Growth-promoting and abdominal fat-reducing chicken-derived lactobacillus agilis and screening and application thereof

Abstract

The present application belongs to the field of agricultural microorganism application, and particularly relates to a growth-promoting and abdominal fat-reducing chicken-derived Ligilactobacillus agilis and application of the same in screening and improving growth performance of broilers and reducing abdominal fat deposition. A strain of Ligilactobacillus agilis with significant growth-promoting and abdominal fat-reducing effects on Cobb broilers is screened from the intestinal tract of fecal microbiota transplantation positive (recipient is high weight and low abdominal fat phenotype) broilers. After isolation and identification, probiotic performance detection and broiler feeding test verification, it is shown that the strain has good stress resistance, antibacterial effect and significant growth-promoting and abdominal fat-reducing effect on broilers. The Ligilactobacillus agilis CIM-3 strain of the present application is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20231947. The probiotic strain has the effects of improving growth performance of broilers and reducing abdominal fat deposition, and can be applied to broiler breeding as a microecological feed additive, thereby providing a good foundation for development of a poultry feed microecological additive.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial applications, and more specifically relates to an isolated and screened strain of *Ligilactobacillus agilis* CIM-3 with growth-promoting, abdominal fat-reducing, and antibacterial abilities. It also relates to a screening method for *Ligilactobacillus agilis* CIM-3 that promotes broiler growth and reduces abdominal fat deposition, and particularly relates to the use of *Ligilactobacillus agilis* CIM-3 in broiler feed microecological additives. Background Technology

[0002] Modern broiler chickens, through continuous selective breeding, are known for their rapid growth and high meat production capacity (BARBUT S, LEISHMAN EM. Quality and Processability of Modern Poultry Meat[J]. Animals (Basel), 2022). However, this has also led to many developmental and metabolic diseases, among which excessive fat deposition is a problem that cannot be ignored in the poultry industry (MOREIRA GCM et al. Integration of genome wide association studies and wholegenome sequencing provides novel insights into fat deposition in chicken[J]. Sci Rep, 2018). Excessive fat deposition affects the taste of meat, reduces feed conversion rate, and thus increases breeding costs (WEN CL et al. Joint contributions of the gut microbiota and host genetics to feedefficiency in chickens[J]. Microbiome, 2021). Moreover, fat is usually disposed of as low-economic-value waste, resulting in resource waste and environmental pollution.

[0003] Studies have shown that gut microbes can participate in lipid metabolism, reduce fat deposition, and improve meat quality (D'OPLINTERAD et al. Gut microbes participate in food preference alterations during obesity[J]. Gut Microbes, 2021; MARTINEZ-GURYN K et al. Small Intestine Microbiota Regulate Host Digestive and Absorptive Adaptive Responses to Dietary Lipids[J]. Cell Host Microbe, 2018). Gordon's team (TURNBAUGH PJ et al. Anobesity-associated gut microbiome with increased capacity for energy harvest[J]. Nature, 2006) found that germ-free mice transplanted with feces from mice on a high-fat diet had stronger lipid absorption capacity than mice transplanted with feces from mice on a low-fat diet, demonstrating that gut microbes play an important role in lipid metabolism.

[0004] The gut microbiota is incredibly diverse (Xiang, Hai, et al. Specific Microbial Taxa and Functional Capacity Contribute to Chicken Abdominal Fat Deposition. Frontiers in Microbiology, 2021), leading many studies to focus on identifying key microorganisms that regulate lipid metabolism. Xie Chunlin et al. (Xie, Chunlin, et al. Multi-omics analysis reveals gut microbiota-induced intramuscular fat deposition via regulating expression of lipogenesis-associated genes. Animal Nutrition, 2022) remodeled the gut environment of commercial pigs to improve meat quality, screening for four key candidate strains (B. uniformis, S. globosa, H. saccharovorans, and P. piscolens) that reduce fat deposition and improve pork quality. Chen et al. compared the cecal flora of chickens with high and low abdominal fat deposition and found that Sphaerochaeta was more abundant in chickens with low abdominal fat deposition (Chen, Y., et al. Chicken cecal microbiota induces abdominal fat deposition by regulating fat metabolism. NPJ BiofilmsMicrobiomes, 2023). There are reports that feeding high-cholesterol rats with Lactobacillus plantarum significantly reduces cholesterol levels and improves the intestinal microecological environment (LI Y et al. Regulation of viable / inactivated / lysed probiotic Lactobacillus plantarum H6 on intestinal microbiota and metabolites in hypercholesterolemic mice[J]. Npj Sci Food, 2022). Currently, there are few reports on strains in broilers that both promote growth and reduce abdominal fat.

[0005] The applicant of this invention utilized fecal microbiota transplantation (FMT) technology to transplant feces from high-weight, low-abdominal-fat broiler chickens into recipient chickens, and found that the recipient chickens exhibited the same traits as the donor chickens. To increase the probability of obtaining the target bacterial strain that promotes growth and reduces abdominal fat, the applicant isolated and screened a strain of *Lactobacillus argylis* from the intestines of high-weight, low-abdominal-fat recipient chickens. This strain significantly degrades cholesterol, promotes broiler growth, and reduces abdominal fat deposition. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of *Ligilactobacillus agilis* CIM-3, which has good stress resistance, antibacterial effect, and significant growth-promoting and abdominal fat-reducing effects on broilers.

[0007] Another objective of this invention is to provide a method for screening Ligilactobacillus agilis CIM-3 that promotes broiler growth and reduces abdominal fat deposition. The screening method is based on cholesterol degradation efficiency, and is easy to implement and operate.

[0008] Another objective of this invention is to provide an application of Lactobacillus Argyllis CIM-3 in broiler feed microecological additives. It is convenient to use, simple to operate, does not cause stress to chicks, and improves the production efficiency of broiler farming.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A screening method for Ligilactobacillus aglilis CIM-3 that promotes broiler growth and reduces abdominal fat deposition, the steps of which are as follows:

[0011] A. The samples were taken from the intestinal contents of high-weight, low-fat fecal microbiota transplant recipient broilers (Cobb broilers) at the experimental chicken farm of Huazhong Agricultural University. The collected samples were temporarily stored in a 4°C refrigerator for later use.

[0012] B. Take 1 mL of the collected sample and perform a 10-fold serial dilution (add 100 μL of culture medium to 900 μL of sterile water for serial dilution), diluting to a final volume of 10. -7 .

[0013] C. From 10 -4 10 -5 10 -6 10 -7Four dilution gradients were prepared, and 200 μl of bacterial suspension was evenly spread on MRS solid medium: 10 g peptone, 8 g beef extract powder, 4 g yeast extract, 20 g glucose, 2 g K2HPO4, 5 g sodium acetate, 2 g diammonium citrate, 1 ml Tween-80, 0.58 g MgSO4·7H2O, 0.25 g MnSO4·7H2O, and 1.5% agar powder were added. The volume was then adjusted to 1000 ml with distilled water.

[0014] D. Incubate in an anaerobic incubator at 37℃ with the colonies upside down for 36-48 hours, and then select single colonies of different morphologies for pure culture.

[0015] E. The pure culture strain was inoculated onto MRS plates with added CaCO3 and MRS liquid medium with added cholesterol for screening in sequence to obtain a strain of Lactobacillus agellis CIM-3.

[0016] The facultative anaerobic bacterium obtained above is a short rod-shaped, cylindrical, Gram-positive bacterium (Lactobacillus argylis CIM-3, see...). Figure 1 ).

[0017] The *Ligilactobacillus agilis* CIM-3 strain contains the nucleotide sequence shown in SEQ ID NO: 1, which is 1463 bp in length.

[0018] Based on the screening criteria of cholesterol-lowering and antibacterial properties of probiotics, this invention isolated and screened a candidate strain from the intestines of high-weight, low-abdominal-fat recipient broilers. The inventors identified the candidate strain as *Ligilactobacillus agilis* CIM-3. It was deposited on October 19, 2023, at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20231947.

[0019] Bacteriological characteristics of Ligilactobacillus agilis CIM-3 strain:

[0020] Ligilactobacillus agilis CIM-3 strain is a Gram-positive, facultative anaerobic bacterium, appearing as short, rod-shaped or cylindrical rods (see...). Figure 1 On MRS screening plates for lactic acid bacteria, colonies grow as round, milky-white raised, smooth surfaces, with a diameter of approximately 3 mm (see...). Figure 2The 16S rRNA gene was cloned and sequenced. Blast comparison was performed using NCBI, and it was found that the 16S rDNA sequence of the strain of this invention had the highest homology (99.66%) with Lactobacillus argylis (MT573793.1).

[0021] Lactobacillus agellis CIM-3 strain was isolated and screened on MRS plates supplemented with CaCO3, exhibiting a distinct calcium dissolution zone (see...). Figure 3 When cultured anaerobically on MRS plates, its reproductive capacity is strong. It is in a lag phase from 0-5 hours, enters the logarithmic growth phase after 5 hours, and then enters the stationary phase after 13 hours (see...). Figure 4 Lactobacillus argylis CIM-3 exhibits good resistance, tolerating acidic and bile salt environments. After incubation for 2 hours in a pH 2.5 solution, the survival rate was 118.78%. After incubation for 2 hours in a 0.3% bile salt solution, the survival rate was 77.00%. This indicates that this strain has good resistance to acidic and high bile salt environments, demonstrating good resilience and adaptability to the gastrointestinal environment.

[0022] Lactobacillus argylis CIM-3 cultured in MRS medium supplemented with cholesterol solution achieved a cholesterol removal rate of 16.37% (see...). Figure 5 This suggests that the strain can degrade cholesterol. CIM-3 has significant inhibitory effects on Escherichia coli K88, Salmonella, and Staphylococcus aureus (see [link to article]). Figure 6 It is sensitive to commonly used veterinary antibiotics such as penicillin, erythromycin, cephalexin, and chloramphenicol, but not sensitive to streptomycin, gentamicin, tetracycline, norfloxacin, and lincomycin (see...). Figure 7 ).

[0023] The application of Lactobacillus argiraceae CIM-3 in livestock and poultry feed microecological additives involves the following steps:

[0024] A. A total of 72 healthy 1-day-old broiler chickens (Cobbler chickens) were selected and randomly divided into two groups: a blank control group and an Argyllis Lactobacillus group, with 3 replicates in each group and 12 chickens in each replicate.

[0025] Groups B and C served as blank control groups, fed with a basal diet; the CIM-3 group consisted of *Lactobacillus argylis*, fed with a basal diet plus 1 ml / (animal·day) of CIM-3 liquid fermentation broth. The viable count of the liquid fermentation preparation (bacterial culture for 13 hours, centrifuged to remove supernatant, and resuspended with an equal volume of sterile water) was 1.04 × 10⁻⁶. 9 CFU / ml.

[0026] C. The chickens were raised for a total of 42 days. From 1 to 21 days old, they were fed a brooding diet, and from 22 to 42 days old, they were fed a growing diet. The diet consisted of broiler compound feed (510, 511), which was purchased from CP Group Limited.

[0027] D. During the experiment, broilers had free access to water and feed, and were fed and had their water changed twice a day (at 9:30 am and 3:00 pm). The broilers were allowed to acclimatize for three days. Afterward, 1 mL of bacterial suspension was added to the water trough each morning. The temperature was maintained at approximately 35℃ at the beginning of the rearing period, decreasing by 2-3℃ each week until reaching 25℃ in the fourth week. Temperature and humidity were adjusted according to the actual growth conditions.

[0028] The applicant conducted a broiler feeding trial using CIM-3 strain via drinking water and found that it had a significant effect on promoting growth and reducing abdominal fat in broilers (see...). Figure 8 and Figure 9 This achieved the expected results, thus fulfilling the task of the present invention.

[0029] This probiotic strain can improve the growth performance of broilers and reduce abdominal fat deposition. It can be used as a microecological feed additive in broiler farming, providing a good foundation for the development of a microecological feed additive for poultry.

[0030] Compared with the prior art, the present invention has the following advantages and effects:

[0031] (1) The Lactobacillus Argyllis CIM-3 strain isolated from high-weight, low-abdominal-fat receptor broilers can degrade cholesterol in vitro, with a cholesterol removal rate as high as 16.37%.

[0032] (2) The CIM-3 strain of the present invention has strong reproductive capacity and good stability; it can tolerate strong acid and high concentration of bile salts. When incubated in a solution with pH 2.5 for 2 hours, the survival rate is 118.78%; when incubated in a 0.3% bile salt solution for 2 hours, the survival rate is 77.00%, which is conducive to adapting to the gastrointestinal environment of broilers and being used as a microecological feed additive.

[0033] (3) The CIM-3 strain of the present invention has a strong inhibitory effect on pathogenic Gram-positive and Gram-negative bacteria, which is beneficial to antagonizing harmful bacteria in the intestines of broilers.

[0034] (4) Animal experiments have shown that the microecological preparation CIM-3 of the present invention has a significant effect on promoting growth and reducing abdominal fat in broilers, thus achieving the ultimate goal of the present invention. Attached Figure Description

[0035] Figure 1 A schematic diagram of Gram staining results for a chicken-derived Lactobacillus Argyllius CIM-3 strain that promotes broiler growth and reduces abdominal fat deposition (oil immersion; ×1000).

[0036] Figure 2This is a schematic diagram of the growth status of a chicken-derived Lactobacillus Argyllius strain CIM-3, which promotes broiler growth and reduces abdominal fat deposition, on an MRS agar plate. Note: im3 refers to the strain CIM-3 of this invention.

[0037] Figure 3 This is a schematic diagram of the growth curve of Lactobacillus argiris CIM-3, a chicken-derived strain that promotes broiler growth and reduces abdominal fat deposition.

[0038] Figure 4 This is a schematic diagram showing the growth of a chicken-derived Lactobacillus Argyllius CIM-3 strain that promotes broiler growth and reduces abdominal fat deposition on screening medium (MRS+CaCO3).

[0039] Figure 5 This is a schematic diagram illustrating the cholesterol-lowering results of CIM-3, a strain of Lactobacillus argiris from chickens that promotes broiler growth and reduces abdominal fat deposition, as detected by the phthalic acid method.

[0040] From left to right: positive control; CIM-3; CI-3 (another strain of lactic acid bacteria selected); negative control.

[0041] Figure 6 This is a schematic diagram of the in vitro antibacterial effect of the fermentation broth supernatant of Lactobacillus Argyllis CIM-3, a strain that promotes broiler growth and reduces abdominal fat deposition.

[0042] Explanation of reference numerals in the attached figures: Figure 6 The inhibition zone marker is the CIM-3 strain isolated in this invention. The indicator strains used from left to right are: Escherichia coli K88, Salmonella, Staphylococcus aureus, Figure 6 The strain CIM-3 in this invention is IM-3.

[0043] Figure 7 This is a schematic diagram illustrating the antibiotic susceptibility test results of a chicken-derived Lactobacillus Argyllius CIM-3 strain that promotes broiler growth and reduces abdominal fat deposition. Figure 7 The strain CIM-3 in this invention is IM-3.

[0044] Figure 8 This is a schematic diagram showing the body weight and average daily weight gain of chickens in an experiment with Lactobacillus argiris CIM-3, a strain that promotes growth and reduces abdominal fat deposition in broilers.

[0045] CIM-3 group: fed with Lactobacillus argylis CIM-3 via drinking water; Ctrl group: fed with sterile water via drinking water.

[0046] Figure 9 This is a schematic diagram of the abdominal fat dissection results in broiler chickens using Lactobacillus Argyllius CIM-3, a strain that promotes broiler growth and reduces abdominal fat deposition.

[0047] 3-18: CIM-3 group, fed with Lactobacillus agilis CIM-3 via drinking water; C-18: blank control group, fed with sterile water via drinking water.

[0048] Figure 10 This is a schematic diagram of the bacterial phylogenetic tree constructed using the neighbor-joining method for Lactobacillus argiris CIM-3, a strain that promotes broiler growth and reduces abdominal fat deposition.

[0049] Note: im3 in the figure is the strain CIM-3 of this invention. Detailed Implementation

[0050] Example 1: Isolation and identification of Lactobacillus argylis CIM-3 strain

[0051] Strain isolation:

[0052] The screening method for a strain of *Ligilactobacillus aglilis* CIM-3 that promotes broiler growth and reduces abdominal fat deposition includes the following steps:

[0053] A. The samples were taken from the intestinal contents of high-weight, low-fat fecal microbiota transplant recipient broilers (Cobb broilers) at the experimental chicken farm of Huazhong Agricultural University. The collected samples were temporarily stored in a 4°C refrigerator for later use.

[0054] B. Take 1 mL of the collected sample and perform a 10-fold serial dilution (add 100 μL of culture medium to 900 μL of sterile water for serial dilution), diluting to a final volume of 10. -7 .

[0055] C. From 10 -4 10 -5 10 -6 10 -7 Four dilution gradients were prepared, and 200 μl of bacterial suspension was evenly spread on MRS solid medium: 10 g peptone, 8 g beef extract powder, 4 g yeast extract, 20 g glucose, 2 g K2HPO4, 5 g sodium acetate, 2 g diammonium citrate, 1 ml Tween-80, 0.58 g MgSO4·7H2O, 0.25 g MnSO4·7H2O, and 1.5% agar powder were added. The volume was then adjusted to 1000 ml with distilled water.

[0056] D. Incubate in an anaerobic incubator at 37℃ with the colonies upside down for 36-48 hours, and then select single colonies of different morphologies for pure culture.

[0057] E. The pure culture strains were inoculated onto MRS plates with added CaCO3 and MRS liquid medium with added cholesterol for screening in sequence.

[0058] F. The pure culture strain was inoculated onto MRS plates supplemented with CaCO3 and then onto MRS liquid medium supplemented with cholesterol for screening. The isolated strain grew into milky white, smooth, round colonies with a diameter of about 3 mm on MRS plates supplemented with CaCO3, with a transparent calcium dissolution zone around them;

[0059] G. After anaerobic culture in MRS liquid medium with added cholesterol for 12 hours, the color in the EP tube was significantly lighter when detected by the o-phthalaldehyde method.

[0060] The *Ligilactobacillus agilis* CIM-3 strain contains the nucleotide sequence shown in SEQ ID NO: 1.

[0061] Strain species identification:

[0062] Based on the above identification, the 16S rRNA gene sequence of *Lactobacillus argyris* CIM-3 was further detected to confirm the species to which *Lactobacillus argyris* CIM-3 belongs. The specific steps are as follows:

[0063] A. Genome extraction of the target strain:

[0064] (1) Take 1 mL of culture medium, centrifuge at 10000 rpm for 30 s, discard the supernatant as much as possible, and collect the bacterial cells.

[0065] (2) Add 200 μL of buffer RB to resuspend, centrifuge at 10,000 rpm for 30 s, and discard the supernatant.

[0066] (3) Add 120 μL of lysozyme (20 mg / mL in 10 mM Tris-HCl, pH 8.0), invert and mix well, incubate at 37 °C for 30-60 min. Centrifuge at 12000 rpm for 2 min, discard the supernatant and resuspend in 180 μL of buffer RB.

[0067] (4) Add 20 μL of proteinase K (20 mg / mL) solution, mix thoroughly, then add 200 μL of binding solution CB, vortex immediately to mix thoroughly, and place at 70°C for 10 min.

[0068] (5) After cooling, add 100 μL of isopropanol and immediately vortex to mix thoroughly. At this time, flocculent precipitate may appear.

[0069] (6) Add the mixture from the previous step (including any possible precipitate) to an adsorption column AC (place the adsorption column in the collection tube), centrifuge at 13000 rpm for 30-60 seconds, and discard the waste liquid in the collection tube.

[0070] (7) Add 500 μL of inhibitor removal solution IR, centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0071] (8) Add 700 μL of washing buffer WB (with anhydrous ethanol added), centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0072] (9) Add 500 μL of rinsing solution WB (with anhydrous ethanol added), centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0073] (10) Place the adsorption column AC back into the empty collection tube, centrifuge at 13000 rpm for 2 min, and remove as much of the washing liquid as possible to avoid the residual ethanol in the washing liquid inhibiting the downstream reaction.

[0074] (11) Remove the adsorption column AC and place it in a clean centrifuge tube. Add 50 μL of elution buffer EB (preheated in a 67-70℃ water bath beforehand) to the middle of the adsorption membrane. Incubate at room temperature (20-25℃, the same applies below) for 3-5 min, then centrifuge at 12000 rpm for 1 min. Add the resulting solution back to the adsorption column, incubate at room temperature for 2 min, then centrifuge at 12000 rpm for 1 min.

[0075] (12) Store the DNA sample at -20℃ for later use.

[0076] Amplification of the B.16S rRNA gene:

[0077] The 16S rRNA sequence of *Lactobacillus argyris* CIM-3 isolate was amplified using universal bacterial primers. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their DNA sequences are as follows:

[0078] Forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3,

[0079] Reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3';

[0080] The PCR amplification system is shown in Table 1. Amplification conditions: 94℃ pre-denaturation for 5 min, 94℃ for 1 min, 50℃ for 30 s, 72℃ for 1.5 min, 30 cycles, followed by a 72℃ extension for 10 min. The PCR amplification products were detected by electrophoresis on a 0.8% agarose gel (containing ethidium bromide). The amplified fragment size was consistent with expectations, approximately 1500 bp.

[0081] Table 1. PCR amplification system of Lactobacillus argyris CIM-3 strain

[0082]

[0083] C. Constructing a bacterial phylogenetic tree to determine species and genus:

[0084] Bacterial genomic DNA was extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were analyzed using BLAST on NCBI, and a bacterial phylogenetic tree was constructed using MEGA 7.0 software via neighbor-joining. The analysis results showed that strain CIM-3 is *Lactobacillus argylis* (see...). Figure 10 ).

[0085] Example 2:

[0086] Prebiotic properties of Lactobacillus argylis CIM-3 strain:

[0087] Antibacterial activity test of Lactobacillus argylis CIM-3 strain:

[0088] Using Escherichia coli K88, Salmonella, and Staphylococcus aureus as indicator bacteria, and the supernatant of the fermentation broth of strain CIM-3 as an antibacterial agent, the in vitro antibacterial activity of the strain was detected. The specific operation steps are as follows:

[0089] A. Preparation of indicator bacteria (Escherichia coli K88, Salmonella, Staphylococcus aureus) suspension: Streak the indicator bacteria on agar plates and incubate at 36°C for 20 h; pick single colonies and incubate on LB broth at 37°C with shaking for 16 h to revive the indicator bacteria. Adjust the bacterial count of the cultured indicator bacteria suspension to 1.0 × 10⁻⁶. 7 CFU / mL. (Note: Dilute with 0.01 mol / L PBS during the dilution process.)

[0090] B. Preparation and treatment of bacterial fermentation broth: The bacterial strain of this invention was streaked onto plates and cultured at 37°C for 16 h. The isolated strain was picked and cultured in 50 mL of LB liquid medium at 37°C and 200 rpm for 24 h to obtain the seed culture. The seed culture was inoculated into 100 mL of LB liquid medium at a 1% inoculum rate and anaerobically cultured at 37°C and 200 rpm for 48 h to obtain the fermentation broth. The prepared fermentation broth was centrifuged at 10000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm sterile filter for later use.

[0091] C. Antibacterial test: Take 200 μL of indicator bacterial suspension and drop it onto an LB agar plate. Spread it evenly with a spreader until no visible water droplets remain. Then, use sterile forceps to pick up a sterile Oxford cup and gently place it on the surface of the LB solid medium, placing 3 Oxford cups evenly on each medium. Use a micropipette to draw 200 μL of the supernatant from the bacterial fermentation broth into each stable Oxford cup (add carefully, avoiding spillage onto the medium outside the Oxford cup); then replace the original glass plate lid with a sterile ceramic lid (to absorb any water that evaporates from the medium during the subsequent test).

[0092] D. Incubation: Place the culture medium containing the supernatant in a 4°C refrigerator for 8 hours, and then place the plate after complete diffusion treatment in a 37°C constant temperature incubator for 16-24 hours. Observe and record the experimental results.

[0093] The experimental results show that the strains of this invention have strong inhibitory ability against pathogenic bacteria. Among them, CIM-3 has a very strong inhibitory effect on K88 and a high degree of inhibition against Staphylococcus aureus and Salmonella. The results are shown in the figure. Figure 6 And Table 2.

[0094] Table 2. Antibacterial test of fermentation supernatant of bacterial strains

[0095]

[0096] Example 3: Safety evaluation of Lactobacillus argylis CIM-3 strain

[0097] Drug sensitivity test:

[0098] Nine antimicrobial susceptibility test discs (purchased from Hangzhou Microbial Reagent Co., Ltd.) were selected for testing, including penicillins, cephalosporins, quinolones, aminoglycosides, tetracyclines, and sulfonamides. The interpretation criteria were based on the latest version of the NCCLS standards provided by the World Health Organization (WHO) (2020 edition). The specific steps are as follows:

[0099] A. Inoculate 1% of the bacterial culture into LB liquid medium and incubate overnight at 37°C and 200 rpm / min on a shaker.

[0100] B. Using a white paper with black text as a background, adjust the turbidity to 0.5 McFarland standard turbidity tube turbidity. If the bacterial suspension concentration is too high, it can be diluted with physiological saline.

[0101] C. Dip a sterile cotton swab in the bacterial solution, press it against the tube wall to squeeze out the excess solution, and then spread it onto an LB agar plate. Rotate the plate 60 degrees each time, and finally spread it around the inner edge of the plate twice. Repeat this process several times to ensure even spreading.

[0102] D. After the moisture on the plate has been completely absorbed by the agar, use sterile tweezers to pick up the drug sensitivity test strips and attach them to the plate surface. Once the strips are attached, they should not be removed. Attach 5 strips to each plate, with a spacing of at least 24 mm between each strip and a center distance of at least 15 mm from the edge of the plate.

[0103] E. Place the plates in a 37℃ constant temperature incubator for 12-16 hours and observe the results.

[0104] The experimental results are shown in Table 3. The Lactobacillus argylis CIM-3 strain of the present invention is sensitive to commonly used veterinary antibiotics such as penicillin, erythromycin, cephalexin, and chloramphenicol, but not sensitive to streptomycin, gentamicin, tetracycline, norfloxacin, and lincomycin.

[0105] Table 3. Results of antibiotic susceptibility testing of Lactobacillus argiris CIM-3 strain

[0106]

[0107] Note: S indicates sensitive, M indicates moderately sensitive, and R indicates resistant.

[0108] Example 4: Stress resistance and growth characteristics of Lactobacillus argylis CIM-3

[0109] 1. Stress resistance test:

[0110] (1) Acid resistance test:

[0111] A. Prepare an acidic solution by adjusting the pH to 2.5 using sodium hydroxide solution and dilute hydrochloric acid.

[0112] B. Preparation of bacterial culture: Use a sterile inoculation loop to pick up a small amount of isolated and preserved bacterial strain and streak it on an MRS agar plate. Incubate at 37°C for 24 hours. Pick a single colony and inoculate it into MRS liquid medium. Incubate anaerobically at 37°C for 18 hours.

[0113] C. Take 1 mL of the cultured bacterial solution, centrifuge at 6000 r / min and 4℃ for 20 min, discard the supernatant, resuspend twice with 50 mmol / L K2HPO4 (pH 6.5), add 1 mL of hydrochloric acid solution with pH 2.5, use PBS as a control, and incubate at 37℃ for 2 h after resuspending.

[0114] D. Take 200 μL of the incubated bacterial solution and add it to 5 ml of LMR S medium. Incubate anaerobicly at 37°C for 16 h.

[0115] E. Microplate reader assay of strains at OD 600 The absorbance value below.

[0116] 2. Bile salt tolerance test:

[0117] A. Prepare a solution containing 0.3% bile salts.

[0118] B. Preparation of bacterial culture: Use a sterile inoculation loop to pick up a small amount of isolated and preserved bacterial strain and streak it on an MRS agar plate. Incubate at 37°C for 24 hours. Pick a single colony and inoculate it into MRS liquid medium. Incubate anaerobically at 37°C for 18 hours.

[0119] C. Take 1 mL of the cultured bacterial solution, centrifuge at 6000 r / min and 4℃ for 20 min, discard the supernatant, resuspend twice with 50 mmol / L K2HPO4 (pH 6.5), add 1 mL of 0.3% bile salt solution, use PBS as a control, and incubate at 37℃ for 2 h after resuspending.

[0120] D. Take 200 μL of the incubated bacterial solution and add it to 5 ml of LMR S medium. Incubate anaerobicly at 37°C for 16 h.

[0121] E. Microplate reader assay of strains at OD 600 The absorbance value below.

[0122] The survival rate of the strains to withstand acid / bile salts was calculated using the following formula: Survival rate (%) = Hydrochloric acid solution / Bile salt solution treated bacterial solution OD 600 OD value of PBS-treated bacterial culture 600 Value × 100%. The results are shown in Table 4.

[0123] Table 4 Results of acid / bilirubin resistance tests on strains

[0124]

[0125] 3. Determination of growth curve:

[0126] A. Preparation of seed culture: Pick a single colony and inoculate it into 3 mL of LB liquid medium, and culture at 37℃ with shaking for 24 h to obtain seed culture;

[0127] B. Preparation of fermentation broth: The seed culture was inoculated into 100 mL of LB liquid medium at an inoculation rate of 1%, and cultured at 37 °C with shaking for 24 h to obtain the fermentation broth;

[0128] C. Determination of growth curve: The fermentation broth was inoculated into 50 mL of LB liquid medium at an inoculum rate of 1%. The bacterial suspension was collected at 0 h, 2 h, 4 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, and 17 h, and its growth curve was measured at OD. 600 The absorbance values ​​were measured. Growth curves for each strain were determined using the pour method after 10-fold serial dilutions, with three replicates for each experiment. The OD values ​​were plotted with incubation time on the x-axis. 600 The values ​​are plotted on the ordinate to create a growth curve for the strain.

[0129] Experiments show that the strain isolated in this invention has strong reproductive capacity. It is in a lag phase from 0-5 hours, enters the logarithmic growth phase after 5 hours and lasts until 13 hours, after which it enters the stationary phase. Its growth curve is shown in [Figure number missing]. Figure 4 .

[0130] Example 5: Broiler Feeding Trial

[0131] The liquid fermentation broth of *Lactobacillus argylis* CIM-3 of this invention was fed to broiler chickens (Cobb broilers) in drinking water to verify the effects of the probiotic strain of this invention on promoting growth and reducing abdominal fat in broilers. An application of *Lactobacillus argylis* CIM-3 in livestock and poultry feed microecological additives includes the following steps:

[0132] A. Seventy-two 1-day-old healthy broiler chickens (Cobbler broilers) were randomly divided into two groups: a blank control group and an *Lactobacillus argyris* group, with three replicates per group and 12 chickens per replicate. Group C was the blank control group, fed with a basal diet; Group C was the *Lactobacillus argyris* group, fed with a basal diet plus 1 ml / (chicken·day) of CIM-3 liquid fermentation broth.

[0133] B. Liquid fermentation preparation (the bacterial culture for 13 hours was centrifuged to remove the supernatant, and the bacterial cells were resuspended in an equal volume of sterile water with the supernatant) had a viable count of 1.04 × 10⁻⁶. 9 CFU / ml.

[0134] C. The chickens were raised for a total of 42 days. From 1 to 21 days old, they were fed a brooding diet, and from 22 to 42 days old, they were fed a growing diet. The diet consisted of broiler compound feed (510, 511), which was purchased from CP Group Limited.

[0135] D. The broiler feeding experiment was conducted at the experimental chicken farm of Huazhong Agricultural University in Wuhan, Hubei Province. The experimental broilers were raised in three-tiered stacked cages. The feeding period was from July 15, 2023 to August 26, 2023, for a total of 42 days.

[0136] E. During the experiment, broilers had free access to water and feed. They were fed and had their water changed twice a day (at 9:30 am and 3:00 pm). The broilers were allowed to adapt for three days. After that, the bacterial suspension was added to the water trough at a rate of 1 mL per bird every morning.

[0137] F. When starting to raise the animals, keep the temperature at around 35℃, and lower it by 2-3℃ each week until it reaches 25℃ in the fourth week. Adjust the temperature and humidity according to the actual growth situation.

[0138] Measurement indicators:

[0139] (1) Production performance:

[0140] The production performance indicators of broilers, including average daily gain (ADG) and final body weight, were statistically analyzed at 1 day, 14 days, and 42 days.

[0141] (2) Organ Index:

[0142] Twenty chickens of similar weight were selected from each group for dissection and sampling to observe abdominal fat and weigh them.

[0143] Results analysis:

[0144] As shown in Table 5, after 42 days of the trial, the average daily weight gain in group C was 1.24g, and the average daily weight gain in group CIM-3 was 1.37g. Compared with group C, the daily weight gain in group CIM-3 was significantly higher (P<0.05). The abdominal fat percentage in group C was 1.17%, and the abdominal fat percentage in group CIM-3 was 0.96%. Compared with group C, the abdominal fat percentage in group CIM-3 was significantly lower (P<0.05).

[0145] Table 5 Comparison of growth performance of broilers

[0146]

[0147] During the experiment, the addition of Lactobacillus argylis increased the weight of broilers by 5.00%, indicating that adding the strain CIM-3 of the present invention to the drinking water can significantly improve the growth performance of broilers; the abdominal fat percentage of broilers decreased by 17.95%, indicating that the Lactobacillus argylis CIM-3 strain of the present invention can significantly improve abdominal fat deposition in broilers.

Claims

1. A growth-promoting and fat-reducing chicken-derived Lactobacillus agellis, characterized in that: The growth-promoting and fat-reducing chicken-derived Lactobacillus Argyllis is Lactobacillus Argyllis ( Ligilactobacillus agilis The CIM-3 strain, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20231947; *Lactobacillus aeruginosa* (… Ligilactobacillus agilis CIM-3 is derived from the intestinal contents of high-weight, low-fat fecal microbiota transplant recipient broilers.

2. The *Lactobacillus argylis* strain from chickens that promotes growth and reduces abdominal fat according to claim 1, characterized in that: The aforementioned Lactobacillus aureus ( Ligilactobacillus agilis The CIM-3 strain contains the nucleotide sequence shown in SEQ ID NO:

1.

3. The use of Lactobacillus argylis as described in claim 1 or 2 in the preparation of a feed microecological additive for promoting broiler growth and reducing abdominal fat deposition in broilers.

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