Ostrich-derived bacillus safensis for promoting growth of broiler chickens, screening method and application thereof

Abstract

The application discloses an ostrich-derived bacillus safensis for promoting the growth of broiler chickens and a screening method and application thereof, and steps are as follows: A, taking the ostrich feces as a sample, and bringing back to a laboratory for treatment in an ice box; B, taking 1g of the ostrich feces and adding sterile normal saline to mix uniformly; C, gradient dilution is carried out; D, taking 10 ‑1 ~10 ‑4 gradient dilution; E, taking 10 ‑2 , 10 ‑3 , 10 ‑4 three dilution culture solutions and coating on LB culture medium added with 2.5% skimmed milk powder, and placing the plate in a culture box for culture, picking single colonies around transparent circles to carry out purification culture; and F, carrying out gram staining test, and obtaining gram staining of the strain being positive, short rod-shaped and single or double. The application of the bacillus safensis O-6 in livestock and poultry feed micro-ecological additives. The bacillus safensis produces protease and cellulase, which improves the ability of broiler chickens to digest protease and cellulase. The bacillus safensis has strong acid resistance, and the survival rate of the spore body is 72.56% after being treated at pH 3.0, which resists the gastric acid in the intestinal tract of broiler chickens and is beneficial to the colonization of the strain in the intestinal tract.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial application technology, and more specifically relates to a strain of *Salmonella ostrichella* that promotes broiler growth, a screening method for a strain of *Salmonella ostrichella* that promotes broiler growth, and the application of a strain of *Salmonella ostrichella* that promotes broiler growth in poultry farming. Background Technology

[0002] my country is a major livestock and poultry farming country. The use of antibiotics in feed has led to a series of problems, including antibiotic resistance in pathogenic microorganisms, reduced immunity in livestock and poultry, and drug residues [Ruan Chen. 2018. Dynamic effects of probiotics on the production performance and gut microbiota of Wuliangshan black-bone chicken chicks [D / OL]. Kunming University of Science and Technology, 2023.]. To address these issues, researchers have explored probiotics as an alternative to antibiotics, primarily for improving animal production performance and promoting animal health [Li Xiuyu. 2021. Effects of adding probiotics to the diet on growth performance, blood indicators, intestinal morphology, and cecal microbiota of Lingshan native chickens [D / OL]. Guangxi University, 2023.]. Studies have shown that adding Bacillus subtilis preparations to chicken diets can significantly increase the activity of jejunal digestive enzymes, enhance intestinal digestive function, promote the production of short-chain fatty acids in the cecum, and promote the development of immune organs [Zhu Peiji. Effects and mechanisms of Bacillus subtilis on the production performance, intestinal health and immune function of Snow Mountain chickens [D]. Yangzhou University, 2017.].

[0003] In large-scale broiler farming, the feed used to feed broilers is rich in nutrients, with protein accounting for more than 20%. However, compared with other farm animals (pigs, cattle, sheep), chickens have shorter digestive tracts and smaller digestive tract volumes, resulting in insufficient digestion of nutrients and low feed utilization. Studies have shown that adding a certain amount of digestive enzymes (protease, amylase, cellulase, etc.) to the feed can improve feed utilization. In particular, adding protease can also improve the meat quality of chicken [Fang Lichao, Song Daijun, Kan Ning, et al. Effects of dietary energy and protein levels on broiler meat quality [J]. Southwest China Journal of Agricultural Sciences] [Li Dongdong, Li Zongrui, Ding Xuemei, et al. Effects of adding exogenous protease to diets with different crude protein levels on broiler production performance, serum biochemical indicators and antioxidant function [J]. Journal of Animal Nutrition, 2015.].

[0004] Ostriches are the largest birds in the world, with advantages such as rapid growth and strong disease resistance. Furthermore, ostrich meat is characterized by high protein, low fat, low cholesterol, and low calories [Han Zhanbing, Hu Xiaoyang & Zhang Liheng. (2018). Analysis of the economic value and breeding benefits of ostriches]. The growth, development, and disease resistance of an animal are not only controlled by its own genes but also closely related to the large number of microorganisms present in its gut [Francisco Guarner; Juan-R Malagelada. Gut flora in health and disease[J].The Lancet,2003(9356)]. Therefore, we have reason to believe that the advantages of ostriches, such as rapid growth, strong disease resistance, and good meat quality, depend not only on the ostrich's genes but also on the regulation of its gut microbiota.

[0005] Inspired by the modern animal microecology theory that gut microbiota constitutes a second genome for animals, this study links the large size, strong disease resistance, and high-quality meat of ostriches to the digestive enzymes of their gut microbiota. Focusing on addressing the problem of incomplete digestion and absorption of the high protein content in broiler feed, this study creatively selected beneficial microorganisms from the ostrich gut, using digestive enzyme activity as the standard for screening. Through antibacterial experiments, stress resistance experiments (acid, bile salt, and heat tolerance), and detection of antibiotic resistance and virulence genes, a probiotic strain with protease production, good stress resistance, and safety and efficacy was screened. Feeding trials with broilers confirmed that this strain aids digestion and promotes growth, achieving the objectives of this invention. The method of this invention is easy to implement, simple to operate, and highly efficient. However, the strain's tolerance to high temperatures is relatively poor, and further research is needed to determine how to apply it in actual production. Summary of the Invention

[0006] The purpose of this invention is to provide an ostrich-derived *Bacillus sarcodactylis* strain that promotes broiler growth. This strain produces proteases and cellulases, enhancing the broiler's ability to digest these enzymes. Furthermore, this strain exhibits relatively strong acid resistance; after treatment at pH 3.0 for 2 hours, the survival rate of the spores is 72.56%, indicating its tolerance to gastric acid in the broiler's intestines, which is beneficial for colonization within the gut.

[0007] Another objective of this invention is to provide a screening method for *Bacillus sarcodactylis* from ostriches that promotes broiler growth. This method is easy to implement and simple to operate, and the isolated strain is safe, efficient, and environmentally friendly. Through enzyme production tests, antibacterial tests, acid, bile salt, and high-temperature resistance tests, as well as the detection of drug resistance and virulence genes, a probiotic strain with protease production capabilities, good stress resistance, and safety and efficacy was screened using the most suitable method.

[0008] Another objective of this invention is to provide the application of a strain of Bacillus safensis O-6 in livestock and poultry feed microecological additives, which is applied to broiler farming and improves its production efficiency.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] The screening method for an ostrich-derived *Bacillus sarcodactylis* strain that promotes broiler growth involves the following steps:

[0011] A. The samples were taken from ostrich droppings at Jiufengshan Forest Zoo in Wuhan, Hubei Province, and brought back to the laboratory for processing in an ice box.

[0012] B. Take 1g of ostrich droppings and add 9mL of sterile saline solution. Shake the bottle appropriately to mix it evenly.

[0013] C. Perform 10 -1 ~10 -4 Serial dilution (100 μL of culture medium is added to 900 μL of sterile water for serial dilution);

[0014] D. Take 10 -2 10 -3 10 -4 Three dilutions of the culture medium were spread on LB medium supplemented with 2.5% skim milk powder. The plates were incubated at 37°C for 23–25 hours. Single colonies with a clear zone around the colony were picked for purification culture.

[0015] E. Gram staining of the purified strain yields Gram-positive, short rod-shaped individuals, arranged singly, in pairs, or in chains (see...). Figure 2 ).

[0016] This invention uses enzyme production performance as a standard to isolate and screen a strain with strong protease and cellulase production capabilities from ostrich feces. After identification, the inventors determined that the obtained strain is Bacillus safensis, which they named Bacillus safensis O-6. On August 31, 2023, it was deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20231572.

[0017] Bacteriological characteristics of Bacillus safensis O-6:

[0018] Bacillus safensis O-6 grows into round colonies with smooth surfaces, regular edges, and a diameter of about 2 mm on LB medium (see...). Figure 1This bacterium is Gram-positive, and its cells are single, straight rods with blunt, rounded ends, and spores (see...). Figure 2 The 16S rRNA gene was cloned and sequenced. Blast comparison was performed using NCBI, and it was found that the 16S rRNA sequence of the strain of this invention had the highest homology (99.86%) with Bacillus sabovella (HQ699512.1).

[0019] Bacillus safensis O-6 exhibits strong reproductive capacity. It is in a lag phase from 0-1 hour, enters the logarithmic growth phase after 1-27 hours, stabilizes at 27-33 hours, rapidly increases again between 33-41 hours, and then gradually decreases after 41 hours, entering a plateau phase. The pH of the bacterial culture is 6.33. Bacillus safensis O-6 produces spores. The spores survive 72.65% after 2 hours of incubation in LB medium at pH 3.0; however, the survival rate is 0% after 2 hours of incubation in 0.3% bile salts. In the spore state, it is relatively acid-tolerant but intolerant of bile salts.

[0020] Bacillus safensis O-6 grew on a milky white LB agar plate containing 2.5% skim milk powder, with clear zones forming around the colonies, indicating that this strain produces protease (see...). Figure 3 Colonies grown by this strain on plates with sodium carboxymethyl cellulose (CMC-Na) as the sole carbon source, when stained with Congo red, showed pale yellow hydrolysis zones around the colonies (see...). Figure 4 This indicates that the strain can secrete cellulase to hydrolyze cellulose.

[0021] Bacillus safensis O-6 showed no significant antibacterial activity against Staphylococcus aureus ATCC25923, Salmonella C78-1, and Escherichia coli K88 in vitro (see [link to article]). Figure 5 It is highly sensitive to commonly used veterinary antibiotics such as penicillin, oxacillin, ampicillin, ofloxacin, norfloxacin, ceftriaxone, doxycycline, tetracycline, erythromycin, gentamicin, streptomycin, and amikacin, proving that there is no risk of spreading drug resistance genes.

[0022] The application of an ostrich-derived *Bacillus salsa* strain that promotes broiler growth in livestock and poultry feed microecological additives involves the following steps:

[0023] A. Preparation of seed culture: Streaking of the strain on a plate and incubation at 37℃ for 24h. Pick the isolated strain and incubate in 3mL of LB liquid medium at 37℃ and 200r / min for 12h to obtain the seed culture.

[0024] B. Preparation of fermentation broth: Inoculate the seed culture at a rate of 1% into 3 mL of LB liquid medium and incubate at 37℃ and 200 r / min for 12 h to obtain the fermentation broth. The viable cell count is 4.74 × 10⁻⁶. 7 CFU / mL.

[0025] C. Add 1 ml of the fermentation liquid per chick per day to their daily drinking water for free consumption. This method can reduce the stress response caused by gavage to chicks.

[0026] The applicant conducted a broiler chicken feeding experiment using the strain of this invention through drinking water, and found that it has a significant growth-promoting effect on broilers.

[0027] A strain with strong enzyme-producing ability (especially protease production) was isolated and screened from the feces of ostriches, a large bird species, for application in broiler farming to improve production efficiency. This isolated strain is safe, efficient, and environmentally friendly. The probiotic strain isolated in this invention was identified as *Bacillus safensis* by 16S rRNA testing. The enzyme production performance (protease, cellulase, amylase, etc.), safety, stress resistance, and growth-promoting effect on broilers as a feed additive were studied.

[0028] This invention screened a strain of Bacillus safensis O-6 (capable of producing both protease and cellulase) from fresh feces of healthy ostriches, which has a significant growth-promoting effect on broilers. Experiments such as probiotic performance testing, stress resistance testing, safety evaluation, and broiler feeding showed that this strain can be used as a growth-promoting microecological feed additive in broiler farming.

[0029] Compared with the prior art, the present invention has the following effects and advantages:

[0030] (1) It is a strain that was isolated from the feces of large birds, such as ostriches, and has a strong ability to produce cellulase and protease.

[0031] (2) The strain of the present invention produces spores and has strong stability; after treatment at pH 3.0 for 2 hours, the survival rate is 72.65%, which is beneficial for preparing microecological preparations and for use as feed additives in production.

[0032] (3) Animal experiments have shown that the strain of the present invention has a significant growth-promoting effect on broiler chicks, thus achieving the ultimate goal of the present invention.

[0033] For more detailed technical solutions, please refer to the "Specific Implementation Plan". Attached Figure Description

[0034] Figure 1This is a schematic diagram of the growth status of Bacillus safensis O-6, an ostrich-derived bacillus that promotes broiler growth, on LB agar plates.

[0035] Figure 2 This is a schematic diagram (×1000) of Gram staining results of Bacillus safensis O-6, an ostrich-derived bacillus that promotes broiler growth.

[0036] Figure 3 This is a schematic diagram showing the protease production detection results of Bacillus safensis O-6, an ostrich-derived bacterium that promotes broiler growth.

[0037] Figure 4 This is a schematic diagram showing the cellulase production detection results of Bacillus safensis O-6, an ostrich-derived bacterium that promotes broiler growth.

[0038] Figure 5 This is a schematic diagram showing the in vitro antibacterial results of Bacillus safensis O-6, an ostrich-derived bacterium that promotes broiler growth.

[0039] Figure 5 A: Escherichia coli K88ac is an indicator bacterium. Figure 5 B: Staphylococcus aureus ATCC25923 is an indicator bacterium. Figure 5 C: Salmonella C78-1 is an indicator bacterium.

[0040] Figure 6 This is a bacterial phylogenetic tree constructed using the neighbor-joining method for Bacillus safensis O-6, an ostrich-derived bacterium that promotes broiler growth. T-6 is the strain O-6 of this invention.

[0041] Figure 7 A gel image for detecting the virulence gene of Bacillus safensis O-6, an ostrich-derived bacterium that promotes broiler growth.

[0042] M: DL2000 DNA molecular weight standard; Lane 1: Positive control; Lane 2: Bacillus sabolicii O-6 of the present invention; Lane K: Negative control. Figure 7 A: Results of detection of the virulence gene nheA; Figure 7 B: Results of detection of the virulence gene nheB; Figure 7 C: Results of detection of the virulence gene nheC; Figure 7 D: Results of detection of the virulence gene entFM.

[0043] Figure 8 This is a schematic diagram of the growth curve of Bacillus safensis O-6, an ostrich-derived bacillus that promotes the growth of broilers. Detailed Implementation Plan

[0044] Example 1: Isolation and identification of Bacillus safensis O-6

[0045] A screening method for ostrich-derived *Bacillus sarcodactylis* that promotes broiler growth, comprising the following steps:

[0046] A. The samples were taken from ostrich droppings at Jiufengshan Forest Zoo in Wuhan, Hubei Province, and brought back to the laboratory for processing in an ice box.

[0047] B. Take 1g of ostrich droppings and add 9mL of sterile saline solution. Shake the bottle appropriately to mix it evenly.

[0048] C. Perform 10 -1 ~10 -4 Serial dilution (100 μL of culture medium is added to 900 μL of sterile water for serial dilution);

[0049] D. Take 10 -2 10 -3 10 -4 Three dilutions of the culture medium were spread on LB medium supplemented with 2.5% skim milk powder. The plates were incubated at 37°C for 23–25 hours. Single colonies with a clear zone around the colony were picked for purification culture.

[0050] E. Gram staining of the purified strain yields Gram-positive, short rod-shaped individuals, arranged singly, in pairs, or in chains (see...). Figure 2 A strain of Bacillus safensis O-6 was obtained.

[0051] Strains and species identification

[0052] Based on the above identification, the 16S rRNA gene sequence of the isolate O-6 of this invention was further detected. Bacillus safensis O-6 contains the nucleotide sequence shown in SEQ ID NO: 1, with a length of 1453 bp. The species to which strain O-6 belongs was confirmed and identified, and the specific steps are as follows:

[0053] (I) Amplification of the 16S rRNA gene:

[0054] The 16S rRNA sequence of isolate O-6 was amplified using universal bacterial primers. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their DNA sequences are as follows:

[0055] Forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3,

[0056] Reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3';

[0057] The PCR amplification system is shown in Table 1. Amplification conditions: 95℃ pre-denaturation for 5 min, 95℃ for 50 s, 55℃ for 35 s, 72℃ for 1 min, 30 cycles, followed by 72℃ extension for 10 min. The PCR amplification products were analyzed by electrophoresis on a 0.8% agarose gel (containing ethidium bromide). The amplified fragment size was consistent with expectations, approximately 1500 bp.

[0058] Table 1. PCR amplification system of strain O-6

[0059]

[0060] (II) Constructing a bacterial phylogenetic tree to determine species and genus:

[0061] Bacterial genomic DNA was extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were BLASTed on NCBI, and a bacterial phylogenetic tree was constructed using MEGA 7.0 software via neighbor-joining (see [link to MEGA 7.0]). Figure 6 Analysis results showed that strain O-6 was Bacillus safensis.

[0062] Example 2:

[0063] The application of Bacillus safensis O-6 in livestock and poultry feed microecological additives involves the following steps:

[0064] A. Preparation of seed culture: Streaking of the strain on a plate and incubation at 37℃ for 24h. Pick the isolated strain and incubate in 3mL of LB liquid medium at 37℃ and 200r / min for 12h to obtain the seed culture.

[0065] B. Preparation of fermentation broth: Inoculate the seed culture at a rate of 1% into 3 mL of LB liquid medium and incubate at 37℃ and 200 r / min for 12 h to obtain the fermentation broth. The viable cell count is 4.74 × 10⁻⁶. 7 CFU / mL.

[0066] C. Add 1 ml of the fermentation liquid per chick per day to their daily drinking water for free consumption. This method can reduce the stress response caused by gavage to chicks.

[0067] Example 3: Detection of the probiotic properties of Bacillus safensis O-6

[0068] I. Enzyme production test

[0069] (I) Detection of protease activity

[0070] Bacillus safensis O-6 was inoculated onto a plate with milk as the sole carbon source and incubated at 37°C for 24 hours. If protease was produced, the proteins surrounding the colonies would decompose, resulting in a clear zone on the plate. The results were as follows: Figure 3 As shown in Table 2, the results of the protease activity detection of the strain are presented.

[0071] Table 2. Results of protease production by Bacillus safensis O-6

[0072]

[0073] (II) Detection of cellulase activity

[0074] Bacillus safensis O-6 was inoculated onto a plate using sodium carboxymethyl cellulose (CMC-Na) as the sole carbon source and incubated at 37°C for 24 hours. Staining with Congo red revealed pale yellow hydrolysis zones around the colonies (see...). Figure 4 This indicates that the strain can secrete cellulase to hydrolyze cellulose. The cellulase activity detection results of Bacillus safensis O-6 are shown in Table 3.

[0075] Table 3. Detection results of cellulase production by Bacillus safensis O-6

[0076]

[0077] (III) Bacillus safensis O-6 does not produce amylase and lipase.

[0078] II. Antibacterial activity test of Bacillus safensis O-6

[0079] Table 4. Antibacterial test of fermentation supernatant of bacterial strains

[0080]

[0081] Example 4: Safety evaluation of Bacillus safensis O-6

[0082] I. Drug sensitivity test

[0083] Twelve antimicrobial susceptibility test discs (purchased from Hangzhou Microbial Reagent Co., Ltd.) were selected for testing, including penicillins, cephalosporins, quinolones, aminoglycosides, tetracyclines, and sulfonamides. The test interpretation criteria were based on the latest version of the NCCLS standards provided by the World Health Organization (WHO) (2018 edition). The specific steps are as follows:

[0084] (1) The bacterial culture was inoculated into LB liquid medium at a rate of 1% and cultured overnight at 37°C and 200 rpm / min on a shaker.

[0085] (2) Use an OD meter to measure the OD of the bacterial culture. 600 Set the value to 1;

[0086] (3) Use a sterile cotton swab to dip the bacterial solution and spread it on the LB agar plate. Rotate the plate 60 degrees each time, and finally spread it around the inner edge of the plate twice. Repeat this several times to ensure that the solution is evenly spread.

[0087] (4) 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 stick them on the plate surface. Once the test strips are stuck on, they should not be picked up again. Stick 4 test strips on each plate, with a spacing of not less than 45 mm between each test strip and a center distance of not less than 20 mm from the edge of the plate.

[0088] (5) Place the plate in a 37℃ constant temperature incubator for 10-12 hours and observe the results.

[0089] The experimental results are shown in Table 5. The Bacillus safensis O-6 strain of the present invention is relatively sensitive to commonly used veterinary drugs such as penicillin, oxacillin, ampicillin, ofloxacin, norfloxacin, ceftriaxone, doxycycline, tetracycline, erythromycin, gentamicin, streptomycin, and amikacin.

[0090] Table 5. Results of antibiotic susceptibility testing of Bacillus safensis strain O-6.

[0091]

[0092] Note: S indicates sensitive, M indicates moderately sensitive, and R indicates resistant.

[0093] II. Virulence Gene Detection Test

[0094] Bacillus cereus AH7-7, containing the non-hemolytic enterotoxin Nhe gene (nheA, nheB, nheC) and the enterotoxin FM gene entFM, was used as a positive control strain to detect the virulence genes related to the target strain.

[0095] Using the genomes of the target strains prepared above as templates, PCR amplification was performed using specific primers for each virulence gene. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PCR reaction conditions were: 95℃ for 10 min; 95℃ for 15 s, 55℃ for 15 s, 72℃ for 40 s, 35 cycles; 72℃ for 5 min (Rowan 2003). The primer sequences and expected PCR product sizes for the virulence genes are shown in Table 6, and the PCR amplification system for the virulence genes is shown in Table 7.

[0096] Table 6 Primer sequences for PCR amplification of virulence genes

[0097]

[0098] Table 7. PCR amplification system for virulence genes

[0099]

[0100] Note: The negative control bacterial solution was replaced with 1 μL ddH2O.

[0101] PCR products were electrophoresed on a 1.0% agarose gel (containing ethidium bromide), and observed and photographed using a gel imaging system. The electrophoresis results showed that positive strains amplified four virulence genes: nheA, nheB, nheC, and entFM. The sizes of the amplified fragments were consistent with expectations. No enterotoxin-related virulence genes were detected in strain O-6. (See attached results). Figure 7 .

[0102] The results of the above-mentioned drug susceptibility tests and virulence gene detection collectively demonstrate the safety of Bacillus safensis O-6 and its applicability in actual production.

[0103] Example 5: Stress resistance and growth characteristics of Bacillus safensis O-6

[0104] I. Stress Resistance Test

[0105] (a) Bile salt tolerance test

[0106] (1) Prepare 0.3% bile salt medium: Add 0.3% porcine bile salt to LB liquid culture and sterilize at 121℃ for 15 min;

[0107] (2) Spore preparation: A small amount of isolated and preserved strains were picked with a sterile inoculation loop, streaked on LB agar plates, and incubated at 37°C for 24 h. Single colonies were picked and inoculated into LB liquid medium and incubated at 37°C until the bacteria reached the logarithmic growth phase. The culture was incubated in an 80°C water bath for 15 min, and the spores were collected by centrifugation (3000 r / min, 5 min). The bacterial cells were washed twice with phosphate buffer and finally reconstituted in PBS buffer to obtain the spore suspension. (3) 100 μL of the spore suspension was added to 900 μL of LB liquid medium containing 0.3% porcine bile salt and blank LB medium, respectively, and incubated at 37°C for 2 h.

[0108] (4) Remove the bacterial culture, vortex it for 40 seconds, and dilute it to 10-fold serial dilution. -5 Take 10 -3 10 -4 10 -5 For each of the three dilution gradients, take 50 μL of each and add it evenly to a 60 mm Petri dish. Then, pour 6–8 mL of LB solid medium cooled to approximately 50 °C into the Petri dish. Gently shake the Petri dish back and forth and side to side to ensure even distribution of the bacterial culture in the solid medium. At least three replicate Petri dishes are needed for each dilution gradient to reduce errors caused by random factors. Incubate at 37 °C for 24 hours.

[0109] (5) The colonies cultured on ordinary LB medium and those treated with 0.3% porcine bile salts and then cultured on LB medium were plate counted and the survival rate was calculated.

[0110] (II) Acid Resistance Test

[0111] (1) Prepare acidic LB medium: Adjust the pH of LB liquid medium to 3.0 with sodium hydroxide solution and dilute hydrochloric acid, and sterilize at 121℃ for 15 min;

[0112] (2) Preparation of bacterial suspension: A small amount of isolated and preserved bacterial strain was picked up with a sterile inoculation loop and streaked on an LB agar plate. The plate was incubated at 37°C for 24 h. A single colony was picked and inoculated into LB liquid medium. The culture was incubated at 37°C until the bacteria reached logarithmic growth. The culture was incubated in an 80°C water bath for 15 min. The spores were collected by centrifugation (3000 r / min, 5 min). The bacterial cells were washed twice with phosphate buffer and finally reconstituted in PBS buffer to obtain the spore suspension. (3) 100 μL of the spore suspension was added to 900 μL of LB liquid medium (pH 3.0) and blank LB medium, respectively. The plates were incubated at 37°C for 2 h.

[0113] (4) Remove the bacterial culture, vortex it for 40 seconds, and dilute it to 10-fold serial dilution. -5 Take 10 -3 10 -4 10-5 For each of the three dilution gradients, take 50 μL of each and add it evenly to a 60 mm Petri dish. Then, pour 6–8 mL of LB solid medium cooled to approximately 50 °C into the Petri dish. Gently shake the Petri dish back and forth and side to side to ensure even distribution of the bacterial culture in the solid medium. At least three replicate Petri dishes are needed for each dilution gradient to reduce errors caused by random factors. Incubate at 37 °C for 24 hours.

[0114] (5) The colonies cultured on ordinary LB medium and on LB medium at pH 3.0 were plate counted and the survival rate was calculated.

[0115] The survival rate of the strains to bile salts / acids was calculated using the following formula: Survival rate (%) = Number of viable bacteria after treatment / Number of viable bacteria without treatment × 100%; the results are shown in Table 8.

[0116] Table 8 Results of Bacillus safensis O-6 bile salt / acid resistance test

[0117]

[0118] II. Determination of Growth Curve

[0119] (1) 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;

[0120] (2) Preparation of fermentation broth: The seed liquid was inoculated into 3 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;

[0121] (3) Determination of growth curve: In a 100-well honeycomb plate, one well was inoculated with 150 μL of LB liquid medium as a blank control. Seed culture was inoculated at a rate of 1% into 150 μL of LB liquid medium, with five replicates. The plates were placed in an automated growth curve analyzer. The bacterial suspension was automatically measured at OD values ​​every hour. 600 The absorbance value is calculated by subtracting the OD value of the control group from the OD value of the experimental group. The OD value of the bacterial culture is obtained by plotting the culture time on the x-axis. 600 The values ​​are plotted on the ordinate to create a growth curve for the strain.

[0122] Experiments show that the strain isolated in this invention has strong reproductive capacity. It is in a lag phase from 0-1 h, enters the logarithmic growth phase after 1-27 h, stabilizes at 27-33 h, rapidly increases again between 33-41 h, and then gradually decreases in growth rate after 41 h, entering a plateau phase. This strain has strong reproductive capacity, which is beneficial for large-scale industrial fermentation production. Its growth curve is shown in [Figure showing growth curve]. Figure 8 .

[0123] Example 6: Broiler Feeding Trial

[0124] The liquid fermentation preparation of the probiotic strain O-6 of the present invention was fed to broilers via drinking water to verify the growth-promoting effect of the probiotic strain of the present invention on broilers.

[0125] I. Experimental Animals and Grouping

[0126] Seventy-two one-day-old healthy Kuangda Yellow Chickens (a hybrid of Sanhuang Chicken and white-feathered broiler chicken) were selected as experimental animals. A single-factor completely randomized design was used, randomly dividing the chickens into two groups, with four replicates per group and nine chickens per replicate. Group C served as the blank control group, fed a basal diet; the Bacillus safensis O-6 group was fed a basal diet plus 1 ml / (chicken·day) of O-6 liquid fermentation preparation.

[0127] Preparation of liquid fermentation broth for *Bacillus sabolicii* O-6: The strain was streaked onto plates and incubated at 37°C for 24 h. The isolated strain was picked and incubated in 3 mL of LB broth at 37°C and 200 rpm for 12 h to obtain the seed culture. The seed culture was then inoculated at a rate of 1% into 3 mL of LB broth and incubated at 37°C and 200 rpm for 12 h to obtain the fermentation broth. The viable cell count was 4.74 × 10⁻⁶. 7 CFU / mL.

[0128] The trial period was 35 days. Chickens were fed a brooding diet from 1 to 21 days old and a growing diet from 22 to 35 days old. The diet consisted of broiler compound feed (510, 511) purchased from CP Group Co., Ltd.

[0129] II. Experimental Procedure

[0130] This experiment was conducted at the experimental chicken farm of Huazhong Agricultural University in Wuhan, Hubei Province. The broilers were raised in four-tiered stacked cages, and the feeding period was from March 2, 2023 to April 6, 2023, a total of 35 days. During the experiment, the chickens had free access to water and feed. Probiotic liquid was fed at 8:30 AM daily, and chicken feed was added at 3:00 PM. In the early stages of rearing, the broilers were chicks, and the ambient temperature was controlled at around 35℃, decreasing by 2-3℃ weekly until it reached around 24℃. Humidity and temperature were adjusted according to the actual growth conditions during the experiment.

[0131] III. Measurement Indicators

[0132] 1. Production performance

[0133] The production performance indicators of broilers, including average daily weight gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR), were collected at 7d, 14d, 21d, 28d, and 35d.

[0134] The effect of Bacillus safensis O-6 of the present invention on broiler growth was examined by measuring changes in body weight. The weight was measured every 7 days during the feeding period, and the average daily weight gain and feed conversion ratio were calculated.

[0135] 2. Organ Index

[0136] Broilers were euthanized on day 35, and their livers, spleens, thymus, bursa of Fabricius, pectoral muscles, and leg muscles were collected, weighed, and their organ indices were calculated. The duodenum, jejunum, ileum, and cecum were collected for length measurement.

[0137] 3. Intestinal tissue morphology

[0138] Paraffin sections were prepared and stained with hematoxylin and eosin (HE). The morphology of the intestinal tissue was observed under an electron microscope. The height of the intestinal villi and the depth of the intestinal crypts were measured, and the ratio of villi height to crypt depth was calculated.

[0139] IV. Results Analysis

[0140] 1. Production performance

[0141] Table 9 shows that after 35 days of experimentation, the average daily weight gain in the control group was 26.63g, while the average daily weight gain in the experimental group was 28.54g. The average daily weight gain in the experimental group was significantly higher than that in the control group (P<0.05). Table 10 shows that there was no significant difference in the feed conversion ratio between the control group and the blank control group.

[0142] Table 9 Comparison of growth performance of broilers

[0143]

[0144] Note: If the same letter in the same row has the same or no letter inscription, the difference is not significant (P>0.05); if the letter inscription is different (lowercase), the difference is significant (p<0.05); and if the letter inscription is different (uppercase), the difference is extremely significant (p<0.01). The same applies to the following table.

[0145] Table 10 Feed conversion ratio

[0146]

[0147] 2. Organ Index

[0148] After 35 days of experimentation, the spleen index, bursa of Fabricius index, ileum length and pectoral muscle index of the experimental group were significantly higher than those of the blank control group (p<0.05) (see Table 111).

[0149] Table 11 Comparison of broiler slaughter performance

[0150]

[0151] Note: If the same letter in the same row has the same or no letter inscription, the difference is not significant (P>0.05); if the letter inscription is different (lowercase), the difference is significant (p<0.05); and if the letter inscription is different (uppercase), the difference is extremely significant (p<0.01). The same applies to the following table.

[0152] 3. Intestinal tissue morphology

[0153] After 35 days of experimentation, the height of the duodenal villi in the experimental group was significantly higher than that in the blank control group (p<0.05) (see Table 12).

[0154] Table 12. Duodenal villus height in broiler chickens

[0155]

[0156] Throughout the trial period, compared with the control group, the addition of probiotic strains resulted in an average daily weight gain increase of 7.16%, spleen weight increase of 31.18%, bursa of Fabricius weight increase of 44.20%, pectoral muscle weight increase of 28.53%, ileum length increase of 15.02%, and duodenal villus height increase of 13.88% in the experimental group. This demonstrates that the addition of the probiotic Bacillus safensis O-6 of this invention to the diet has a significant effect on improving the growth performance of broilers.

Claims

1. A strain of *Bacillus sarcodactylis* derived from ostriches that promotes broiler growth, characterized by: The Bacillus safensis O-6 strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20231572; the Bacillus safensis O-6 strain contains the nucleotide sequence shown in SEQ ID NO:

1.

2. The application of *Bacillus sarcodactylis* from ostriches according to claim 1 in the preparation of a microecological additive for chicken feed, comprising the following steps: A. Preparation of seed culture: streaking the strain on a plate, culturing at 37℃ for 24h, picking the isolated strain and culturing in 3mL of LB liquid medium at 37℃ and 200r / min for 12h to obtain the seed culture; B. Preparation of fermentation broth: inoculating the seed culture at a 1% inoculum into 3mL of LB liquid medium, culturing at 37℃ and 200r / min for 12h to obtain the fermentation broth, with a viable count of 4.74×10⁻⁶. 7 CFU / mL.

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