A strain of Lysinibacillus louembei SCAUH16 and its application

Abstract

The present invention discloses a strain of Lysinibacillus louembei SCAUH16 and its application, which belongs to the field of microorganisms. The strain is preserved in the Guangdong Provincial Microbial Culture Collection Center with a preservation number of GDMCC NO: 65794. The strain of the present invention uses the residue after chemical treatment of dead pigs as an isolation source, and isolates and screens a strain with protease and lipase activity under high temperature conditions. The strain can quickly degrade the residue after chemical treatment of dead pigs, and the degradation rate reaches 61.37%. Furthermore, aerobic composting fermentation is carried out with the residue after chemical treatment of dead pigs as the main raw material. After adding the strain, the temperature of the pile body is increased, the material conversion is accelerated, the composting time is shortened, and the maturity of the compost is promoted, which is beneficial to reducing excessive nutrient loss during the composting process, maintaining the nutrients in the compost material, and improving the quality of the compost. It has good application prospects in the resource utilization of agricultural waste.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a Lysinibacillus louembei SCAUH16 strain and an application thereof. Background Art

[0002] With economic development and the improvement of residents' living standards, the consumption of livestock products such as pork continues to rise, and the demand for pig farming continues to increase. However, during the pig farming process, a large number of dead pigs will be produced due to stillbirths, diseases, natural disasters and other reasons. The carcasses of dead pigs pose serious hazards to economic production, ecological environment, food hygiene and human health. Therefore, it is particularly important to harmlessly dispose of the carcasses of dead pigs. The chemical treatment of dead pigs has the advantages of simple operation, good sterilization effect, short treatment cycle, and no smoke generation. It is currently one of the main ways to harmlessly treat dead pigs and an important way to achieve harmless treatment and resource utilization of dead livestock and poultry. However, after chemical treatment, the carcasses of dead pigs will produce by-products mainly composed of oils and solid materials. Currently, this problem is mainly solved by incineration, which will cause serious secondary pollution problems.

[0003] The residue from the composting of dead pigs, consisting primarily of fat and solid materials, contains large amounts of organic matter, nitrogen, phosphorus, and calcium, and has the potential to be used as an organic fertilizer. However, if the residue is directly returned to the fields, it takes a long time to decompose and poses the risk of secondary pollution. Therefore, the residue is used as an organic solid waste for high-temperature aerobic composting to produce organic fertilizer, thereby achieving resource recycling. Because the residue contains large amounts of protein, fat, and other substances, its complex composition makes rapid composting difficult. Numerous studies have shown that inoculating high-temperature, high-efficiency functional microbial agents during the composting process can not only accelerate the decomposition of substances, promote rapid temperature rise of the pile, and effectively kill pathogenic microorganisms in the pile, but also control the generation of odor during the composting process, promote the transformation of substances, and shorten the composting process. Currently, there are many reports on the addition of functional microbial agents to high-temperature aerobic composting, mainly focusing on the development and application of cellulase-producing agents under high-temperature conditions. However, the screening and application of high-temperature strains with protease and lipase degradation capabilities are relatively limited. Therefore, in order to achieve the harmless treatment and resource utilization of the residues after chemical processing of dead pigs, high-temperature bacterial strains with high efficiency in degrading protein and fat were screened and applied to the high-temperature aerobic composting of the residues after chemical processing of dead pigs to prepare organic fertilizer. This not only improved the quality of the compost, but most importantly, promoted the resource utilization of the residues after chemical processing of dead pigs and reduced the risk of secondary pollution, which is of great significance for achieving sustainable development. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: in order to isolate and screen a high-temperature bacterial strain capable of decomposing protein and fat, and apply it to the decomposition of residues after dead pig processing and its high-temperature aerobic composting treatment, one of the purposes of the present invention is to provide a Bacillus strain with the ability to decompose protein and fat, and another purpose is to provide an application of the strain.

[0005] The technical solution of the present invention is: a Lysinibacillus strain Lysinibacillus louembei SCAUH16, referred to as SCAUH16, which was deposited in the Guangdong Provincial Microbiological Culture Collection on January 15, 2025, with a deposit number of GDMCC NO: 65794, and the deposit unit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0006] A microbial inoculant containing the strain Lysinibacillus louembei SCAUH16.

[0007] Application of the above-mentioned strain Lysinibacillus louembei SCAUH16 or microbial agent in the treatment of residues after dead pig processing.

[0008] Furthermore, the treatment is high-temperature aerobic composting.

[0009] Furthermore, the high-temperature aerobic composting refers to mixing the residue after the dead pigs are processed with sawdust, inoculating the above-mentioned strain Lysinibacillus louembei SCAUH16 or microbial agents, and performing high-temperature composting fermentation.

[0010] Furthermore, the high-temperature aerobic composting method is as follows: sawdust is used as an auxiliary material and mixed with the residue after the dead pig is processed, the moisture of the material is adjusted to 50-60%, the initial carbon-nitrogen ratio is controlled at 25-30, and the above-mentioned microbial agent is added to the material at a weight ratio of 0.5% for high-temperature aerobic composting for 30 days. When the pile temperature reaches above 50°C, the first turning of the pile is carried out; when the temperature reaches 60°C, the second turning of the pile is carried out.

[0011] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0012] The present invention uses dead pig composting residue as a source for separation and screening. The strain, Lysinibacillus louembei SCAUH16, obtained through separation and screening, can grow under high-temperature conditions and has the ability to decompose protein and fat, showing potential for application in high-temperature aerobic composting. This strain, belonging to the genus Lysinibacillus, has an optimal growth temperature range of 35-70°C and exhibits high protease and lipase activities of 56.9 μmol / mL and 28.6 μmol / mL, respectively. Inoculation with this strain significantly reduces the volume of dead pig composting residue, lowers the protein content by 25.56%, and achieves a degradation rate of 61.37%. The microbial agent prepared with this strain is applied to high-temperature aerobic composting fermentation with dead pig residue as the main material, thereby achieving the effect of promoting the degradation and composting of dead pig residue. The present invention not only shortens the fermentation cycle of composting of such materials, but also improves the overall quality of the compost, provides strain resources and technical references for the resource utilization of dead pig residue, is beneficial to the harmless treatment and resource utilization of agricultural waste, and has good application prospects.

[0013] The preservation information is as follows:

[0014] The Lysinibacillus strain Lysinibacillus louembei SCAUH16, referred to as SCAUH16, was deposited in the Guangdong Provincial Microbiological Culture Collection on January 15, 2025, with the deposit number GDMCC NO: 65794, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a photo of the colony morphology of Lysinibacillus louembei SCAUH16 on tryptic soy agar medium (TSA medium);

[0016] Figure 2 This is the phylogenetic tree of Lysinibacillus louembei SCAUH16 constructed based on 16S rRNA sequence results;

[0017] Figure 3 This is the preliminary screening result of protease produced by Lysinibacillus louembei SCAUH16;

[0018] Figure 4 This is the preliminary screening result of lipase production by Lysinibacillus louembei SCAUH16;

[0019] Figure 5 The pH changes of the culture medium of the residual material after the treatment of dead pigs with Lysinibacillus louembei SCAUH16 and without bacteria (CK) (1-9 days);

[0020] Figure 6 The degradation effect of Lysinibacillus louembei SCAUH16 on dead pig residues; a is the blank control without inoculation, b is the experimental group inoculated with Lysinibacillus louembei SCAUH16, and from left to right are the degradation effect graphs from 1 to 9 days;

[0021] Figure 7 To compare the degradation rate of the residue after the treatment of dead pigs with Lysinibacillus louembei SCAUH16 and without bacteria (CK) (1-9 days);

[0022] Figure 8 To study the changes in protein content in the residues of dead pigs treated with Lysinibacillus louembei SCAUH16 and without bacteria (CK) (1-9 days).

[0023] Figure 9 The temperature change of the compost pile;

[0024] Figure 10 is the C / N change of compost;

[0025] Figure 11 It is the change of germination index (GI) of compost seeds;

[0026] Figure 12 For compost pH changes;

[0027] Figure 13 is the change of compost moisture content;

[0028] Figure 14 is the change in electrical conductivity of the compost;

[0029] Figure 15 For the changes of total phosphorus in compost;

[0030] Figure 16 For the changes of total potassium in compost;

[0031] Figure 17 This is a Venn diagram analysis of bacterial flora in samples of dead pig residue compost at different fermentation stages;

[0032] Figure 18 This is the bacterial community composition of samples from different fermentation stages of composting residues from dead pigs. DETAILED DESCRIPTION

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0034] In this application:

[0035] Tryptone soy peptone agar medium (TSA medium): tryptone 15 g, soy peptone 5 g, NaCl 5 g, agar 20 g, distilled water 1000 mL.

[0036] TSB medium: tryptone 17 g, soy peptone 3 g, NaCl 5 g, K2HPO4 2.5 g, glucose 2.5 g, agar 20 g, distilled water 1000 mL.

[0037] Protease seed medium (g / L): glucose 10 g, yeast powder 20 g, NaCl 5 g, K2HPO4 1 g, MgSO4 0.4 g, distilled water 1000 mL, pH 7.0;

[0038] Protease basal fermentation medium (g / L): glucose 5 g, yeast powder 10 g, KH2PO4 0.5 g, MgSO4.7H2O0.3 g, (NH4)2SO4 1 g, CaCL2 1 g, NaCl 1 g, distilled water 1000 mL, pH 7.2;

[0039] Carcass residue culture medium (g / L): 5g glucose, 10g solid residue from harmless carcasses, 0.5g KH2PO4, 0.3g MgSO4.7H2O, 1g (NH4)2SO4, 1g CaCl2, 1g NaCl, 1000mL distilled water, pH 7.2. The solid residue from harmless carcasses used in the fermentation medium is chemically treated carcasses.

[0040] Skim milk solid culture medium: yeast extract 2.5 g, peptone 2 g, acid hydrolyzed casein 1.5 g, casein 1.5 g, skim milk 40 g, agar 20 g, glucose 1 g, distilled water 1000 mL.

[0041] Protease seed medium: glucose 10 g, yeast powder 20 g, NaCl 5 g, K2HPO4 1 g, MgSO4 0.4 g, pH 7.0, distilled water 1000 mL.

[0042] Victoria Blue B solid medium: peptone 10 g, beef extract 3 g, sodium chloride 5 g, agar 20 g, olive oil 25 mL, distilled water 1000 mL, Victoria Blue (4 mg / 100 mL).

[0043] Example 1 Isolation and Identification of Lysinibacillus louembei SCAUH16

[0044] 1.1 Isolation of Lysinibacillus louembei SCAUH16

[0045] Weigh 10g of fresh dead pig residue after processing and add 90mL of sterile water into a 250mL sterile conical flask, put it into a shaker, set the temperature at 50℃ and the speed at 180r / min, incubate for 30min, mix well and let it stand, take the supernatant for gradient dilution, the gradient dilution factor is 10 -1 to 10 -5 , from 10 -3 , 10 -4 , 10 -5 Take 100 μL of each of the liquids and spread evenly on casein agar. Place the plate in a constant temperature incubator at 50°C for 48 hours. Select strains with rapid growth and large colonies and purify them using the dilution plate streak method until a purified strain is obtained. Transfer the purified strain to trypticase soy broth (TSB) and culture at 50°C with shaking for 7 days. After preservation, add 30% glycerol and freeze at -80°C.

[0046] 1.2 Identification of Lysinibacillus louembei SCAUH16

[0047] 1.2.1 Strain morphology

[0048] A single colony of strain SCAUH16 was picked and inoculated on tryptic soy agar medium (TSA medium), cultured at 50°C for 24 hours, and the colony morphology of the strain was observed.

[0049] 1.2.2 DNA extraction and PCR amplification

[0050] (1) Reagents: Sterile double-distilled water, Mix (Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0051] (2) Primers

[0052] 27F:5'-AGAGTTTGATCCTGGCTCAG-3

[0053] 1492R:5'-TACGACTTAACCCCAATCGC-3'.

[0054] (3) Bacterial DNA extraction

[0055] Use a disposable inoculating loop to pick a small amount of bacteria into a sterile 1.5mL centrifuge tube, then add 500μL of sterile double-distilled water, quickly freeze the centrifuge tube with liquid, then place it in a 99℃ metal bath for 5min, take it out and vortex for 30s, repeat the above operation 1-2 times, then centrifuge the obtained bacteria and sterile double-distilled water mixture at 12000rpm for 3min, take the supernatant as a template, and store it at -20℃; detect by 1% agarose gel electrophoresis.

[0056] (4) Bacterial 16S rRNA gene amplification

[0057] 16S rRNA gene amplification conditions included 30 cycles of initial denaturation at 95°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1.5 minutes, with a total extension at 72°C for 20 minutes. PCR products were purified using the Shanghai Sangon EZ Spin Column PCR Product Purification Kit UN1Q-1 (SK1142-N) according to the manufacturer's instructions and sent to Sangon Biotechnology Co., Ltd. for sequencing.

[0058] 1.2.3 16S rRNA gene sequence analysis and phylogenetic tree construction

[0059] The sequence obtained after sequencing (SEQ ID No. 1) was searched for similarity in NCBI using BLAST software. The 16S rRNA gene sequence of the most similar published standard strain of the same genus was selected as the reference sequence. The sequence of the standard strain of Bacillus subtilis (AB042061) was used as the outgroup. Multiple sequence alignment analysis was performed using Clustal X software, and a phylogenetic tree was constructed using the Newton-Jones method using MEGA7.0 software to determine the taxonomic status of the strain.

[0060] 1.3 Experimental Results

[0061] Lysinibacillus louembei SCAUH16 was isolated from the residue of dead pigs. After culturing on TSA medium at 50℃ for 1 day, the colonies were 4-5 mm in size, round and convex, with irregular edges and smooth surface. The colonies were beige in color and easy to pick. They were Gram-positive, aerobic, rod-shaped, and had spores. The size of the bacteria was 0.6×5.0μm ( Figure 1 ).

[0062] The 16S rRNA sequence of strain SCAUH16 was determined, and BLAST homology comparison was performed in the NCBI database. Phylogenetic analysis was performed using MEGA7.0 software. The 16S rRNA nucleotide sequence of this strain was 99.93% identical to that of Lysinibacillus louembei. Based on the morphological characteristics, physiological and biochemical characteristics, and 16S rRNA sequence homology analysis, strain SCAUH16 was identified as Lysinibacillus louembei ( Figure 2 ).

[0063] Example 2 Growth Temperature and Enzyme Production Capacity Determination of Lysinibacillus louembei SCAUH16

[0064] 2.1 Determination of temperature range

[0065] The strain Lysinibacillus louembei SCAUH16 was inoculated on TSA medium and cultured in incubators at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 48 hours. Its growth was observed and recorded. Based on the measurement results, the temperature detection range was further narrowed in units of 5℃ until the maximum and minimum growth temperatures of the strain were determined.

[0066] 2.2 Protein degradation ability determination

[0067] 2.2.1 Preliminary screening of protein degradation ability

[0068] The strain was evenly spread on tryptic soy peptone agar medium (TSA medium) and placed in a 50℃ biochemical incubator to culture until the strain grew evenly on the entire medium surface. A bacterial cake was made with a 5mm diameter puncher and inverted on a skim milk medium. After culture in a 50℃ biochemical incubator for 2 to 4 days, the hydrolysis zone diameter (D) and colony diameter (d) were measured respectively, and the ratio of the two (D / d) was calculated. The larger the ratio, the stronger the strain's ability to degrade protein. Three replicates were set.

[0069] 2.2.2 Determination of protease activity of strains

[0070] (1) Drawing of standard curve

[0071] After preparing 100 μg / mL L-tyrosine standard liquid, dilute the 100 μg / mL L-tyrosine standard liquid to obtain L-tyrosine solutions with concentrations of 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. Take 1 mL of each diluted solution, add 5 mL of 0.4 mol / L sodium carbonate solution and 1 mL of 1 mol / L Folin-phenol reagent solution, shake well, and develop color in a 40±0.2°C water bath for 20 minutes. Then take out and measure the absorbance using a spectrophotometer at a wavelength of 680 nm and a 10 mm cuvette, using tube 0 without tyrosine as the blank. Draw a standard curve with absorbance A as the ordinate and tyrosine concentration c as the abscissa. Use the regression equation to calculate the amount of tyrosine (μg) when the absorbance is 1, which is the absorption constant K value. The K value should be within the range of 95 to 100. Draw an L-tyrosine standard curve based on the concentration-absorbance data.

[0072] (2) Preparation of crude enzyme solution.

[0073] Pick one loop of pure culture stored on the slant and inoculate it into protease seed medium, culture it at 50℃ on a shaker at 150 rpm for 12 h, inoculate it into protease basal fermentation medium at a 1% (v / v) inoculation rate, culture it at 50℃ on a shaker at 150 rpm for 48 h, then centrifuge and collect the supernatant for enzyme activity determination.

[0074] (3) Enzyme activity determination

[0075] The crude enzyme solution was assayed for enzyme activity using the Folin-phenol method. One unit of enzyme activity is defined as the production of 1 μg of tyrosine per minute of casein substrate hydrolysis at 40°C. A 2% (mass fraction) casein solution was placed in a 40°C constant temperature water bath and preheated for 5 minutes. 1 mL of crude enzyme solution was added to 1 mL of preheated casein solution, mixed thoroughly, and then placed in a 40°C water bath for 10 minutes. The reaction was terminated by adding 2 mL of 0.4 mol / L trichloroacetic acid. 1 mL of supernatant was added to 5 mL of 0.4 mol / L Na2CO3, followed by 1 mL of Folin-phenol reagent. The mixture was then colorized at 40±0.2°C in a water bath for 20 minutes. The absorbance was then measured at 660 nm, with the water-added reaction system serving as the blank.

[0076] (4) Calculation of enzyme activity: Read the enzyme activity of the final dilution from the standard curve in U / mL. The enzyme activity of the original solution is calculated according to the following formula:

[0077] Protease activity (U / mL) = A × K × 4 ÷ 10 × n

[0078] Where: A: OD value of parallel experiments of fermentation stock solution;

[0079] K: absorption constant;

[0080] n: dilution factor of protease solution;

[0081] 4: Total volume of reaction reagents;

[0082] 10: Reaction time 10 min; the results are expressed to the nearest integer.

[0083] 2.3 Determination of fat degradation ability

[0084] 2.3.1 Preliminary screening of fat degradation ability

[0085] The strain was evenly spread on tryptic soy peptone agar medium (TSA medium) and placed in a 50℃ biochemical incubator to culture until the strain grew evenly on the entire surface of the medium. A bacterial cake was made with a 5mm diameter puncher and inverted on Victoria Blue B solid medium. After culturing in a 50℃ biochemical incubator for 2 to 4 days, the culture medium was observed to see whether blue was produced at the inoculation site. The color change zone diameter (D) and colony diameter (d) were measured respectively, and the ratio of the two (D / d) was calculated. The larger the ratio, the stronger the strain's ability to degrade fat. Three replicates were set.

[0086] 2.3.2 Lipase activity assay of strains

[0087] Take several 100mL conical flasks, one as the control flask and the others as the test flasks. The specific method is shown in Table 1.

[0088] Table 1 Lipase activity determination method

[0089]

[0090] Titrate with 0.05M standard sodium hydroxide solution until the solution turns slightly red, and record the volume of NaOH solution used for titration.

[0091] Calculation: The specific activity of lipase is defined as the amount of enzyme that produces 1 μmol of fatty acid per minute when 1 g of lipase hydrolyzes fat at pH 7.5 and 40°C.

[0092]

[0093] Where: A is the alkali solution consumed by the sample (ml); B is the alkali solution consumed by the control group (ml); N is the concentration of the alkali solution, i.e. 0.05 μmol; f is the final dilution factor of the crude enzyme solution; t is the action time (min).

[0094] 2.4 Experimental Results

[0095] It was determined that the growth range of Lysinibacillus louembei SCAUH16 is 35-70°C and it has the ability to produce protease and lipase ( Figure 3 and Figure 4 The relative enzyme activities of protease and lipase were 56.9 μmol / mL and 28.6 μmol / mL, respectively (Table 2). These results indicate that Lysinibacillus louembei SCAUH16 exhibits excellent thermostability and the ability to produce functional enzymes, suggesting potential for further development and application.

[0096] Table 2 Enzyme production capacity of Lysinibacillus louembei SCAUH16

[0097]

[0098] Example 3 Preparation of microbial agent

[0099] This embodiment provides a microbial agent, and the preparation method of the microbial agent is as follows:

[0100] Lysinibacillus louembei SCAUH16 was streaked onto TSA medium, and a single colony was picked and inoculated into TSB medium. The culture was carried out at 50-60°C and 160-200 rpm for 1 day to obtain a seed solution. The seed solution was inoculated into a fermentation medium at a 5% (v / v) inoculum and cultured at 50-60°C and 160-200 rpm for 24-48 hours to obtain a microbial inoculum with an effective viable count of 1.3×10 8 CFU / g, pH value is 6.0~8.0.

[0101] Fermentation medium formula: peptone 7g, beef extract 1g, sodium chloride 5g, glucose 10g, distilled water 1000mL.

[0102] Example 4 Fermentation culture of strain Lysinibacillus louembei SCAUH16

[0103] 4.1 Experimental materials and process

[0104] Preparation of solid residue from pig carcass chemical treatment: After crushing the pig carcasses, they were treated in a high-temperature, high-pressure chemical treatment tank (temperature ≥ 140°C, pressure ≥ 0.5 MPa (absolute pressure)) for 4 hours, then dried in a high-temperature, normal-pressure drying tank. Finally, the liquid was separated in an oil press to obtain the pig carcass residue. The pig carcass residue used in this invention was provided by Chengdu Kenong Animal Harmless Disposal Co., Ltd.

[0105] Experimental setup: A blank control group (CK group) without the addition of any microbial agents and a microbial agent containing Lysinibacilluslouembei SCAUH16 (SCAUH16 group, added at 0.5% (v / v)) were added. Both were cultured at 50°C and 180r / min. Sampling was continued for 9 days, and the physical and chemical properties of the samples were analyzed to determine the degradation effect of strain SCAUH16.

[0106] 4.2 Analysis of physical and chemical properties

[0107] 4.2.1 Degradation rate of dead pig residue

[0108] The degradation rate of solid residues after harmless treatment of dead pigs was calculated according to the weight loss method: the residues after the dead pigs were cultured in shake flasks were filtered through filter paper, the residues were rinsed with distilled water for 2 to 3 times, dried at 65℃ for 24h, weighed, and the weight loss and degradation rate were calculated.

[0109]

[0110] Where: A is the initial dry weight of solid residue after harmless treatment of dead pigs (g);

[0111] B is the dry weight (g) of the solid residue after the degradation and harmless treatment of dead pigs.

[0112] 4.2.2 Determination of pH value of culture medium containing dead pig residue

[0113] Refer to the pH determination method in the Agricultural Industry Standard of the People's Republic of China, NY / T 525-2021. Transfer the culture medium containing dead pig residue from a 250mL Erlenmeyer flask to a 250mL beaker, let it stand for 30 minutes, and then measure the pH using a pH meter. Read and record the pH value. Before measurement, calibrate the pH meter with a standard buffer solution (temperature compensation set to 25°C).

[0114] 4.2.3 Protein content of dead pig residue substrate

[0115] Following the method outlined in the Agricultural Industry Standard of the People's Republic of China, NY / T 525-2021, "Determination of Total Nutrient Content in Fertilizers," weigh 0.5-1.0 g (accurate to 0.0001 g) of air-dried sample of dead pig residue and place it at the bottom of an appropriately sized Erlenmeyer flask. Add 5 mL of sulfuric acid and 1.5 mL of hydrogen peroxide, shake carefully, and place a small curved-necked funnel at the top of the flask. Leave overnight. The next day, slowly heat the sulfuric acid until it fumes. Once cooled slightly, add 15 drops of hydrogen peroxide. Gently shake the Erlenmeyer flask or digestion tube and heat for 10 minutes. Once cooled slightly, add another 5-10 drops of hydrogen peroxide and digest in portions until the solution is colorless or light yellow. Continue heating for 15 minutes to remove any remaining hydrogen peroxide. Remove the flask, cool it, and carefully add water to 20-30 mL. Gently shake the Erlenmeyer flask or digestion tube. Rinse the small curved-necked funnel with a small amount of water and collect the washings in the Erlenmeyer flask. Transfer the digestion solution to a 50 mL volumetric flask, cool to room temperature, and dilute to volume with water. Allow to settle or dry filter with phosphate-free filter paper into a stoppered Erlenmeyer flask for later use. For a blank run, use the same reagent amounts as for the experimental and operational samples, except that no sample is added. Analyze and calculate the protein content in the sample using an automated Kjeldahl nitrogen analyzer and express it as mass fraction (%).

[0116]

[0117] Where:

[0118] C is the molar concentration of the calibration standard solution, in moles per liter (mol / L);

[0119] V0 is the volume of the calibration standard solution consumed during the blank test, in milliliters (mL);

[0120] V is the volume of the standard solution consumed during sample determination. The unit is milliliter (mL);

[0121] 14 is the molar mass of nitrogen in grams per mole (g / moL);

[0122] M is the mass of the air-dried sample, in grams (g);

[0123] X is the value of the moisture content of the air-dried sample;

[0124] D is the aliquot multiple, constant volume / aliquot volume;

[0125] 6.25 is the factor for converting nitrogen to protein.

[0126] 4.3 Experimental Results

[0127] 4.3.1 pH changes in dead pig residues

[0128] The pH values of the samples in the two treatment groups were measured. It was found that the pH values of the CK group samples did not change significantly, and the pH value of the fermentation broth was between 5.41 and 5.73. However, the pH value of the SCAUH16 group samples showed a significant upward trend from the second day. From the first day to the sixth day, the pH value of the SCAUH16 group samples increased from 6.19 to 8.97, and then stabilized at 8.95-9.00 ( Figure 5 The results showed that Lysinibacillus louembei SCAUH16 decomposed proteins during metabolism to produce alkaline substances, which led to changes in pH.

[0129] 4.3.2 Changes in the degradation rate of dead pig residue

[0130] The amount of dead pig residue after adding strain SCAUH16 was significantly reduced compared with the CK group ( Figure 6 ), the degradation rate of dead pig residue samples was calculated by weight loss method and it was found that the dead pig residue samples of the uninoculated group (CK group) degraded slowly. From the first day to the ninth day, the degradation rate of the dead pig residue samples of the CK group did not change significantly, and the degradation rate was between 31.64% and 36.83%. However, the degradation rate of the dead pig residue in the bacteria-added group (SCAUH16 group) increased from 34.41% on the first day to 61.37%, an increase of 26.96%, and the upward trend was obvious. The degradation rate of dead pig residue ( Figure 7 The results showed that the addition of strain SCAUH16 could effectively improve the degradation rate of dead pig residues and had the potential to be used as a microbial fermentation agent.

[0131] 4.3.3 Protein changes in dead pig residues

[0132] The protein content of the dead pig residue samples in the CK group did not change significantly, and the protein content was 60.04%-62.00%. However, the protein content of the dead pig residue samples in the SCAUH16 group continued to decrease from 60.30% on the first day of culture to 25.69% on the ninth day, a decrease of 25.56% ( Figure 8 The results showed that the protease produced by Lysinibacillus louembei SCAUH16 can effectively degrade proteins in dead pig residues, increase the degradation rate of proteins, and promote the rapid conversion of organic matter, showing that it has the potential to be used as a microbial fermentation agent.

[0133] Example 5 Application of Lysinibacillus louembei SCAUH16 in Composting of Residue from Dead Pigs

[0134] 5.1 Compost preparation and sample collection

[0135] 5.1.1 Compost preparation

[0136] The pig carcass residues were provided by Chengdu Kenong Animal Harmless Disposal Co., Ltd., and fresh cattle dung was sourced from a large state-owned farm in Zhaojue County, Sichuan Province, China. Aerobic composting of the pig carcass residues was performed using a stacking system, with sawdust as an auxiliary material. The moisture content of the compost was adjusted to 50-60%, and the initial carbon-nitrogen ratio (C / N) was controlled at 25-30. The microbial inoculant described in Claim 2 and Example 3 was added to the adjusted moisture and C / N ratio at a weight ratio of 0.5%, and the compost was fermented for 30 days. The experiment was divided into two groups: the first group received no microbial inoculant (a blank control group (CK), and the second group received a 0.5% (v / v) inoculant containing Lysinibacillus louembei SCAUH16 (SCAUH16 group). Each treatment was replicated three times.

[0137] 5.1.2 Compost sample collection

[0138] An automatic thermometer was used to monitor and record daily compost temperature (Tm) at 8:00 AM and 6:00 PM. Compost samples (three samples / group) were collected daily for analysis of their physical and chemical properties and microbial flora. During the composting process, the first turning of the pile was performed when the pile temperature reached above 50°C, and the second turning was performed when the temperature reached 60°C to maintain aerobic conditions and uniformity, promoting biodegradation and compost maturity.

[0139] 5.2 Analysis of compost physical and chemical properties

[0140] Compost samples were analyzed for their physical and chemical properties, including moisture content, pH, conductivity, organic matter (OM), total nitrogen (N), total phosphorus (P2O5), total potassium (KO), and seed germination index (GI), according to the Chinese Organic Fertilizer Agricultural Industry Standard (NY / T 525-2021). The moisture content of the compost samples was determined by drying. A pH meter (INESAPHSJ-3F, China) was used to measure the pH of a 1:5 aqueous solution. A conductivity meter (DDS-307A, China) was used to measure the conductivity of a 1:10 aqueous solution. The organic matter content of the compost samples was determined by potassium dichromate titration. 0.5 g of air-dried manure was digested in concentrated H2SO4 and H2O2, and total N, P2O5, and KO were determined. Total nitrogen was determined by the Kjeldahl method, total P2O5 by ammonium molybdate spectrophotometry, and total KO by flame photometry.

[0141] 5.3 Compost microbial flora analysis

[0142] Take 0.5g of compost sample and use Total DNA was extracted using the SPIN Kit for Soil (MP BIO Laboratories, California, USA), and DNA purity and concentration were determined using an ultramicro-UV spectrophotometer. Qualified DNA samples were amplified using primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R (5'-CCGTCAATTCCTTTGAGTTT-3') to amplify the V4-V5 region of bacterial 16S. After amplification, library construction, MiSeq sequencing, and sequence assembly were all performed on the Parnoson Illumina MiSeq sequencing platform. Bioinformatics analysis of high-throughput sequencing data was performed on the Parnoson Gene Cloud (https: / / www.genescloud.cn / home). Based on sequence similarity, valid sequences were clustered into OTUs (97% similarity), and species annotation of the OTUs was performed to obtain taxonomic information for each sample. Bacterial diversity was calculated and correlation indices were obtained, and the species composition of the bacteria in the samples was analyzed.

[0143] 5.4 Experimental Results

[0144] 5.4.1 Temperature changes of compost samples

[0145] The temperatures of the piles in both the untreated and treated groups changed after the start of composting. The temperature of the pile in the treated group (SCAUH16 group) rose more rapidly and entered the thermophilic stage quickly on the second day after the start of composting. It reached the thermophilic stage faster than the CK (sterile group) and was able to maintain a longer high temperature period ( Figure 9 The results showed that in this study, the addition of SCAUH16 bacterial preparation to aerobic composting could accelerate the composting of dead pig residue into the thermophilic stage.

[0146] 5.4.2 Changes in C / N ratio of compost samples

[0147] like Figure 10 As shown in the data, the C / N ratio in the CK group decreased from 25.21 to 24.69, while the C / N ratio in the SCAUH16 group decreased from 25.28 to 20.16. This may be because the addition of exogenous microbial agents promoted the decomposition of protein in the dead pig residues, resulting in a decrease in the C / N ratio of the compost and promoting the analysis of organic matter in the compost.

[0148] 5.4.3 Changes in seed germination rate (GI) of compost samples

[0149] GI is an indicator of compost maturity. Figure 11As shown, the GI values of both the CK and SCAUH16 groups initially decreased and then increased throughout the composting process, indicating that as organic matter decomposed and transformed, harmful substances to plant growth were gradually removed. At the end of composting, the GI value of the CK group was 52.26%, while that of the SCAUH16 group was 80.36%. According to the national standard "NY / T525-2021," a GI value ≥70 indicates compost maturity. This indicates that the compost without the addition of the microbial agent did not meet the required maturity and could not be used in field production. However, the addition of the SCAUH16 microbial agent accelerated the composting process of the pig carcass residue compost, ensuring its safety for subsequent use.

[0150] 5.4.4 pH changes in compost samples

[0151] pH is one of the important factors affecting the growth of microorganisms. Generally, the suitable pH for microorganisms is neutral and slightly alkaline. Too high or too low pH will affect the smooth progress of composting. According to the China Organic Fertilizer Agricultural Industry Standard (NY / T 525-2021), the pH value of the compost pile should be between 5.5 and 8.5. Figure 12 As shown, the pH value of the CK group was between 7.8-8.02, and the pH value of the SCAUH16 group was between 7.88 and 8.24, both of which were in line with the national standards.

[0152] 5.4.5 Changes in moisture content of compost samples

[0153] The moisture content of compost is an important parameter that affects the composting process. If the moisture content is too high, the ventilation of the entire pile will be poor, which may easily cause local anaerobic conditions in the pile, produce acidic and smelly odors, and hinder the exudation of nutrients, resulting in nutrient loss. If the moisture content is too low, it may also increase the mass transfer resistance of nutrients and reduce the metabolism of microorganisms. Figure 13 As shown in the figure, affected by the temperature of the pile, the moisture content of the compost in the CK group and the SCAUH16 group was slowly decreasing, and there was no significant change between the two groups.

[0154] 5.4.6 Changes in conductivity of compost samples

[0155] The electrical conductivity reflects the nutrient decomposition effect and product quality during the composting process. Figure 14 As shown in the figure, the conductivity of the CK group and the SCAUH16 group were between 2.77 and 5.39, with no significant changes.

[0156] 5.4.7 Changes in Total Phosphorus and Total Potassium in Compost Samples

[0157] like Figures 15-16As shown, total nutrients (total phosphorus and total potassium) gradually increased throughout the composting process, with the compost samples treated with the SCAUH16 inoculant exhibiting the richest nutrient content. Total phosphorus and total potassium in the SCAUH16 group were significantly higher than those in the CK group on day 25, indicating that the addition of the inoculant facilitated nutrient retention in the compost.

[0158] 5.4.8 Compost Microbial Community Diversity Analysis

[0159] Special annotation analysis was performed on the representative sequences of bacterial OTUs in samples of dead pig residues at different aerobic composting stages. The results are as follows Figure 17 As shown in the data, there were 29 bacterial OTUs in different fermentation periods of composting. The unique OTUs in the SCAUH16 inoculation and non-inoculation treatments at different periods were 3689, 1852, 1843, 910, 797 and 3592, 1639, 1347, 559, 791, respectively. This indicates that there is a complex and diverse original bacterial community in the dead pig residue samples, and the microbial community in the compost changes significantly with the advancement of composting fermentation. Compared with the treatment without the addition of microbial agents, the number of OTUs in the treatment with SCAUH16 addition was always higher than that in the group without the addition of microbial agents, indicating that the SCAUH16 group with the addition of high-temperature degradation microbial agents can quickly adapt to the rapid temperature rise of the pile and the high-protein and high-fat dead pig residue materials. Its proliferation increases the abundance of microbial communities in the compost. SCAUH16 can quickly adapt to the environment and quickly become the dominant microbial community. Therefore, the addition of SCAUH16 microbial agent can drive the transformation of materials and promote the maturity of compost.

[0160] The analysis of the bacterial community composition in samples of dead pig residues at different aerobic composting stages found that at the phylum level, the dominant bacterial groups were mainly Firmicutes, Proteobacteria, Actinobacteria, and Bacteridetes, but their relative abundance varied at different composting stages. In the early stage of fermentation, Firmicutes, Proteobacteria, Actinobacteria and Bacteridetes were the main dominant bacterial phyla in the compost. As the composting time went on and the temperature increased, the relative abundance of Firmicutes increased significantly in the treatment with the addition of the bacterial agent SCAUH16 on day 1, while Proteobacteria increased significantly in the group without the addition of the bacterial agent. However, Bacteridetes, which was an important microbial component in the early stage of fermentation, was almost undetectable at the end of composting. Firmicutes was the absolute dominant bacterial phylum in the compost. Figure 18The increase in composting temperature also led to significant changes in the composition of the microbial community at the genus level in compost at different fermentation stages. The addition of the inoculant SCAUH16 also led to significant differences in the composition of the microbial community at the genus level in the compost compared to the compost without the addition of the inoculant ( Figure 18 The results showed that as composting progressed, microbial decomposition and transformation of materials, coupled with rising temperatures, significantly impacted the composition of the microbial flora in the compost. Changes in compost temperature led to significant differences in the microbial flora at each stage of composting. The addition of SCAUH16 reshaped the microbial composition in the compost, affected the transformation of materials in the compost, and increased the compost temperature, all of which facilitated compost maturity.

Claims

1. A Lysinibacillus strain, Lysinibacillus louembeiSCAUH16, was deposited in Guangdong Provincial Microbiological Culture Collection with the deposit number GDMCC NO: 65794.

2. A microbial agent containing the strain Lysinibacillus louembei SCAUH16 according to claim 1.

3. Use of the strain Lysinibacillus louembei SCAUH16 according to claim 1 or the microbial agent according to claim 2 in treating residues after processing dead pigs.

4. The use according to claim 3, characterized in that The treatment is high temperature aerobic composting.

5. The use according to claim 4, characterized in that The high-temperature aerobic composting is to mix the residue after the dead pigs are processed with sawdust, inoculate the strain Lysinibacillus louembei SCAUH16 according to claim 1 or the microbial agent according to claim 2, and perform high-temperature composting fermentation.

6. The use according to claim 4, characterized in that The high-temperature aerobic composting method comprises the following steps: using sawdust as an auxiliary material and mixing it with residues after the treatment of dead pigs; adjusting the moisture content of the material to 50-60%, controlling the initial carbon-nitrogen ratio to 25-30, adding the microbial agent according to claim 2 to the material at a weight ratio of 0.5%, and performing high-temperature aerobic composting for 30 days; performing the first turning of the pile when the pile temperature reaches above 50°C; and performing the second turning of the pile when the temperature reaches 60°C.

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