A Paenibacillus alvei strain SCAUF07 and its applications

Abstract

The present invention discloses a strain of Paenibacillus alvei SCAUF07 and its application, belonging to the field of microorganisms. This strain is preserved in the Guangdong Microbial Culture Collection Center, with the preservation number GDMCC NO: 65793. The Paenibacillus alvei SCAUF07 of the present invention uses the residue after the rendering treatment of dead pigs as the isolation source, and isolates and screens a strain with protease and lipase activities under high-temperature conditions. This strain can degrade the residue after the drying treatment of dead pigs, and the degradation rate of the dead pig residue reaches 63.15% after 6 days of cultivation. Further, in the aerobic composting with the residue after the rendering of dead pigs as the main raw material, adding the Paenibacillus alvei SCAUF07 bacterial agent can not only promote material transformation, accelerate the composting process, shorten the composting time, but also maintain the nutrients in the composting materials, improve the quality of the compost, and provide a reference for the application of strain resources for the harmless utilization of the residue after the rendering of dead pigs.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a Bacillus sporothermodurans SCAUF07 from milk and its application. Background Art

[0002] With the economic development and the improvement of residents' living standards, the consumption of livestock products such as pork continues to rise, and the demand for the amount of live pig breeding is constantly increasing. However, during the breeding process of live pigs, a large number of dead pigs will be produced due to reasons such as stillbirths, diseases, and natural disasters. The corpses of dead pigs pose serious hazards in aspects such as economic production, ecological environment, food hygiene, and human health. Therefore, it is particularly important to harmlessly treat the corpses of dead pigs. The chemical treatment method for dead pigs has the advantages of relatively simple operation, good sterilization effect, short treatment cycle, and no generation of flue gas. It is one of the main ways to harmlessly treat dead pigs at present and an important way to realize the harmless treatment and resource utilization of dead livestock and poultry. However, after the chemical treatment of dead pig corpses, by-products mainly composed of grease and solid materials will be produced, and currently, it is mainly solved by incineration, which will cause serious secondary pollution problems.

[0003] The residue mainly composed of grease and solid materials after the chemical treatment of dead pigs contains a large amount of organic matter, nitrogen, phosphorus, calcium, etc., and has the potential to be used as organic fertilizer. However, if the residue after the chemical treatment of dead pigs is directly used for returning to the field, there is a risk of long decomposition time and secondary pollution. Therefore, the residue after the chemical treatment of dead pigs is used as an organic solid waste for high-temperature aerobic composting to produce organic fertilizer, so as to achieve the recycling of resources. Since the residue after the chemical treatment of dead pigs contains a large amount of protein, fat, etc., its composition is complex and it is difficult to achieve rapid composting. A large number of studies have shown that inoculating high-temperature and efficient functional microbial agents during the composting process can not only accelerate the decomposition of substances, promote the rapid heating of the compost pile, effectively kill pathogenic microorganisms in the compost materials, but also control the generation of odors during the composting process, promote the transformation of substances, and shorten the composting and ripening process. At present, there have been many reports on the addition of functional microbial agents in high-temperature aerobic composting, mainly focusing on the development and application of cellulase-producing agents under high-temperature conditions, while there are few reports on the screening and application of high-temperature strains with protease and lipase degradation abilities. Therefore, for the harmless treatment and resource utilization of the residue after the chemical treatment of dead pigs, screening high-temperature strains with high-efficiency protein and fat degradation abilities and applying them to the high-temperature aerobic composting of the residue after the chemical treatment of dead pigs to prepare organic fertilizers not only improves the quality of the compost, but most importantly promotes the resource utilization of the residue after the chemical treatment of dead pigs, reduces the risk of secondary pollution, and is of great significance for achieving sustainable development. Summary of the Invention

[0004] The technical problems to be solved by the present invention are as follows: aiming at the nutrient characteristics of the residues after the rendering of dead pigs, isolating and screening high-temperature strains with the ability to decompose proteins and fats, and applying them to the decomposition of the residues after the rendering of dead pigs and their high-temperature aerobic composting treatment. One of the purposes of the present invention is to provide a Bacillus strain with the ability to decompose proteins and fats, and another purpose is to provide the application of this strain.

[0005] The technical solution of the present invention is as follows: a Paenibacillus lactis strain SCAUF07, hereinafter referred to as SCAUF07, which was deposited at the Guangdong Microbial Culture Collection Center on January 15, 2025, with the deposit number GDMCC NO: 65793, and the address of the deposit unit is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Guangdong Province.

[0006] A microbial inoculum containing Paenibacillus lactis SCAUF07.

[0007] The application of Paenibacillus lactis SCAUF07 or a microbial inoculum containing this strain in the treatment of the residues after the rendering of dead pigs.

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

[0009] Furthermore, the high-temperature aerobic composting means mixing the residues after the rendering of dead pigs with sawdust, inoculating the above-mentioned Paenibacillus lactis SCAUF07 or microbial inoculum, and carrying out composting high-temperature fermentation.

[0010] Furthermore, the high-temperature aerobic composting method is as follows: using sawdust as an auxiliary material to mix with the residues after the rendering of dead pigs, adjusting the moisture content of the material to 50-60%, controlling the initial carbon-nitrogen ratio at 25-30, adding the above-mentioned microbial inoculum to the material according to a weight ratio of 0.5% for high-temperature aerobic composting for 30 days. When the temperature of the compost pile reaches above 50°C, the first turning is carried out; when the temperature reaches 60°C, the second turning is carried out.

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

[0012] The present invention uses the residue after rendering of dead pigs as the separation source. The isolated and screened Paenibacillus lactis SCAUF07 can grow under high-temperature conditions, and at the same time has the ability to decompose proteins and fats, and has the potential for application in high-temperature aerobic composting. The suitable temperature range for the growth of this strain is 35-70 °C, and it has relatively high protease and lipase activities, and their enzyme activities are 61.2 μmol / mL and 22.9 μmol / mL respectively. Inoculating this strain can significantly reduce the volume of the residue after rendering of dead pigs, reduce the protein content by 28.56%, and the degradation rate reaches 63.15%. The microbial inoculant prepared with this strain is applied in high-temperature aerobic composting fermentation with the residue of dead pigs as the main material, and the effect of promoting the degradation of the residue of dead pigs and the maturity of composting can be obtained. The present invention not only shortens the fermentation cycle of composting of this kind of material, but also improves the overall quality of composting, provides strain resources and technical references for the resource utilization of the residue after rendering of dead pigs, is conducive to the harmless treatment and resource utilization of agricultural waste, and has good application prospects.

[0013] The preservation information is as follows:

[0014] Paenibacillus lactis SCAUF07, hereinafter referred to as SCAUF07, was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 15, 2025, with the deposit number GDMCC NO: 65793, and the deposit address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Guangdong Province. Description of the Drawings

[0015] Figure 1 It is a colony morphology photo of Paenibacillus lactis SCAUF07 on tryptic soy agar medium (TSA medium);

[0016] Figure 2 It is a phylogenetic tree diagram of Paenibacillus lactis SCAUF07 constructed based on the 16S rRNA sequence results;

[0017] Figure 3 It is the primary screening result of protease production by Paenibacillus lactis SCAUF07;

[0018] Figure 4 It is the primary screening result of lipase production by Paenibacillus lactis SCAUF07;

[0019] Figure 5pH change of the residue medium after rendering of dead pigs in the treatment group with Paenibacillus lactis SCAUF07 added and the non-bacterial treatment group (CK) (1 - 9 days);

[0020] Figure 6 Degradation effect diagram of the residue of dead pigs by Paenibacillus lactis SCAUF07; among them, a is the blank control without inoculating the strain, b is the experimental group inoculated with Paenibacillus lactis SCAUF07, and the degradation effect diagrams from left to right are for 1 - 9 days;

[0021] Figure 7 Degradation rate change of the residue after rendering of dead pigs in the treatment group with Paenibacillus lactis SCAUF07 added and the non-bacterial treatment group (CK) (1 - 9 days);

[0022] Figure 8 Protein content change of the residue after rendering of dead pigs in the treatment group with Paenibacillus lactis SCAUF07 added and the non-bacterial treatment group (CK) (1 - 9 days);

[0023] Figure 9 Temperature change of the compost heap body;

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

[0025] Figure 11 Change of the germination index (GI) of the compost seeds;

[0026] Figure 12 pH change of the compost;

[0027] Figure 13 Change of the water content of the compost;

[0028] Figure 14 Change of the electrical conductivity of the compost;

[0029] Figure 15 Change of the total phosphorus of the compost;

[0030] Figure 16 Change of the total potassium of the compost;

[0031] Figure 17 Venn diagram analysis of the bacterial flora of the samples at different fermentation stages of the compost of the residue after rendering of dead pigs;

[0032] Figure 18 Bacterial community composition of the samples at different fermentation stages of the compost of the residue after rendering of dead pigs. Detailed implementation manners

[0033] In the following experimental methods in the embodiments, unless otherwise specified, they are all conventional methods. Unless otherwise specified, the test materials used in the following embodiments are all obtained from commercial channels.

[0034] In this application:

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

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

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

[0038] Protease basic fermentation medium (g / L): 5 g of glucose, 10 g of yeast powder, 0.5 g of KH2PO4, 0.3 g of MgSO4·7H2O, 1 g of (NH4)2SO4, 1 g of CaCl2, 1 g of NaCl, 1000 mL of distilled water, pH 7.2;

[0039] Dead pig residue medium (g / L): 5 g of glucose, 10 g of solid residue after harmless treatment of dead pigs, 0.5 g of KH2PO4, 0.3 g of MgSO4·7H2O, 1 g of (NH4)2SO4, 1 g of CaCl2, 1 g of NaCl, 1000 mL of distilled water, pH 7.2. Among them, the solid residue after harmless treatment of dead pigs used in the fermentation medium is the residue of dead pigs after rendering harmless treatment.

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

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

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

[0043] Example 1 Isolation and Identification of Paenibacillus lactis SCAUF07

[0044] 1.1 Isolation of Paenibacillus lactis SCAUF07

[0045] Weigh 10 g of the residue after fresh dead pigs are processed by rendering and put it into a 250 mL sterilized Erlenmeyer flask containing 90 mL of sterile water. Place it on a shaker, set the temperature at 50 °C, the rotation speed at 180 r / min, culture for 30 min, mix well and then let it stand. Take the supernatant for gradient dilution, and the gradient dilution factor is from 10 -1 to 10 -5 . Take 100 μL from the liquids of 10 -3 , 10 -4 , 10 -5 respectively, spread them evenly on the casein agar medium, and place the plate in a constant temperature incubator and culture at 50 °C for 48 h. Pick out the strains with rapid growth and large colonies, and use the dilution plate streaking method to purify until pure strains are obtained. Transfer the pure strains to tryptic soy broth liquid medium (TSB), culture them with shaking at 50 °C for 7 d, then preserve them with 30% glycerol, and store them frozen at -80 °C.

[0046] 1.2 Identification of Paenibacillus lactis SCAUF07

[0047] 1.2.1 Strain Morphology

[0048] Pick a single colony of strain SCAUF07 and inoculate it on tryptic soy agar medium (TSA medium), place it at 50 °C and culture for 24 h, and observe the colony morphology of the strain.

[0049] 1.2.2 Strain 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 inoculation loop to pick up a small amount of bacterial cells and transfer them into a sterile 1.5 mL centrifuge tube. Then add 500 μL of sterile double-distilled water. Immediately freeze the centrifuge tube in liquid nitrogen, and then place it in a 99 °C metal bath for 5 min. After taking it out, vortex for 30 s. Repeat the above operation 1 - 2 times. Then centrifuge the mixture of bacterial cells and sterile double-distilled water at 12000 rpm for 3 min. Take the supernatant as the template and store it at -20 °C; detect it by 1% agarose gel electrophoresis.

[0056] (4) Amplification of bacterial 16S rRNA gene

[0057] Amplification conditions of 16S rRNA gene: pre-denaturation at 95 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 1.5 min, 30 cycles, and final extension at 72 °C for 20 min. The PCR products are purified by Shanghai Sangon EZ Spin Column PCR Product Purification Kit (SK1142-N) according to the operation manual, and the purified products are sent to Sangon Biotech Co., Ltd. for sequencing.

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

[0059] Use the obtained sequenced sequence (SEQ ID No.1) to perform similarity search in NCBI using BLAST software. Select the 16S rRNA gene sequence of the published standard strain with the highest similarity in the same genus as the reference sequence. Use the standard strain sequence of Bacillus subtilis (AB042061) as the outgroup. Perform multiple sequence alignment analysis using Clustal X software, and construct a phylogenetic tree by the N-J method using MEGA7.0 software to determine the taxonomic status of the bacteria.

[0060] 1.3 Experimental results

[0061] Paenibacillus lactis SCAUF07 was isolated from the residue after rendering of dead pigs. After culturing on TSA medium at 50 °C for 48 h, the colony size is 2 - 3 mm, round and slightly convex, with a rough edge, an uneven surface, and a light yellow colony color. It is easy to pick, Gram-positive, aerobic, rod-shaped, with spores, and the cell size is 0.6 × 3.0 μm( Figure 1 ).

[0062] The 16S rRNA sequence of strain SCAUF07 was determined, BLAST homology alignment was performed in the NCBI database, and phylogenetic analysis was carried out using MEGA7.0 software. The homology of the 16S rRNA nucleotide sequence of this strain with that of Paenibacillus lactis was 100%. Based on the experimental results of comprehensive morphological characteristics, physiological and biochemical characteristics, and 16S rRNA sequence homology analysis, strain SCAUF07 was identified as Paenibacillus lactis( Figure 2 ).

[0063] Example 2 Determination of the growth temperature and enzyme-producing function of Paenibacillus lactis SCAUF07

[0064] 2.1 Determination of temperature range

[0065] Strain Paenibacillus lactis SCAUF07 was inoculated on TSA medium and cultured, and placed in incubators at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C respectively for 48 h. Observe and record its growth conditions. According to the measurement results, with 5°C as a unit, further narrow the temperature detection range until the highest and lowest growth temperatures are determined.

[0066] 2.2 Determination of protein degradation ability

[0067] 2.2.1 Preliminary screening of protein degradation ability

[0068] The strain was evenly spread on tryptic soy agar medium (TSA medium) and cultured in a biochemical incubator at 50°C until the strain grew evenly on the entire surface of the medium. A bacterial cake was made with a punch with a diameter of 5 mm and inverted on skim milk medium. After culturing in a biochemical incubator at 50°C for 2 - 4 d, the diameters of the hydrolysis zone (D) and the colony (d) were measured respectively, and the ratio of the two (D / d) was calculated. The larger the ratio, the stronger the protein degradation ability of the strain. Set 3 replicates.

[0069] 2.2.2 Determination of protease activity of the strain

[0070] (1) Drawing of the standard curve

[0071] After preparing a standard L-tyrosine solution at a concentration of 100 μg / mL, dilute the 100 μg / mL standard L-tyrosine solution. Finally, 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 to each. After shaking well, place in a water bath at 40 ± 0.2 °C for color development for 20 min. Then take out and measure the absorbance with a spectrophotometer at a wavelength of 680 nm using a 10 mm cuvette, with the 0 tube without tyrosine as the blank. Using the absorbance A as the ordinate and the tyrosine concentration c as the abscissa, plot the standard curve. Using the regression equation, calculate the amount of tyrosine (μg) when the absorbance is 1, which is the absorbance constant K value. The K value should be in the range of 95 - 100. Draw the L-tyrosine standard curve based on the concentration-absorbance data of each concentration.

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

[0073] Pick 1 pure culture preserved on the slant and inoculate it into the protease seed medium. Culture it at 50 °C on a shaker at 150 rmp for 12 h. Then inoculate it into the protease basic fermentation medium at an inoculation amount of 1% (v / v) and culture it at 50 °C on a shaker at 150 rmp for 48 h. After centrifugation, take the supernatant for enzyme activity determination.

[0074] (3) Enzyme activity determination

[0075] Use the Folin-phenol method to determine the enzyme activity of the crude enzyme solution. Definition of enzyme activity unit: At 40 °C, hydrolyzing 1 μg of tyrosine from the casein substrate per minute is defined as one protease activity unit. Put the 2% (mass fraction) casein solution into a 40 °C constant temperature water bath and preheat it for 5 min. Take 1 mL of the crude enzyme solution and add it to 1 mL of the preheated casein solution, mix well, then place it in a 40 °C water bath for incubation for 10 min. Then add 2 mL of 0.4 mol / L trichloroacetic acid to terminate the reaction. Take 1 mL of the supernatant, add 5 mL of 0.4 mol / L Na2CO3, add 1 mL of Folin-phenol reagent, and place it in a 40 ± 0.2 °C water bath for color development for 20 min. Then measure the absorbance at 660 nm, with the reaction system with water added as the blank.

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

[0077] Activity of protease (U / mL) = A × K × 4 ÷ 10 × n

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

[0079] K: Absorbance constant;

[0080] n: Dilution factor of the protease solution;

[0081] 4: Total volume of the reaction reagent;

[0082] 10: Reaction time is 10 min; The result is expressed as an 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 the tryptic soy agar medium (TSA medium), placed in a biochemical incubator at 50 °C until the strain grew evenly on the entire surface of the medium, made into a bacterial cake with a puncher with a diameter of 5 mm, inverted on the Victoria blue B solid medium, and cultured in a biochemical incubator at 50 °C for 2 - 4 d. Then observe whether blue color appears at the inoculation site of the medium, measure the diameter of the discoloration circle (D) and the diameter of the colony (d) respectively, calculate the ratio of the two (D / d). The larger the ratio, the stronger the ability of the strain to degrade fat. Set 3 replicates.

[0086] 2.3.2 Determination of lipase activity of the strain

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

[0088] Table 1 Method for determining lipase activity

[0089]

[0090]

[0091] Titrate with 0.05 M standard sodium hydroxide solution until it turns slightly pink, and record the volume of the NaOH solution used for titration.

[0092] Calculation: The specific activity of lipase is defined as the amount of enzyme that hydrolyzes fat to produce 1 μmol of fatty acid per minute by 1 g of lipase under the conditions of pH 7.5 and 40 °C as one enzyme activity unit.

[0093]

[0094] In the formula: A is the volume of the alkali solution consumed by the sample (ml); B is the volume of the alkali solution consumed by the control group (ml); N is the concentration of the alkali solution, that is, 0.05 μmol; f is the final dilution factor of the crude enzyme solution; t is the reaction time (min).

[0095] 2.4 Experimental results

[0096] It was determined that the growth range of Paenibacillus lactis SCAUF07 is 35-70°C and has the ability to produce protease and lipase ( Figure 3 and 4 , Table 2). The relative enzyme activities of protease and lipase were 61.2μmol / mL and 22.9μmol / mL, respectively. (Table 2). The results showed that Paenibacillus lactis SCAUF07 had good thermostability and ability to produce functional enzymes, and had the potential for further development and application.

[0097] Table 2 Enzyme production capacity of Paenibacillus lactis SCAUF07

[0098]

[0099] Example 3 Preparation of microbial inoculants

[0100] This embodiment provides a microbial agent, and the preparation method of the microbial agent is as follows:

[0101] The Paenibacillus lactis SCAUF07 plate was streaked and inoculated on TSA medium, and a single colony was picked and placed in TSB medium, and cultured at 50-60°C and 160-200rpm for 1 day to obtain a seed solution. The seed solution was inoculated into a fermentation medium at an inoculum of 5% (v / v), and cultured at 50-60°C and 160-200rpm for 24-48 hours to obtain a microbial agent with an effective viable count of 1.3×10 8 CFU / g, pH value is 6.0~8.0.

[0102] Fermentation medium formula: peptone 7g, beef extract 1g, sodium chloride 5g, glucose 10g, distilled water 1000mL.

[0103] Example 4 Fermentation culture of strain Paenibacillus lactis SCAUF07

[0104] 4.1 Experimental materials and process

[0105] Preparation of solid residue after chemical treatment of dead pigs: After the dead pig carcasses are crushed, they are treated in a high temperature and high pressure chemical tank (temperature ≥ 140°C, pressure ≥ 0.5MPa (absolute pressure)) for 4 hours, and then dried in a high temperature and normal pressure drying tank, and finally the dead pig residue is obtained after the liquid is separated by an oil press. The dead pig residue used in the present invention is provided by Chengdu Kenong Animal Harmless Disposal Co., Ltd.

[0106] Experimental setup: A blank control group (CK group) without adding any microbial inoculant, and a microbial inoculant containing Paenibacillus lactis SCAUF07 (SCAUF07 group, addition amount 0.5% (v / v)) were placed in an incubator at 50 °C and 180 r / min for continuous sampling for 9 days. The physical and chemical properties of the samples were analyzed to determine the degradation effect of strain SCAUF07.

[0107] 4.2 Physical and chemical property analysis

[0108] 4.2.1 Determination of the degradation rate of the residue of dead pigs

[0109] The degradation rate of the solid residue after harmless treatment of dead pigs was calculated by the weight loss method: After the residue of the dead pigs processed by shaking flask culture was filtered through filter paper, the residue was rinsed with distilled water 2 - 3 times, dried at 65 °C for 24 h, weighed, and the weight loss and degradation rate were calculated.

[0110]

[0111] In the formula: A is the initial dry weight (g) of the solid residue after harmless treatment of dead pigs;

[0112] B is the dry weight (g) of the solid residue after harmless treatment of dead pigs after degradation.

[0113] 4.2.2 Determination of the pH value of the culture medium of the residue of dead pigs

[0114] Referring to the determination of acidity and alkalinity (pH meter method) in the agricultural industry standard NY / T 525 - 2021 of the People's Republic of China, the culture medium of the residue of dead pigs was transferred from a 250 mL Erlenmeyer flask to a 250 mL beaker, allowed to stand for 30 min, and measured with a pH meter. The pH meter reading was recorded. Before measurement, the pH meter was calibrated with a standard buffer solution (temperature compensation set at 25 °C).

[0115] 4.2.3 Protein content of the substrate of the residue of dead pigs

[0116] Refer to the method 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 g to 1.0 g (accurate to 0.0001 g) of the air-dried sample of the dead pig residue substrate, place it at the bottom of a conical flask with an appropriate volume, add 5 mL of sulfuric acid and 1.5 mL of hydrogen peroxide, shake gently, place a small bent-neck funnel at the mouth of the flask, and let it stand overnight. The next day, slowly heat until sulfuric acid fumes, cool slightly, add 15 drops of hydrogen peroxide, gently shake the conical flask or digestion tube, heat for 10 min, cool slightly, then add 5 to 10 drops of hydrogen peroxide and digest in portions until the solution becomes colorless or light yellow clear liquid, and then continue to heat for 15 min to remove the remaining hydrogen peroxide. Take it down and cool, carefully add water to 20 mL to 30 mL, gently shake the conical flask or digestion tube, rinse the small bent-neck funnel with a small amount of water, and collect the washing liquid into the conical flask. Transfer the digestion solution into a 50 mL volumetric flask, cool to room temperature, add water to make up the volume, let it stand and clarify or filter it dry through a phosphorus-free filter paper into a stoppered conical flask for standby. For the blank experiment, except without adding the sample, the reagent dosage, operation, and experimental sample are the same. Use the automatic Kjeldahl method to analyze and calculate the protein content in the sample, and express it as a mass fraction (%).

[0117]

[0118] Wherein:

[0119] C is the molar concentration of the calibrated standard solution, with the unit of mole per liter (mol / L);

[0120] V0 is the volume of the calibrated standard solution consumed during the blank experiment, with the unit of milliliter (mL);

[0121] V is the volume of the calibrated standard solution consumed during the sample determination. The unit is milliliter (mL);

[0122] 14 is the molar mass of nitrogen, with the unit of gram per mole (g / moL);

[0123] M is the numerical value of the mass of the air-dried sample, with the unit of gram (g);

[0124] X is the numerical value of the water content of the air-dried sample;

[0125] D is the dilution factor, fixed volume / dilution volume;

[0126] 6.25 is the coefficient for converting nitrogen to protein.

[0127] 4.3 Experimental Results

[0128] 4.3.1 pH Change of Dead Pig Residue

[0129] The pH of the samples from the two treatment groups was measured, and it was found that the pH of the samples in the CK group did not change significantly. The pH of the fermentation broth was between 5.26 and 5.53, while the pH of the samples in the SCAUF07 group showed an obvious upward trend starting from the 2nd day. From the 1st day to the 6th day, the pH of the samples in the SCAUF07 group increased from 5.73 to 8.45, and then tended to be stable, stabilizing at 8.45 - 8.48( Figure 5 ). The results showed that Paenibacillus lactis SCAUF07 decomposed proteins during metabolism to produce alkaline substances, resulting in changes in pH.

[0130] 4.3.2 Changes in the degradation rate of the residues of dead pigs

[0131] After adding strain SCAUF07, the amount of the residues of dead pigs was significantly reduced compared with the CK group( Figure 6 ). By calculating the degradation rate of the residues of dead pigs samples using the weight loss method, it was found that the degradation of the residues of dead pigs samples in the non - inoculated group (CK group) was slow. From the 1st day to the 9th day, there was no obvious change in the degradation rate of the residues of dead pigs samples in the CK group, and the degradation rates were all between 24.95% and 27.03%. While in the inoculated group (SCAUF07 group), the degradation rate of the residues of dead pigs increased from 27.12% on the 1st day to 63.15% on the 9th day, an increase of 36.03%, and the upward trend was obvious( Figure 7 ). The results showed that adding strain SCAUF07 could effectively improve the degradation rate of the residues of dead pigs and had the potential to be used as a microbial fermentation inoculant.

[0132] 4.3.3 Changes in the protein of the residues of dead pigs

[0133] There was no obvious change in the protein content of the residues of dead pigs samples in the CK group, and the protein contents were all between 53.94% and 57.35%. While the protein content of the residues of dead pigs in the SCAUF07 group decreased continuously from 56.31% on the 1st day of cultivation to 27.75% on the 9th day, a decrease of 28.56%( Figure 8 ). The results showed that the protease produced by Paenibacillus lactis SCAUF07 could effectively degrade the protein in the residues of dead pigs, improve the degradation rate of the protein, promote the rapid conversion of organic substances, and had the potential to be used as a microbial fermentation inoculant.

[0134] Example 5 Application of Paenibacillus lactis SCAUF07 in the composting of the residues after rendering of dead pigs

[0135] 5.1 Compost preparation and sample collection

[0136] 5.1.1 Preparation of compost

[0137] The residue after rendering the dead pigs was provided by Chengdu Kenong Animal Harmless Disposal Co., Ltd., and the fresh cow dung came from a large state-owned farm in Zhaojue County, Sichuan Province, China. Aerobic composting of the residue after rendering the dead pigs was carried out in a stack, with sawdust as the auxiliary material, adjusting the moisture content of the material to 50-60%, controlling the initial carbon-nitrogen ratio (C / N) at 25-30, adding the microbial inoculant described in Example 3 to the material with the moisture and carbon-nitrogen ratio already adjusted according to a weight ratio of 0.5%, and composting and fermenting for 30 days. The experiment was divided into two groups, that is, the first group without adding any microbial inoculant was used as the blank control group (CK), and the second group added a microbial inoculant containing Paenibacillus lactis SCAUF07 according to an addition amount of 0.5% (v / v) (SCAUF07 group), with 3 replicates for each treatment.

[0138] 5.1.2 Compost sample collection

[0139] An automatic thermometer was used to monitor and record the daily temperature (Tm) changes of the compost at 8:00 am and 6:00 pm, and compost samples (3 samples / group) were collected every day for analyzing their physical and chemical properties and microbial flora characteristics. During the composting process, when the stack temperature reached above 50 °C, the first turning was carried out; when the temperature reached as high as 60 °C, the second turning was carried out to maintain aerobic conditions and uniformity, and to promote material degradation and compost maturity.

[0140] 5.2 Analysis of compost physical and chemical properties

[0141] According to the agricultural industry standard of organic fertilizers in China (NY / T 525-2021), the physical and chemical properties of the compost samples such as moisture content, pH, electrical conductivity, organic matter (OM), total nitrogen (N), total phosphorus (P2O5), total potassium (K2O) and seed germination index (GI) were analyzed. The moisture content of the compost samples was determined by the drying method. The pH value of the 1:5 aqueous solution was detected with a pH meter (INESAP HSJ-3F, China). The electrical conductivity of the 1:10 aqueous solution was detected with a conductivity meter (DDS-307A, China). The organic matter content in the compost samples was determined by the dichromate titration method. 0.5 g of air-dried manure was digested in concentrated H2SO4 and H2O2 to determine the total N, P2O5 and K2O. The total nitrogen was determined by the Kjeldahl method, the total P2O5 was determined by the ammonium molybdate spectrophotometric method, and the total K2O was determined by the flame photometric method.

[0142] 5.3 Analysis of the microbial flora of the compost

[0143] Take 0.5 g of the compost sample and use The total DNA was extracted using the SPIN Kit for Soil (MP BIO Laboratories, California, USA), and the purity and concentration of the DNA were detected using a ultra-micro ultraviolet spectrophotometer. For the qualified DNA samples, the V4-V5 region of bacterial 16S was amplified using the primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R (5'-CCGTCAATTCCTTTGAGTTT-3'). After amplification, subsequent library construction, Miseq sequencing, and sequence splicing were all performed on the PacBio Illumina MiSeq sequencing platform. The bioinformatics analysis of the high-throughput sequencing data was carried out on the PacBio Gene Cloud (https: / / www.genescloud.cn / home). According to the sequence similarity, the valid sequences were clustered into OTUs (97% similarity), taxonomic information of each sample was obtained by annotating the OTUs, bacterial diversity was calculated and related indices were obtained, and the species composition of bacteria in the samples was analyzed.

[0144] 5.4 Experimental results

[0145] 5.4.1 Temperature change of compost samples

[0146] The temperatures of the compost piles in the non-inoculated group and the inoculated group changed after the start of composting. Among them, the compost pile in the experimental group with inoculant (SCAUHF07 group) heated up more rapidly, quickly entered the thermophilic stage on the 3rd day after the start of composting, reached the thermophilic stage faster than CK (sterile group), and was able to maintain a longer high-temperature period ( Figure 9 ). The results showed that in this study, adding the SCAUF07 bacterial preparation to aerobic composting could accelerate the compost of the residue after the rendering of dead pigs into the thermophilic stage.

[0147] 5.4.2 Change of C / N in compost samples

[0148] As Figure 10 shown, the C / N in the compost pile of the CK group increased from 25.73 to 33.41, while the C / N in the compost pile of the SCAUF07 group decreased from 26.19 to 23.43. This may be because the addition of exogenous microbial inoculants promoted the decomposition of proteins in the residue of dead pigs, resulting in a decrease in the C / N of the compost and promoting the decomposition of organic matter in the compost.

[0149] 5.4.3 Change of germination index (GI) of compost samples

[0150] GI is an index reflecting the maturity of compost. As Figure 11As shown, the GI values of the CK group and the SCAUF07 group both showed a trend of first decreasing and then increasing during the entire composting process, indicating that as the organic matter in the compost decomposed and transformed, the harmful substances to plant growth in the compost were gradually removed. At the end of composting, the GI value of the CK group was 55.47%, while the GI value of the SCAUF07 group was 93.28%. According to the national standard "NY / T525-2021", when the GI value ≥ 70, the compost is considered mature. This shows that the compost in the group without adding the inoculant did not meet the maturity requirements and could not be further used for field production, while adding the SCAUF07 microbial inoculant could accelerate the composting process of the residue source after the rendering of dead pigs and ensure the safety of subsequent use.

[0151] 5.4.4 pH change of compost samples

[0152] 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 Chinese organic fertilizer agricultural industry standard (NY / T 525-2021), the pH value of the compost pile should be between 5.5 and 8.5. As Figure 12 shown, the pH value of the CK group was between 8.3 and 8.7, and the pH value of the SCAUF07 group was between 6.92 and 8.29. The pH value of the CK group without adding the inoculant was slightly higher than the national standard, while the pH value of the group adding the SCAUF07 inoculant met the national standard of the pH value of the compost pile.

[0153] 5.4.5 Moisture content change of compost samples

[0154] The moisture content of the compost is an important parameter affecting the composting process. If the moisture content is too high, the ventilation of the whole pile is poor, which is easy to cause local anaerobic conditions in the pile, produce a sour smell, and at the same time is not conducive to the leaching of nutrients, resulting in nutrient loss. If the moisture content is too low, it is also easy to increase the mass transfer resistance of nutrients and lead to a decrease in microbial metabolism. As Figure 13 shown, affected by the pile temperature, the moisture contents of the composts in the CK group and the SCAUF07 group were both slowly decreasing, and the changes between the two groups were not significant.

[0155] 5.4.6 Electrical conductivity change of compost samples

[0156] The electrical conductivity reflects the nutrient decomposition effect and product quality during the composting process. As Figure 14 shown, the electrical conductivities of the CK group and the SCAUF07 group were both between 3.2 and 4.6, and the changes were not significant.

[0157] 5.4.7 Total phosphorus and total potassium changes of compost samples

[0158] As Figure 15 and Figure 16As shown in the figure, the total nutrients (total phosphorus and total potassium) gradually increased during the composting process, and the nutrient content in the compost samples treated with the SCAUF07 inoculant was richer. The total phosphorus and total potassium in the SCAUF07 group were significantly higher than those in the CK group on the 25th day. Adding the inoculant was beneficial to the retention of nutrients in the compost materials.

[0159] 5.4.8 Analysis of the diversity of compost microbial flora

[0160] Specific annotation analysis was carried out on the representative sequences of bacterial OTUs in samples at different aerobic composting stages of the residues after rendering of dead pigs. The results are as Figure 17 shown. There were 19 common bacterial OTUs in different fermentation periods of the compost. The unique OTUs in the SCAUF07 treatment and the non-inoculated treatment at different times were 3981, 1853, 1317, 515, 781 and 4815, 1924, 2128, 593, 2244 respectively, indicating that there was a complex and diverse original bacterial flora in the residues after rendering of dead pigs, and obvious changes occurred in the microbial groups in the compost with the progress of composting fermentation. Compared with the treatment without inoculant, the number of OTUs in the SCAUF07 treatment was always higher than that in the non-inoculant group, and the number of OTUs in the SCAUF07 treatment was significantly higher than that in the non-inoculant treatment at the maturity stage, indicating that the SCAUF07 group with a high-temperature degradation inoculant could quickly adapt to the rapid temperature rise of the compost pile and the residues of dead pigs with high protein and high fat. Its proliferation increased the abundance of the bacterial community in the compost. The SCAUF07 could quickly adapt to the environment and quickly become the dominant bacterial community at the bottom, promoting the recovery of the original microorganisms or the colonization of new bacteria, and activating the diversity of the microbial community at the end of the composting. Therefore, adding the SCAUF07 inoculant could drive the transformation of substances and promote the maturity of the compost.

[0161] Analysis of the bacterial community composition in samples at different aerobic composting stages of the residues after rendering of dead pigs showed that at the phylum level, the dominant bacterial groups were mainly Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes, but their relative abundances varied at different composting times. In the initial stage of composting, Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the main dominant phyla. As the composting time progressed and the compost temperature increased, the relative abundance of Proteobacteria in the treatment with the inoculant SCAUF07 added on day 1 increased significantly, while in the group without the inoculant, Firmicutes increased significantly. However, as the fermentation time passed, Firmicutes became the absolute dominant phylum in the compost, and the abundance of Proteobacteria in the inoculant-added group increased again at the end of composting ( Figure 18 as shown in A). The increase in compost temperature also led to significant changes in the microbial community composition at the genus level in compost at different fermentation stages, and the addition of the inoculant SCAUF07 also resulted in obvious differences in the composition of the microbial flora in the compost at the genus level compared with that without the inoculant ( Figure 18 as shown in B). The results indicated that with the progress of composting, the decomposition and transformation of materials by microorganisms, and the increase in temperature had a significant impact on the composition of the microbial flora in the compost, and obvious differences in the microbial flora existed in each stage of compost fermentation with the change of the pile temperature. The addition of the SCAUF07 inoculant reshaped the microbial composition in the compost, affected the transformation of substances in the compost, increased the pile temperature, and was beneficial to the maturity of the compost.

Claims

1. A strain of Paenibacillus alvei ( Paenibacillus lactis ), designated as SCAUF07, was deposited at the Guangdong Provincial Culture Collection of Microorganisms with the accession number GDMCC No: 65793.

2. A microbial inoculum containing the Bacillus sporolactis ( Paenibacillus lactis ) SCAUF07 described in claim 1.

3. Use of the Bacillus sporothermodurans ( Paenibacillus lactis ) SCAUF07 as claimed in claim 1 or the microbial inoculum as claimed in claim 2 in the treatment of residues after rendering of dead pigs.

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

5. The application according to claim 4, wherein The high-temperature aerobic composting refers to mixing the residue after rendering the dead pigs with sawdust, inoculating the Bacillus sporothermodurans ( Paenibacillus lactis ) SCAUF07 as described in claim 1 or the microbial inoculum as described in claim 2, and performing high-temperature fermentation of the compost.

6. The application according to claim 4, characterized in that The high-temperature aerobic composting method is as follows: Using sawdust as an auxiliary material, mix it with the residue after the rendering of dead pigs and diseased pigs, adjust the moisture content of the material to 50 - 60%, control the initial carbon-nitrogen ratio at 25 - 30, add the microbial inoculant described in claim 2 to the material according to a weight ratio of 0.5% for high-temperature aerobic composting for 30 days. When the stack temperature reaches 50 °C, conduct the first turning of the pile; when the temperature reaches 60 °C, conduct the second turning of the pile.

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