized water, transfer it to a 100mL volumetric flask and make up to volume to obtain a standard solution with a mass concentration of 10mg / mL. Then measure 10mL of this solution and transfer it to another clean 100mL volumetric flask and make up to volume to obtain a glucose standard solution with a mass concentration of 1mg / mL.

[0112] Glucose standard curve: Take seven 25mL graduated tubes and pipette 0, 0.2, 0.6, 1.4, 1.8, and 2.0mL of glucose standard solution into each tube respectively. Then add distilled water to each tube to a final volume of 2mL. Add 2mL of DNS reagent to each tube and incubate in a boiling water bath for 5 minutes. After cooling to room temperature, dilute with distilled water to a final volume of 25mL. Shake to mix well and then measure the absorbance at 540nm using a spectrophotometer. Plot the absorbance to form a standard curve, with glucose content on the x-axis and absorbance value on the y-axis.

[0113] (3) Determination of cellulase activity in strains

[0114] Take four clean 10mL graduated test tubes and label them. Accurately add 15mL of 1% CMC-Na solution to each tube. Then, add 0.5mL of the crude enzyme solution prepared in step (1) to three of the test tubes respectively, leaving the blank tube untreated. Mix well and place in a 50℃ constant temperature water bath for 30min. Then, add 3mL of DNS reagent to each tube and 0.5mL of the crude enzyme solution to be tested to the blank tube. Mix well and place all four test tubes in a boiling water bath for 10min. Remove and cool to room temperature, then bring the volume to 10mL and mix well. Measure the absorbance at 540nm with the blank as the zero point and calculate the glucose content based on the glucose standard curve.

[0115] Enzyme activity calculation method: Under the above conditions and measurement methods, the amount of enzyme required to produce 1 μmol of glucose per minute from the substrate is taken as one unit of enzyme activity (U), expressed as U / mL.

[0116] Formula for calculating enzyme activity:

[0117]

[0118] (4) Determination of strain filter paper enzyme activity

[0119] Take four clean, graduated 10mL test tubes and label them. Add 1.5mL of citrate-sodium citrate buffer to each test tube, and add a Waterman filter paper strip (6cm long, 1cm wide) to each test tube. Roll up the filter paper strip so that the buffer solution in the test tube does not submerge the filter paper strip. Add 0.5mL of appropriately diluted crude enzyme solution prepared in step (1) to three reaction tubes and mix well. Incubate at 50℃ for 1 hour. Add 0.5mL of the crude enzyme solution to be tested to the blank tube, and then quickly add 3mL of DNS reagent to each of the four test tubes. Place them in a boiling water bath for 10 minutes, then remove and cool to room temperature. After the solution stabilizes, make up to 10mL. Measure the absorbance at 540nm. Calculate the reducing sugar content according to the glucose standard curve, and then calculate the enzyme activity using the enzyme activity calculation formula. The method for calculating enzyme activity is the same as that for cellulase activity.

[0120] (5) Results of cellulase activity and filter paper enzyme activity detection

[0121] The enzyme activity of the enzyme produced by strain YCY1-1 was determined using the above method, and the results are shown in Table 2. The cellulase activity of strain YCY1-1 was 1.836±0.032 U / mL, and the filter paper enzyme activity was 4.314±0.050 U / mL.

[0122] Table 2. Enzyme activity assay results of strain YCY1-1

[0123] strain Cellulase activity (U / mL) Filter paper enzyme activity (U / mL) YCY1-1 1.836±0.032 4.314±0.050

[0124] Note: The data in the table are the mean ± standard deviation of three replicates.

[0125] Example 3: PGPR functional characteristics analysis of strain YCY1-1

[0126] I. Determination of potassium solubilization capacity

[0127] The culture medium for determination was as follows: sucrose 5.0 g; disodium hydrogen phosphate 2.0 g; magnesium sulfate 0.5 g; calcium carbonate 0.1 g; ferric chloride 0.005 g; potassium feldspar powder 1.0 g; agar 18.0 g; dissolved in 1000 mL of distilled water; pH 7.0.

[0128] Inoculate strain YCY1-1 onto the above potassium-solubilizing medium (i.e., the test medium), incubate at 28°C for 3 days, and observe whether transparent oil droplet-like colonies appear on the plate. The presence of transparent oil droplet-like colonies proves that it has potassium-solubilizing ability.

[0129] II. Determination of Iron Production Capacity

[0130] The assay medium (CAS medium) is as follows: (1) The CAS staining solution contains 1 mmol / L chromazurite (CAS), 4 mmol / L hexadecyltrimethylammonium bromide (HDTMA) and 0.1 mmol / L ferric chloride. After thorough mixing, the CAS staining solution is obtained. (2) Weigh 2.427 g of disodium hydrogen phosphate, 0.5905 g of sodium hydrogen phosphate, 0.075 g of potassium hydrogen phosphate, 0.25 g of ammonium chloride, and 0.125 g of sodium chloride. Mix thoroughly to prepare 100 mL of phosphate buffer at pH 6.8. (3) Each 100 mL of CAS assay medium contains 1 mL of 20% sucrose solution, 3 mL of 10% acid-hydrolyzed casein, 100 μL of 1 mmol / L calcium chloride, 2 mL of 1 mmol / L magnesium sulfate, and 1.8 g of agar. Sterilize solutions (1), (2), and (3) respectively, cool them to 50°C, and slowly add 5 mL of solutions (1) and (2) to solution (3) to obtain blue CAS detection medium.

[0131] Inoculate strain YCY1-1 onto CAS detection medium and incubate at 28°C for 24 hours. If a clear zone appears on the CAS detection medium, it indicates that the bacterium can produce siderophores.

[0132] III. IAA Production Capacity Measurement

[0133] Standard curve construction: Prepare standard IAA solutions of 10, 20, 30, 40, and 50 μg / mL in volumetric flasks. Add 100 μL of each concentration of standard IAA solution to a 96-well plate (pre-filled with an equal volume of Salkowski colorimetric solution), in triplicate, and quickly place in the dark for 30 min. Add 100 μL of NA medium (i.e., beef extract peptone medium, formula: 3g beef extract, 10g peptone, 5g sodium chloride, 15-20g agar, 1000mL distilled water, adjusted pH=7.0) to 100 μL of Salkowski colorimetric solution as a negative control. Add 100 μL of 50 μg / mL standard IAA solution to 100 μL of Salkowski colorimetric solution as a positive control. Measure the OD530 absorbance, and average the three measurements. Plot the standard curve with IAA concentration on the x-axis and OD530 value on the y-axis.

[0134] Strain YCY1-1 was inoculated in NA liquid medium (containing 500 mg / L L-tryptophan) and cultured at 180 r / min at 30 ℃ for 48 h. 1 mL of the bacterial culture was transferred to a centrifuge tube, centrifuged at 8000 r / min for 5 min, and 100 μL of the supernatant was added to a 96-well plate. This process was repeated three times. 100 μL of Salkowski colorimetric solution was added, and the plate was immediately placed in the dark for 30 min. The OD530 absorbance was measured, and the average of the three measurements was substituted into the standard curve to calculate the IAA content.

[0135] IV. Results and Analysis

[0136] The results of the assays for potassium solubilization, siderophore production, and IAA production capacity of strain YCY1-1 are as follows: Figure 4 , Figure 5 As shown in Table 3. Figure 4 The results show the potassium solubilizing ability of strain YCY1-1. Figure 5 The results show the siderophore production capacity of strain YCY1-1. The results indicate that strain YCY1-1 possesses potassium solubilization and siderophore production capabilities, with an IAA content of approximately 5.33 ± 0.08 μg / mL.

[0137] Table 3. IAA Production Capacity Determination of Strains YCY1-1

[0138] strain Siderophore (diameter of the clear zone / colony diameter) IAA (μg / mL) YCY1-1 2.17±0.17 5.33±0.08

[0139] Note: The data in the table are the mean ± standard deviation of three replicates.

[0140] Example 4: Determination of the ability of strain YCY1-1 to promote the spoilage of baby bok choy

[0141] The materials used in this embodiment are as follows:

[0142] (1) Baby cabbage.

[0143] (2) Aviation cup (7cm in diameter).

[0144] (3) PDA liquid culture medium: 200g potato; 20g glucose; 20g agar; 5g peptone; 3g potassium dihydrogen phosphate; 1.5g magnesium sulfate; 1000mL distilled water; pH natural.

[0145] Strain YCY1-1 was inoculated into PDA liquid medium and cultured at 37℃ and 120 rpm in a shaker until the bacterial suspension concentration OD600 = 1. 50g of baby bok choy chopped to 1-2cm size was placed in a plastic cup, and the bacterial suspension was inoculated at a rate of 10% (volume percentage) as the experimental group, with water as the control group. Three replicates were set up, and the cups were incubated at 35℃. Samples were taken at 5, 10, and 15 days for decomposition rate determination.

[0146]

[0147] The ability of strain YCY1-1 to promote the decay of baby bok choy is as follows: Figure 6 As shown in Table 4, the results showed that the decomposition rate of baby bok choy inoculated with strain YCY1-1 was significantly higher than that of the control group.

[0148] Table 4. Effect of strain YCY1-1 on the decomposition rate of baby bok choy

[0149] deal with 5d decomposition rate / % 10-day decomposition rate / % 15-day decomposition rate / % control group 11.00±4.00b 22.00±4.00b 55.00±0.00b experimental group 21.00±6.00a 50.00±10.00a 76.00±9.00a

[0150] Note: Different lowercase letters in the same column indicate significant differences (P<0.05). The data in the table are the mean ± standard deviation of three replicates.

[0151] Example 5: Determination of the ability of strain YCY1-1 to promote the decay of sawdust.

[0152] The materials used in this embodiment are as follows:

[0153] (1) Wood chips.

[0154] (2) Plastic cup (7cm in diameter).

[0155] (3) PDA liquid culture medium: 200g potato; 20g glucose; 20g agar; 5g peptone; 3g potassium dihydrogen phosphate; 1.5g magnesium sulfate; 1000mL distilled water; pH natural.

[0156] Strain YCY1-1 was inoculated into PDA liquid medium and cultured at 37℃ in a shaker at 120 rpm until the bacterial suspension concentration (OD600) reached 1. Agricultural sawdust was pulverized into 2-3 cm pieces, and water was added to control the moisture content to 50%. Urea (0.5-2.0 kg / m³) was added per cubic meter to adjust the carbon-to-nitrogen ratio to 30:1. A 10% (volume percentage) inoculation was used as the experimental group, while water was used as the control group. Both groups were incubated at 70℃ for 60 days, and samples were taken for decomposition rate determination.

[0157]

[0158] The ability of strain YCY1-1 to promote the decomposition of sawdust is as follows: Figure 7 As shown in Table 5, the results showed that the decomposition rate of sawdust inoculated with strain YCY1-1 was higher than that of the control group.

[0159] Table 5. Effect of strain YCY1-1 on sawdust decomposition rate

[0160] deal with 60d decomposition rate / % control group 22.29±3.00b experimental group 42.00±5.00a

[0161] Note: Different lowercase letters in the same column indicate significant differences (P<0.05). The data in the table are the mean ± standard deviation of three replicates.

[0162] Example 6: Determination of the ability of strain YCY1-1 to promote spoilage of chili peppers

[0163] The materials used in this embodiment are as follows:

[0164] (1) Chili pepper.

[0165] (2) Plastic cup (7cm in diameter).

[0166] (3) PDA liquid culture medium: 200g potato; 20g glucose; 20g agar; 5g peptone; 3g potassium dihydrogen phosphate; 1.5g magnesium sulfate; 1000mL distilled water; pH natural.

[0167] Strain YCY1-1 was inoculated into PDA liquid medium and cultured at 37℃ and 120 rpm in a shaker until the bacterial suspension concentration OD600 = 1. 50g of chili peppers chopped to 1-2cm in size were placed in plastic cups, with 10% (volume percentage) of bacterial suspension used as the experimental group and water used as the control group. The cups were incubated at 50℃ and the decomposition rate was determined after 15 days.

[0168]

[0169] The ability of strain YCY1-1 to promote the decay of chili peppers is as follows: Figure 8 As shown in Table 6, the results showed that the decomposition rate of peppers inoculated with strain YCY1-1 was significantly higher than that of the control group.

[0170] Table 6. Effects of strain YCY1-1 on the decomposition rate of chili peppers

[0171] deal with 15-day decomposition rate / % control group 3.97±0.30b experimental group 16.67±2.21a

[0172] Note: Different lowercase letters in the same column indicate significant differences (P<0.05). The data in the table are the mean of three replicates.

[0173] Example 7: Deodorization ability test of strain YCY1-1

[0174] The materials used in this embodiment are as follows:

[0175] (1) Baby cabbage.

[0176] (2) Plastic cup (7cm in diameter).

[0177] (3) PDA liquid culture medium: 200g potato; 20g glucose; 20g agar; 5g peptone; 3g potassium dihydrogen phosphate; 1.5g magnesium sulfate; 1000mL distilled water; pH natural.

[0178] Strain YCY1-1 was inoculated into PDA liquid medium and cultured at 37°C in a shaker at 120 rpm until the bacterial suspension concentration (OD600) reached 1. 50g of baby bok choy, chopped to 1-2cm in size, was placed in a plastic cup. A 10% (volume percentage) inoculation of the bacterial suspension served as the experimental group, while a control group was inoculated with water. Three replicates were performed. The plastic cups were sealed with plastic wrap and covered with a sealing film to prevent gas evaporation. The cups were placed in an incubator at 35°C. Every 5 days, the concentrations of ammonia and hydrogen sulfide were measured using a portable gas detector. The inlet was inserted into the cup during each measurement, and the cup was immediately resealed after each measurement.

[0179]

[0180]

[0181] The effects of strain YCY1-1 on ammonia concentration in baby bok choy are shown in Tables 7 and 8. Ten days after inoculation with strain YCY1-1, the ammonia concentration in baby bok choy was significantly lower than that in the control group. At day 10, strain YCY1-1 achieved the highest ammonia removal rate, reaching 49.26%. The effects of strain YCY1-1 on hydrogen sulfide concentration in baby bok choy are shown in Tables 9 and 10. Ten days after inoculation with strain YCY1-1, the hydrogen sulfide concentration in baby bok choy was significantly lower than that in the control group. At days 10, 25, and 30, strain YCY1-1 achieved the highest ammonia removal rate, reaching 100%.

[0182] Table 7. Effects of strain YCY1-1 on ammonia concentration in baby bok choy

[0183]

[0184] Note: The data in the table are the mean ± standard deviation of three replicates.

[0185] Table 8. Determination of ammonia removal rate of strain YCY1-1 on baby bok choy

[0186]

[0187] Table 9. Effects of strain YCY1-1 on hydrogen sulfide concentration in baby bok choy

[0188]

[0189] Note: The data in the table are the mean ± standard deviation of three replicates.

[0190] Table 10. Effects of strain YCY1-1 on hydrogen sulfide concentration in baby bok choy

[0191]

[0192] Note: "-" in the table indicates no data.

[0193] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Proteus, characterized by: The Proteus is Proteus alimentorum Its strain number is YCY1-1, and its registration number at the China General Microbiological Culture Collection Center is CGMCCNo.30359.

2. The culture of Proteus according to claim 1 is a substance obtained by culturing the Proteus according to claim 1 in a bacterial culture medium.

3. A microbial agent, characterized in that: The bacterial agent contains the Proteus strain of claim 1 and / or the culture of claim 2.

4. The microbial agent according to claim 3, characterized in that: The microbial agent is a microbial agent that promotes the decomposition of vegetable waste and / or removes the odor produced by the decomposition of vegetable waste; The vegetables mentioned are baby bok choy or chili peppers; The odor is produced by ammonia and / or hydrogen sulfide.

5. Any of the following applications of the Proteus of claim 1, the culture of claim 2, or the bacterial agent of claim 3 or 4: (A1) Potassium solubilization; (A2) Preparation of potassium-solubilizing products; (A3) Iron-producing carrier; (A4) Products for preparing iron carriers; (A5) Produces IAA; (A6) Prepare products that produce IAA; (A7) Degradation of cellulose; (A8) Prepare products for degrading cellulose; (A9) Prepare cellulase or products with cellulase activity; (A10) Prepare filter paper enzymes or products with filter paper enzyme activity; (A11) Remove ammonia and / or hydrogen sulfide; (A12) Prepare products for removing ammonia and / or hydrogen sulfide.

6. Any of the following applications of the Proteus of claim 1, the culture of claim 2, or the bacterial agent of claim 3 or 4: (B1) Promotes the decomposition of agricultural and forestry waste; (B2) Prepare products that promote the decomposition of agricultural and forestry waste; (B3) Promotes the decomposition of vegetable waste; (B4) Prepare products that promote the decomposition of vegetable waste; (B5) Remove odors produced by the decomposition of agricultural and forestry waste and / or vegetable waste; (B6) Prepare products that remove the odor produced by the decomposition of agricultural and forestry waste and / or vegetable waste; The vegetables mentioned are baby bok choy or chili peppers; The odor is produced by ammonia and / or hydrogen sulfide.

7. The application according to claim 6, characterized in that: The agricultural and forestry waste is wood chips.

8. A method for promoting the decomposition of agricultural and forestry waste and / or vegetable waste, comprising the following steps: treating the agricultural and forestry waste and / or vegetable waste to be decomposed with the Proteus bacteria of claim 1, the culture of claim 2, or the microbial agent of claim 3 or 4; thereby achieving the promotion of the decomposition of agricultural and forestry waste and / or vegetable waste; The vegetables mentioned are baby bok choy or chili peppers.

9. A method for removing odors generated from the decomposition of agricultural and forestry waste and / or vegetable waste, comprising the following steps: treating the agricultural and forestry waste and / or vegetable waste to be decomposed with the Proteus bacteria of claim 1, the culture of claim 2, or the microbial agent of claim 3 or 4, thereby removing the odors generated from the decomposition of agricultural and forestry waste and / or vegetable waste; The vegetables mentioned are baby bok choy or chili peppers; The odor is produced by ammonia and / or hydrogen sulfide.

10. The method according to claim 8 or 9, characterized in that: The agricultural and forestry waste is wood chips.

11. The application of the Proteus of claim 1, the culture of claim 2, the microbial agent of claim 3 or 4, or the method of claim 8 or 9 in the treatment of vegetable waste returning to the field; The vegetables mentioned are baby bok choy or chili peppers.

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