High-yield lysine-providing providencia stuartii, complex microbial inoculant, and uses thereof

Abstract

This invention relates to the field of microbial technology, and more particularly to *Providens skeletalus*, a high-lysine-producing strain, a compound microbial agent, and its applications. This invention isolates *Providens skeletalus* from the intestines of black soldier fly larvae. This strain exhibits high amino acid production, which can improve feed quality and reduce dependence on high-value protein sources. A combination of *Providens skeletalus* with *Enterococcus faecalis* and *Bacillus licheniformis* in a 1:1:1 ratio demonstrates excellent synergistic effects, efficiently degrading organic waste and promoting black soldier fly growth or the synthesis of microbial proteins rich in essential amino acids. This combination has significant application value in the field of organic waste resource utilization.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to *Providens schlegelii*, a high-lysine-producing bacterium, compound bacterial agents, and their applications. Background Technology

[0002] With global urbanization and the expansion of industrial and agricultural scale, the generation of organic waste has exploded, including various types such as agricultural and forestry straw and livestock manure. Improper handling can occupy land, release harmful substances, and cause soil, water, and air pollution, threatening ecology and human health. Therefore, achieving the reduction, harmless treatment, and resource recovery of organic waste is of great significance.

[0003] Among numerous treatment technologies, microbial treatment technology has become one of the mainstream technologies due to its advantages such as low energy consumption, low pollution, and simple operation. It utilizes microbial metabolism to break down large organic molecules in organic waste into smaller molecules, achieving degradation and stabilization. Currently developed processes include composting and anaerobic digestion, employing microbial strains from various groups such as bacteria and fungi, with Bacillus species already in large-scale application. Through these technologies, some organic waste is converted into primary products such as organic fertilizer, achieving initial resource recycling.

[0004] However, existing microbial treatment technologies still face significant technical bottlenecks. The core issue lies in the limited nutritional composition and insufficient overall nutritional value of the treated products. Most current microbial treatment processes can only degrade and transform organic waste, resulting in products with insufficient and unbalanced nutrient content, failing to meet the demands for high-quality and high-yield crops. This deficiency severely limits the application scenarios of the treated products; most can only be used as low-end organic fertilizer with low added value, failing to fully realize the resource potential of organic waste.

[0005] Furthermore, existing microbial strains have relatively limited functions, focusing primarily on improving the efficiency of organic matter degradation while lacking sufficient ability to enhance the nutritional components of the products. With the increasing demand for green agricultural development, the market demand for high-quality organic fertilizers and biostimulants is growing, necessitating the development of microbial technologies that can significantly improve the nutritional richness and balance of products while degrading organic waste. Therefore, screening for highly efficient microbial strains with nutrient-enhancing functions and optimizing microbial treatment processes to transform organic waste into high-value-added nutritional products has become a pressing technical problem in this field, possessing significant technical value and application prospects for promoting the upgrading and development of the organic waste resource utilization industry. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a high-yield lysine-producing *Providens schlegelii*, a compound bacterial agent, and its application.

[0007] Providencia stutii with accession number CCTCC NO: M 20252242.

[0008] The *Providencera spp.* strain provided by this invention is characterized by high production of amino acids, particularly leucine and lysine. Lysine is one of the essential amino acids that animals cannot synthesize on their own, and it is the first limiting amino acid. Its low content severely restricts the efficiency of protein utilization in animal feed. Producing lysine through bacterial fermentation can reduce the amount of expensive fishmeal and soybean meal used in feed, thereby lowering feed costs.

[0009] The present invention also provides a combination of bacteria, characterized in that it includes Providencia squarrosa, Enterococcus faecalis and Bacillus licheniformis as described above.

[0010] Black soldier fly larvae are renowned for their remarkable ability to process organic waste. This invention utilizes bacteria from the gut of black soldier fly larvae as experimental subjects. Combining *Providencera schlegelii*, *Enterococcus faecalis*, and *Bacillus licheniformis* as described above yields a more significant synergistic effect. *Enterococcus faecalis* and *Bacillus licheniformis* can be derived from the gut of black soldier fly larvae or other commercially available strains in the field, all of which can work well with *Providencera schlegelii* to synergistically promote larval weight gain and increase protein content. As a feasible example, the mass ratio of *Providencera schlegelii*, *Enterococcus faecalis*, and *Bacillus licheniformis* in the combined bacteria of this invention is 1:1:1.

[0011] The present invention also provides the application of Providencia schlegelii or the combined bacteria as described above in the treatment of organic waste.

[0012] In this invention, the treatment of organic waste includes, but is not limited to, using organic waste to produce microbial protein or using organic waste to cultivate black soldier flies. The organic waste in this invention includes, but is not limited to, chicken manure, rice husks, agricultural straw, livestock and poultry breeding wastewater, and kitchen waste. Chicken manure or wastewater from cleaning chicken coops contains abundant nitrogen and carbon sources. After treatment with *Providens schlegelii* or a combination of bacteria provided by this invention, it can not only effectively degrade complex organic matter such as crude fiber and protein, reducing the release of odorous substances, but also transform it into microbial protein rich in amino acids through microbial metabolic activities. Rice husks are rich in cellulose and hemicellulose. After adjusting the ratio of C and N sources, treatment with *Providens schlegelii* or a combination of bacteria provided by this invention can also achieve good cultivation of black soldier flies.

[0013] Furthermore, the present invention also provides a method for raising black soldier flies using organic waste, which includes inoculating the organic waste with *Providens schlegelii* or a combination of bacteria as described above and black soldier fly larvae as described above, and culturing it at 30±1℃ for 10 days.

[0014] Preferably, the black soldier fly larvae are 5th instar larvae.

[0015] In some embodiments, the organic waste is chicken manure and / or rice husks. The culture medium for every 600 black soldier fly larvae comprises: 500 g chicken manure and / or rice husks, 200 g glucose, and 2.30 g urea. After homogenization, *Providencera schlegelii* or a combination of bacteria as described above is added. Results show that the addition of *Providencera schlegelii* or the combination of bacteria can promote BSFL growth, increase the protein content of BSFL, and decrease the fat content.

[0016] Furthermore, the present invention also provides a method for preparing microbial protein from organic waste, which includes inoculating organic waste with Providencia schlegelii or a combination of bacteria as described above, and then sealing and fermenting it at 25-30°C until the pH ≤ 5 to obtain a fermentation product containing protein.

[0017] In this invention, the organic waste is chicken manure and / or rice husks.

[0018] In some embodiments, the method for preparing microbial protein includes: sterilizing chicken manure wastewater at 65°C for 12 hours, and then inoculating it with *Providencebringus schlegelii* or a combination of bacteria as described above until the total bacterial count reaches 10. 10 -10 11 / ml, sealed and fermented at 25~30℃ until pH≤5, then the bacterial protein is collected by filtration. The nitrogen content of the chicken manure wastewater is 1.5%.

[0019] In other embodiments, the method for preparing microbial protein includes: sterilizing chicken manure wastewater at 65°C for 12 hours, and then inoculating it with *Providencebringus schlegelii* or a combination of bacteria as described above until the total bacterial count reaches 10. 10 -10 11 / ml, sealed and fermented at 25~30℃ until pH≤5, then 50% of the fermentation liquid was taken out and filtered to obtain microbial protein; the remaining 50% of the fermentation liquid was added with fresh chicken manure wastewater, sealed and fermented at 25~30℃ until pH5-5.5, and 50% of the fermentation liquid was harvested again and filtered to obtain microbial protein, and this process was repeated several times.

[0020] Furthermore, the present invention also provides a microbial protein prepared by the method described above.

[0021] In this invention, the microbial protein is rich in at least one of leucine, lysine, valine, arginine, threonine, isoleucine, phenylalanine, and methionine.

[0022] Furthermore, the present invention also provides a feed comprising the microbial protein as described above.

[0023] The feed provided by this invention is for use in livestock and poultry farming, aquaculture, and other fields. The feed includes the aforementioned microbial protein, as well as conventional feed ingredients (such as corn, soybean meal, wheat bran, fishmeal, etc.). Because the microbial protein in this feed is rich in various essential amino acids such as leucine and lysine, it can significantly improve the protein utilization rate and nutritional value of the feed, reduce animals' dependence on high-priced protein ingredients, and lower feed costs.

[0024] This invention isolates *Providensia spp.* from the intestines of black soldier fly larvae. This strain exhibits high amino acid production, which can improve feed quality and reduce dependence on high-value protein sources. A combination of *Enterococcus faecalis* and *Bacillus licheniformis* in a 1:1:1 ratio demonstrates excellent synergistic effects, efficiently degrading organic waste and promoting black soldier fly growth or the synthesis of microbial proteins rich in essential amino acids. This combination shows significant application value in the field of organic waste resource utilization.

[0025] Biological Preservation Instructions

[0026] Providencia stuartii Eg. was deposited on October 17, 2025, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252242. Attached Figure Description

[0027] Figure 1 The growth fold of each strain relative to CN is represented by *, where p < 0.05.

[0028] Figure 2 The growth curves of individual bacteria for each species are shown.

[0029] Figure 3 The growth rate of the combined bacteria relative to the control group is represented by *, where p < 0.05.

[0030] Figure 4 Results of metagenomic analysis;

[0031] Figure 5 The value of BSFL is given by the inoculum agent on rice husk substrate. * indicates p < 0.05.

[0032] Figure 6 The oil and protein content of BSFL under rice husk substrate conditions is shown in the figure. * indicates p<0.05.

[0033] Figure 7 The value represents the body weight of BSFL (Bacterial Agent for Liver Disease) under chicken manure substrate conditions; * indicates p < 0.05.

[0034] Figure 8 The oil and protein content of BSFL in vitro under chicken manure substrate conditions is shown in the figure. * indicates p<0.05.

[0035] Figure 9 The weight of the bacterial extract protein prepared in each group changed over time, using culture medium as the raw material.

[0036] Figure 10 Using fresh chicken manure wastewater as raw material, the weight of bacterial extract protein changes over time. Detailed Implementation

[0037] This invention provides a high-yield lysine-producing *Providens steudens* bacterium, a compound bacterial agent, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0038] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0039] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0040] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone. A and B can be singular or plural.

[0041] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0042] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have reasonable deviations within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%. The test materials used in this invention are all common commercially available products.

[0043] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0044] This study isolated gut microbiota from the intestines of black soldier fly larvae and, through metabolic analysis, discovered that they exhibited high lysine production. Lysine is one of the essential amino acids that animals cannot synthesize on their own, and it is the first limiting amino acid, present in very low amounts, severely restricting the efficiency of protein utilization in animal feed. Producing lysine through microbial fermentation can reduce the amount of expensive fishmeal and soybean meal in feed, thereby lowering feed costs. The invention is further illustrated below with examples:

[0045] Example 1: Isolation and Identification of Intestinal Microorganisms

[0046] 20 sterile black soldier fly larvae (BSFL) + 2.80 g glucose + 1 mL 0.051 g / mL sterile urea solution + 20 g rice husks + intestinal contents of 20 4-day-old larvae (45% moisture content).

[0047] Remove the dissected larval intestines (5 larvae / tube), add an appropriate amount of sterile quartz sand to the original 1.5 mL centrifuge tube, grind thoroughly, and then serially dilute the homogenate 10-fold with sterile PBS to 10 ... -5 10 -6 10 -7 10 -8 Take 100 μL of the diluted solution and spread it evenly on plates with different nitrogen sources (urea, ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen). Invert the plates and incubate them overnight at 37 ℃. Select plates with colony counts of 30-300 for purification. Pick single colonies with different morphologies and inoculate them again on LB plates for purification. Repeat the purification process at least 3 times until colonies with consistent morphology and uniform size grow on the plates.

[0048] Urea selective medium: Weigh 1.40 g KH2PO4, 2.10 g NaH2PO4, 0.20 g MgSO4·7H2O, 10.00 g anhydrous glucose, and 15.00 g agar powder. Add pure water to a final volume of 1000 mL, adjust the pH to 7.2 ± 0.2, pour into a 250 mL Erlenmeyer flask, autoclave at 115 ℃ for 30 min, cool to room temperature, add 10 mL urea solution (1.00 g / mL urea solution sterilized by filtration through a 0.22 μm filter), pour into sterile Petri dishes, and set aside.

[0049] 1. Screening and identification of strains

[0050] (1) Extraction of bacterial DNA. Extraction was performed according to the instructions for the bacterial genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0051] (2) PCR amplification of the bacterial strain. The extracted DNA was amplified by PCR for 16S rRNA. The primer sequences are shown in Table 3.3. After PCR amplification, the PCR products were detected by gel electrophoresis. The expected size of the bacterial DNA fragment was approximately 1500 bp. Primers used were 27F and 1492R.

[0052] (3) Sequencing and analysis of the strain. PCR products that successfully amplified the expected size fragment were identified by gel electrophoresis and sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared and analyzed with relevant sequences in the NCBI database.

[0053] 2. Identification results and sources of gut microbiota in BSFL

[0054] Eight microbial strains were isolated from urea culture medium, and their species information is shown in Table 1. These strains, capable of growing in urea medium, typically possess the ability to decompose urea as a nitrogen source, and may play an important role in nitrogen cycling and organic waste decomposition. NH4 + Two microorganisms were isolated from the culture medium, NH4 + It is one of the important nitrogen sources for microbial growth. These strains may participate in nitrogen metabolism processes such as ammonia assimilation and nitrification, which is significant for maintaining nitrogen balance in the intestine.

[0055] Table 1

[0056]

[0057] Example 2: Validation of Gut Microbial Function

[0058] To verify the role of the strains described in Table 1 in the conversion of BSFL to urea, a control group (CN, sterile BSFL, sterilized enteric microorganism Escherichia coli) and different single strains were set up in the experimental group. Seed cultures of the single strains were inoculated into fresh LB broth at a volume ratio of 1:100 and cultured at 37 ℃, 210 r / min with shaking for 12-16 h. Twenty bacterial solutions were diluted with an appropriate amount of LB broth, and their OD600 was uniformly adjusted to 1 using a UV spectrophotometer. 20 mL of each diluted bacterial solution was centrifuged at 4000 r / min for 15 min, the supernatant was discarded, and the precipitate was retained for subsequent experiments.

[0059] Prepare clean 150 mL Erlenmeyer flasks and breathable stoppers. Fill each flask with 20 g of rice husks, stopper, and autoclave at 121 °C for 15 min. After cooling and standing for 12 h, autoclave again. After cooling, open the stoppers and add 2.80 g of glucose and an appropriate amount of pure water to each flask (final substrate moisture content 45%). Stir well and let stand for 12 h. Then autoclave at 115 °C for 15 min. After cooling, add sterile urea solution, bacterial precipitate, and sterile larvae in a laminar flow hood. Set up three replicates per group. After incubating at 28 °C for 10 days, open the stoppers, separate the larvae, clean and dry them, and count their weight.

[0060] Growth multiplier = (Final insect weight - Initial insect weight) / (CK Final insect weight - CK Initial insect weight)

[0061] The results are as follows Figure 1 ,Depend on Figure 1It was found that, compared with the control group, *Klebsiella pneumoniae*, *Morganella morganii*, *Citrobacter farmeri*, *Enterococcus faecalis*, *Proteus mirabilis*, *Escherichia coli*, *Bacillus licheniformis*, *Pseudomonas aeruginosa*, *Microbacterium paraoxydans*, and *Providencia stuartii* all increased the fold increase in body weight gain in BSFL, specifically 7.2±0.5, 1.5±0.2, 1.5±0.2, 6.5±0.6, 1.8±0.2, 2.6±0.2, 6±0.2, 1.7±0.3, 3.1±0.5, and 7.5±0.2, respectively. Among these, four strains—*Klebsiella pneumoniae*, *Enterococcus faecalis*, *Bacillus licheniformis*, and *Microbacterium paraoxydans*—showed the most significant increases, all exceeding 6-fold. Klebsiella pneumoniae is a common opportunistic pathogen and will not proceed to the next stage of the experiment.

[0062] Example 3: Single-strain growth curve and amino acid composition

[0063] Escherichia coli (E. coli) was used as a control strain. Enterococcus faecalis, Bacillus licheniformis, and Providencia stuartii were added to urea liquid medium and cultured at 37 °C with shaking at 210 r / min for 36 h. OD values ​​were measured at 0 h, 6 h, 12 h, 24 h, and 36 h. The cultures were then dried in a 60 °C oven to constant weight. The amino acid composition of the bacterial proteins was analyzed using HPLC according to GB / T18246-2019.

[0064] Growth curve of the strain as follows Figure 2 The growth rates of three strains, Enterococcus faecalis (OD600=2.05±0.21), Bacillus licheniformis (OD600=1.98±0.24), and Providencia stuartii (OD600=2.14±0.31), were all higher than those of E. coli (OD600=1.89±0.25).

[0065] The amino acid composition analysis is shown in Table 2. It was found that among the essential amino acids of the cell proteins obtained after culturing the three strains of Enterococcus faecalis, Bacillus licheniformis, and Providencia stuartii, the contents of lysine and methionine were significantly higher than those of E. coli. For resource insects such as black soldier fly, yellow mealworm, and cricket, lysine is a key limiting factor for their growth and development. It can shorten the larval growth cycle, improve production efficiency, increase the protein content of larvae, and enhance their nutritional value as feed. Methionine is an important source of sulfur in chitin synthesis and is crucial for molting, metamorphosis, and cuticle formation in insects (whose exoskeletons are composed of chitin). Supplementing methionine can significantly improve the molting success rate of insects and the vitality of adults. For egg-laying insects, sufficient methionine has a positive impact on the egg production and hatching rate of eggs.

[0066] Table 2 Mass fraction of each amino acid in cell protein

[0067]

[0068] Providencia stuartii (Chinese name: Providencia stuartii, numbered Eg) was deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 20252242, the deposit date was October 24, 2025, and the address: Wuhan University, Wuhan, China.

[0069] Example 4 Verification of the functions of combined microorganisms

[0070] It was attempted to use Providencia stuartii (Providencia stuartii) with the deposit number of CCTCC NO: M 20252242, Enterococcus faecalis, and Bacillus licheniformis as a combined microbial agent.

[0071] The experiment set up a control group (CN, sterile BSFL, sterilized intestinal microorganism Escherichia coli) and an experimental group of combined bacteria.

[0072] The treatment of the control group included sterilized intestinal microorganism Escherichia coli (the seed solution was inoculated at a volume ratio of 1:100).

[0073] The preparation of the combined bacteria included: seed cultures of single bacterial strains (Providencia stuartii, Enterococcus faecalis, and Bacillus licheniformis, preservation numbers: CCTCC NO: M 20252242) were inoculated into fresh LB broth at a volume ratio of 1:100 and cultured at 37 ℃ with shaking at 210 r / min for 12-16 h. Twenty bacterial cultures were diluted with an appropriate amount of LB broth, and their OD600 was uniformly adjusted to 1 using a UV spectrophotometer. Three bacterial strains were mixed in a 1:1:1 ratio, centrifuged in 20 mL at 4000 r / min for 15 min, the supernatant was discarded, and the precipitate was retained for subsequent experiments.

[0074] Prepare clean 150 mL Erlenmeyer flasks and breathable stoppers. Fill each flask with 20 g of rice husks, stopper, and autoclave at 121 °C for 15 min. After cooling and standing for 12 h, autoclave again. After cooling, open the stoppers and add 2.80 g of glucose and an appropriate amount of pure water to each flask (final substrate moisture content 45%). Stir well and let stand for 12 h. Then autoclave at 115 °C for 15 min. After cooling, add sterile urea solution, bacterial precipitate, and sterile larvae in a laminar flow hood. Set up 6 replicates per group. After incubating at 28 °C for 10 days, open the stoppers, separate the larvae, clean and dry them, and count their weight.

[0075] Growth multiplier = (Final insect weight - Initial insect weight) / (CK Final insect weight - CK Initial insect weight)

[0076] The results are as follows Figure 3 Compared with the control group, the combined bacterial agents can increase the weight gain of BSFL by 10.2 ± 0.8 times, which can significantly improve the growth performance of BSFL, and the effect is better than that of the single bacteria in Example 2.

[0077] Intestinal samples from the control group and the combined bacterial agent group of BSFL were sent to Shanghai Meiji Biomedical Technology Co., Ltd. for DNA extraction. After genomic DNA extraction, the extracted genomic DNA was detected by 1% agarose gel electrophoresis. Once the samples were deemed acceptable, PE libraries were constructed, and bridge PCR and Illumina sequencing were performed. Data analysis began with the raw sequences. First, the raw sequences underwent optimization processes such as splitting, quality cutting, and contamination removal. Then, the optimized sequences were used for assembly and gene prediction, and the resulting genes were annotated and classified according to species and function.

[0078] The results are as follows Figure 4Metagenomic results revealed that the lysine pathway was enhanced in the combined bacterial agent group, with the gene abundance of key genes dape, pata and lysc increasing by 5.1±0.2 times, 4.4±0.3 times and 6.2±0.6 times, respectively.

[0079] The *Providencia stuartii*, *Enterococcus faecalis*, and *Bacillus licheniformis* strains used in this embodiment were all isolated from Example 1. Based on this, attempts were made to reproduce the BSFL culture experiment of this embodiment using strains from other sources. For example, the *Enterococcus faecalis* strain was CICC 20175 or CICC 10396 from the China Industrial Microbial Culture Collection Center, and the *Bacillus licheniformis* strain was CICC 21886, CICC 21963, or CICC 21972 from the China Industrial Microbial Culture Collection Center. The results were consistent with... Figure 3 The results are comparable. This indicates that the choice of Bacillus licheniformis and Enterococcus faecalis has little impact on the results. However, when Providencia stuartii is replaced with CICC 21520 or ATCC49809 from the China Industrial Microbial Culture Collection Center, the BSFL growth rate is 1.2 ± 0.33 times, which is lower than the effect of CCTCC NO: M 20252242.

[0080] Example 5: Application of microbial agents in BSFL culture under rice husk substrate conditions

[0081] Control group: 500 g rice husks + 200 g glucose + 2.30 g urea + 600 5-year-old BSFL.

[0082] Microbial agent group: 500 g rice husk + 200 g glucose + 2.30 g urea + 600 5-year-old BSFL + Enterococcus faecalis, Bacillus licheniformis, Providencia stuartii combined microbial agent (seed liquid inoculated at a mass ratio of 1:100).

[0083] Before adding BSFL at the start of the experiment, the system was thoroughly stirred to ensure homogenization. All groups underwent conversion experiments in 2.5 L glass conversion vessels, with six replicates per group, and conversion was performed in a water bath at 30 ± 1 °C for 10 days. After conversion, BSFL and the matrix were manually separated by sieving.

[0084] Insect fat extraction: Petroleum ether was used as the extraction solvent at 65 ℃, ensuring a siphon reflux frequency of 6-8 times / h in the Soxhlet extractor for 8 h. The extracted sample was air-dried overnight to allow residual petroleum ether to evaporate. The extracted sample was then dried in an oven at 65 ℃ until constant weight, and the results were recorded. Crude protein content of the insect body: 1 g of air-dried insect body sample was weighed into a 100 mL Erlenmeyer flask, 5 mL of concentrated hydrogen peroxide was added, and heating was continued for digestion. The hydrogen peroxide addition step was repeated until the solution was clear. After the solution cooled to room temperature, it was diluted to a 50 mL volumetric flask and filtered to remove ash. The protein content was then determined using a semi-automatic Kjeldahl nitrogen analyzer.

[0085] The results are as follows Figure 5-6 Compared with the control group (92±4 mg), the fungal agent group increased the body weight of BSFLs (110±12 mg) and significantly improved their growth performance. Regarding larval weight, the control group had a protein content of 35.1±2.2% and a fat content of 27.8±1.5%. Compared with the control group, the fungal agent group increased the protein content of BSFLs (36.8±4.8%) and decreased the fat content (26.8±5.2%).

[0086] Example 6: Application of microbial agents in BSFL farming under chicken manure substrate conditions

[0087] Control group: 500 g chicken manure + 200 g glucose + 2.30 g urea + 600 5-year-old BSFL.

[0088] Microbial agent group: 500 g chicken manure + 200 g glucose + 2.30 g urea + 600 5-year-old BSFL + Enterococcus faecalis, Bacillus licheniformis, Providencia stuartii combined microbial agent.

[0089] Before adding BSFL at the start of the experiment, the system was thoroughly stirred to ensure homogenization. All groups underwent conversion experiments in 2.5 L glass conversion vessels, with six replicates per group, and conversion was performed in a water bath at 30 ± 1 °C for 10 days. After conversion, BSFL and the matrix were manually separated by sieving.

[0090] Insect fat extraction: Petroleum ether was used as the extraction solvent at 65 ℃, ensuring a siphon reflux frequency of 6-8 times / h in the Soxhlet extractor for 8 h. The extracted sample was air-dried overnight to allow residual petroleum ether to evaporate. The extracted sample was then dried in an oven at 65 ℃ until constant weight, and the results were recorded. Crude protein content of the insect body: 1 g of air-dried insect body sample was weighed into a 100 mL Erlenmeyer flask, 5 mL of concentrated hydrogen peroxide was added, and heating was continued for digestion. The hydrogen peroxide addition step was repeated until the solution was clear. After the solution cooled to room temperature, it was diluted to a 50 mL volumetric flask and filtered to remove ash. The protein content was then determined using a semi-automatic Kjeldahl nitrogen analyzer.

[0091] The results are as follows Figure 7-8 Compared with the control group (122±17 mg), the fungal agent group increased the body weight of BSFLs (157±23 mg), significantly improving their growth performance. Regarding larval weight, the control group had a protein content of 34.2±2.9% and a fat content of 29.2±4.3%. Compared with the control group, the fungal agent group increased the protein content of BSFLs (38.2±3.2%) and decreased the fat content (35.1±2.0%).

[0092] Example 7 Microbial synthesis of protein from high-nitrogen raw materials

[0093] 1. Microbial preparation of proteins using culture media

[0094] Basic culture medium: The reagents and dosages used to prepare the basic culture medium are as follows: peptone 0.5 g / L, yeast extract 0.25 g / L, NaHCO3 2.6 g / L, urea 2 g / L, NaCl 0.1 g / L, MgCl2 0.1 g / L, CaCl2 0.05 g / L, K2HPO4 0.4 g / L, trace element solution 1 mL / L, vitamin solution 1 mL / L.

[0095] The trace element solution composition is as follows: FeCl3·2H2O 2000 mg / L, H3BO3 50 mg / L, ZnCl2 50 mg / L, CuCl2 30 mg / L, MnCl2·4H2O 50 mg / L, CoCl2·6H2O 50 mg / L, (NH4)6Mo7O 24 50 mg / L, AlCl350 mg / L, NiCl2 50 mg / L, H2SeO3 49 mg / L.

[0096] Vitamin solution composition: Biotin 50mg / L, Folic acid 20 mg / L, Vitamin B6 100mg / L, Vitamin B2 50mg / L, Vitamin B1 50mg / L, Niacin 50mg / L, Alpha-lipoic acid 50mg / L, Vitamin B5 50mg / L.

[0097] Take 1000 ml of basal culture medium, sterilize it at 65℃ for 3 hours, and transfer it to a fermenter. Inoculate with 10 g of Enterococcus faecalis, Bacillus licheniformis, Providencia stuartii, and a combined culture (Enterococcus faecalis, Bacillus licheniformis, and Providencia stuartii in a 1:1:1 ratio). The total bacterial count should be 10. 10 -10 11 / ml, sealed fermentation, controlled fermentation temperature at 25-30℃, fermentation cycle of 7 days, fermentation terminal pH≤5; take the remaining fermentation liquid and stir thoroughly to fully stir up the lactic acid bacteria that have settled at the bottom, pump and filter through 400 mesh to obtain bacterial protein. Concentrate the bacterial protein under reduced pressure to obtain active bacterial protein paste, then wash with sterile water, filter, and obtain pure active bacterial protein paste. 5.8g of bacterial protein paste was harvested from Enterococcus faecalis bacterial liquid, 4.9g from Bacillus licheniformis bacterial liquid, 5.2g from Providencia stuartii bacterial liquid, and 7.8g from a combination of bacterial liquids. Figure 9 ).

[0098] Table 3. Mass fraction of each amino acid in the protein paste obtained from the fermentation of Providencia stuartii.

[0099]

[0100] 2. Microbial preparation of protein from chicken manure wastewater

[0101] 1000 g of fresh chicken manure wastewater (nitrogen content approximately 1.5%) was used as raw material. It was sterilized at 65℃ for 12 hours, then transferred to a fermentation tank and inoculated with 10 g of a combined bacterial solution (Enterococcus faecalis, Bacillus licheniformis, and Providenciastuartii in a 1:1:1 ratio). The total bacterial count was 10. 10 -10 11 / ml sealed fermentation, controlled at a temperature of 25-30℃, fermentation cycle of 7 days, with pH ≤5 at the end of fermentation; take the remaining fermentation liquid and stir thoroughly to fully stir up the lactic acid bacteria that have settled at the bottom, pump it out, and filter it through 80 mesh, 200 mesh, and 400 mesh successively to obtain bacterial protein. Concentrate the bacterial protein under reduced pressure to obtain active bacterial protein paste, then wash with sterile water, filter, and obtain approximately 13.5 g of pure active bacterial protein paste. Figure 10 ).

[0102] Table 4. Mass fraction of each amino acid in the protein paste after Providencia stuartii treatment.

[0103]

[0104] 3. Continuous fermentation treatment process for chicken manure wastewater involving microorganisms

[0105] In the first cycle, 1000 kg of fresh chicken manure wastewater (containing approximately 1.5% nitrogen) was used as raw material. It was sterilized at 65℃ for 12 hours, then transferred to a fermentation tank and inoculated with 10 kg of a combined bacterial solution (Enterococcus faecalis, Bacillus licheniformis, and Providencia stuartii in a 1:1:1 ratio), resulting in a total bacterial count of 10... 10 -10 11 / ml sealed fermentation, controlled fermentation temperature at 25-30℃, the first fermentation cycle is 7 days, and the pH at the end of fermentation is ≤5; take 50% of the fermentation liquid and pump it out, filter it through a hollow fiber membrane column (Shandong Bona Biotechnology Group Co., Ltd.) to obtain active bacterial protein paste, then wash it with sterile water, filter it, and obtain about 5.5 kg of pure active bacterial protein paste.

[0106] In the second cycle, the remaining 50% of the fermentation liquid continued to ferment, with 200 kg of fresh chicken manure wastewater added daily for 5 consecutive days. The fermentation temperature was controlled at 25-30℃. The second cycle of fermentation lasted 7 days, with the fermentation pH in the range of 5-5.5. 50% of the fermentation liquid was pumped out and filtered through a hollow fiber membrane column (Shandong Bona Biotechnology Group Co., Ltd.) to obtain an active bacterial protein paste. This paste was then washed with sterile water and filtered to obtain approximately 5.3 kg of pure active bacterial protein paste.

[0107] In the third cycle, the remaining 50% of the fermentation liquid continued to ferment. 200 kg of fresh chicken manure wastewater was added daily as raw material for 5 consecutive days, and the fermentation temperature was controlled at 25-30℃. The fermentation cycle of the third cycle was 7 days, and the fermentation pH was in the range of 5-5.5. 50% of the fermentation liquid was pumped out and filtered through a hollow fiber membrane column (Shandong Bona Biotechnology Group Co., Ltd.) to obtain active bacterial protein paste. It was then washed with sterile water and filtered to obtain about 5.4 kg of pure active bacterial protein paste.

[0108] In the fourth cycle, the remaining 50% of the fermentation liquid continued to ferment. 200 kg of fresh chicken manure wastewater was added daily as raw material for 5 consecutive days, with the fermentation temperature controlled at 25-30℃. The fermentation cycle of the fourth cycle was 7 days, and the fermentation pH was within the range of 5-5.5. 50% of the fermentation liquid was pumped out and filtered through a hollow fiber membrane column (Shandong Bona Biotechnology Group Co., Ltd.) to obtain an active bacterial protein paste. This paste was then washed with sterile water and filtered to obtain approximately 5.2 kg of pure active bacterial protein paste.

[0109] Example 8 Utilization of proteins prepared by microorganisms

[0110] Microbial protein paste is subjected to moist heat treatment, including high-pressure steam treatment at 120-150℃ for 10 minutes, followed by freeze-drying at -40℃ for 24 hours to prepare microbial cell protein powder. This protein powder is added to fish and shrimp feed at a certain proportion (e.g., 3%~10%) to replace part of the fishmeal or soybean meal. The formula is as follows:

[0111] Bass feed: 40.8% fish meal + 8% microbial protein powder prepared in Example 7 + 18% soybean meal + 5% rapeseed meal + 18% wheat flour + 7% soybean oil + 1.2% calcium dihydrogen phosphate + 0.4% choline chloride + 0.8% vitamin premix + 0.8% mineral premix.

[0112] Bass feed: 43.8% fish meal + 5% microbial protein powder prepared in Example 7 + 18% soybean meal + 5% rapeseed meal + 18% wheat flour + 7% soybean oil + 1.2% calcium dihydrogen phosphate + 0.4% choline chloride + 0.8% vitamin premix + 0.8% mineral premix.

[0113] Bass feed: 45.8% fish meal + 3% microbial protein powder prepared in Example 7 + 18% soybean meal + 5% rapeseed meal + 18% wheat flour + 7% soybean oil + 1.2% calcium dihydrogen phosphate + 0.4% choline chloride + 0.8% vitamin premix + 0.8% mineral premix.

[0114] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Providencia stuartii with accession number CCTCC NO: M 20252242.

2. A combination bacterium, characterized in that, Includes Providencia squarrosa, Enterococcus faecalis, and Bacillus licheniformis as described in claim 1.

3. The combined bacteria according to claim 2, characterized in that, The mass ratio of Providencia squarrosa, Enterococcus faecalis, and Bacillus licheniformis is 1:1:

1.

4. The application of *Providensia schoensis* as described in claim 1 or the combined bacteria as described in claim 2 or 3 in the treatment of organic waste; wherein the organic waste is chicken manure and / or rice husks.

5. A method for raising black soldier flies using organic waste, characterized in that, The method includes inoculating organic waste with the Providencia schlegelii of claim 1 or the combined bacteria of claim 2 or 3 and black soldier fly larvae, and culturing it at 30±1℃ for 10 days; wherein the organic waste is chicken manure and / or rice husks.

6. A method for preparing microbial protein from organic waste, characterized in that, The method involves inoculating organic waste with *Providens schlegelii* as described in claim 1 or the combined bacteria as described in claim 2 or 3, and then sealing and fermenting it at 25-30°C until the pH ≤ 5 to obtain a fermentation product containing protein; the organic waste is chicken manure and / or rice husks.

7. Microbial protein, characterized in that, It is prepared by the method described in claim 6.

8. The microbial protein according to claim 7, characterized in that, It is rich in at least one of the following: leucine, lysine, valine, arginine, threonine, isoleucine, phenylalanine, and methionine.

9. Feed, characterized in that, This includes the microbial protein as described in claim 7 or 8.

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