Function and application of a lactic acid bacteria ligilactobacillus salivarius SunLab-GHL02

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a lactic acid bacteria strain and application thereof. The strain is Ligilactobacillus salivarius SunLab-GHL02 (L. salivarius GHL02 for short), which is isolated from the lungs of high-performance Arbor Acres broilers, has been preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20251726. The classification status and functional characteristics of the strain are determined through morphological, physiological and biochemical, 16S rRNA and whole genome identifications. The strain has good acid tolerance, bile salt tolerance and acid production capacity, and can significantly inhibit pathogenic bacteria such as Escherichia coli, pathogenic E. coli, Salmonella, Staphylococcus aureus and Pseudomonas aeruginosa. Animal experiments show that atomization administration of the strain can improve the growth performance and feed conversion rate of poultry, enhance the stress resistance and promote health. The strain and its application can be widely used in the development of feed additives, probiotic preparations and animal health products, and are suitable for improving the health and production performance of poultry under intensive breeding conditions, and have good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a lactic acid bacterium, Ligilactobacillus salivarius SunLab-GHL02, isolated from the lungs of high-productivity broilers, and its application in improving poultry health and production performance. Background Technology

[0002] Under intensive poultry farming conditions, high-density rearing, environmental stress, and excessive pathogen loads often lead to respiratory and digestive diseases, resulting in decreased growth performance, reduced feed utilization, weakened immunity, and increased mortality, severely impacting farming efficiency and industry development. Existing prevention and control measures include vaccination, antibiotic intervention, and improved feeding management; however, under the "antibiotic-free farming" policy, traditional methods relying on antibiotics are no longer applicable.

[0003] Lactic acid bacteria, as typical probiotics, can maintain the balance of the gut microbiota and promote animal health through mechanisms such as acid production, the production of antibacterial substances, competitive rejection of pathogens, and immune regulation. Currently, commercially available lactic acid bacteria preparations primarily focus on improving the intestinal microbial environment and are typically administered via feed or drinking water. However, research on respiratory-derived lactic acid bacteria remains limited, and their potential applications in respiratory colonization, inhibition of respiratory pathogens, and immune regulation have not yet been fully explored.

[0004] Respiratory diseases have a high incidence rate in poultry farming and are easily exacerbated by environmental stress. Current technologies lack effective methods for isolating and screening dominant probiotics from the poultry respiratory tract and directly delivering them to the respiratory tract via atomization or other methods. Therefore, there is an urgent need to develop a strain of lactic acid bacteria derived from the respiratory tract with stable probiotic functions, combined with atomization strategies, to achieve synergistic improvement in respiratory and digestive health, thereby enhancing overall poultry production performance and providing a new technological pathway for antibiotic-free farming. Summary of the Invention

[0005] This invention addresses the problems in intensive poultry farming caused by high-density rearing, environmental stress, and excessive pathogen loads, including frequent respiratory and digestive diseases, reduced growth performance, decreased feed conversion efficiency, and increased mortality. It provides a strain of lactic acid bacteria, *Ligilactobacillus salivarius* SunLab-GHL02, derived from the lungs of high-yield broilers, and its application method. This invention aims to improve poultry health and production performance, thereby increasing farming efficiency and reducing the burden of husbandry and management.

[0006] The core technical solution of this invention lies in: lactic acid bacteria strains isolated from the lungs of high-yield broiler chickens, which, after screening, demonstrate superior characteristics in broad-spectrum antibacterial ability, environmental tolerance, respiratory colonization ability, and immunomodulatory effects, and are then applied to poultry health management. Compared with traditional intestinal-derived lactic acid bacteria, the strains described in this invention exhibit natural adaptability to the respiratory environment, effectively inhibiting the invasion of respiratory pathogens, reducing respiratory damage caused by environmental stress, and promoting overall health by regulating the host's immune response.

[0007] Therefore, the technical problem to be solved by the present invention is to provide a strain of lactic acid bacteria derived from the lungs of poultry. This strain has excellent probiotic characteristics and applicability. Under antibiotic-free farming conditions, it can effectively improve the growth performance and feed conversion rate of poultry, enhance the body's immune function, and regulate and optimize the microbial community structure, thereby providing a safe, effective and scalable technical solution for intensive poultry farming.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] 1. Provide a strain of lactic acid bacteria Ligilactobacillus salivarius SunLab-GHL02 (hereinafter referred to as L. salivarius GHL02) derived from the lungs of high-productivity broiler chickens. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on July 30, 2025, with accession number CCTCC NO: M 20251726.

[0010] 2. Morphological observation, physiological and biochemical characteristic analysis, 16S rRNA sequence identification, and whole-genome sequencing were performed on the strain to confirm its taxonomic classification and functional characteristics. The results showed that *L. salivarius* GHL02 possesses the following advantages:

[0011] (1) Broad-spectrum antibacterial ability: It shows significant inhibitory effects on common poultry pathogens such as Staphylococcus aureus, Salmonella, pathogenic Escherichia coli and Pseudomonas aeruginosa.

[0012] (2) Strong environmental tolerance: It can tolerate acidic conditions (pH≥3.0) and different salt concentrations, and adapt to the complex ecological conditions of feed processing, storage and host respiratory and digestive tracts.

[0013] (3) Suitable acid production level: It can effectively inhibit the growth of harmful bacteria and promote the colonization of beneficial bacteria, thus maintaining the homeostasis of the host's respiratory and digestive tract microecology.

[0014] (4) Probiotic properties and safety: Whole genome analysis shows that it contains abundant probiotic-related genes, does not carry virulence factors or drug resistance genes, and is safe and reliable.

[0015] 3. The present invention also provides a specific technical solution for applying L. salivarius GHL02 to broilers, which is atomized: the live bacteria preparation is atomized into an aerosol with a particle size of 1–5 μm and sprayed evenly into the poultry breeding environment so that the poultry can inhale it through the respiratory tract, thereby achieving direct intervention of respiratory microorganisms.

[0016] 4. Through the implementation of the above technical solutions, the experimental results show that the application of L. salivarius GHL02 can significantly improve the feed conversion rate, production performance and immune function of broilers.

[0017] 5. Beneficial effects

[0018] The present invention has the following beneficial effects:

[0019] (1) Prominent antibacterial effect: It can effectively inhibit a variety of respiratory and digestive pathogens, reducing the risk of disease occurrence and transmission.

[0020] (2) The source is clear and safe: it comes from the lungs of healthy, high-productivity broilers, does not contain toxic factors or drug resistance genes, and meets the requirements for antibiotic-free farming.

[0021] (3) Strong adaptability: It can maintain its activity in acidic and high-salt environments, making it suitable for processing, storage and complex ecological environments of the host.

[0022] (5) Promote production performance and economic benefits: It can significantly increase the daily weight gain of broilers, improve feed utilization, thereby reducing feeding costs and improving overall breeding efficiency.

[0023] (6) Enhances immunity and anti-stress ability: In the LPS challenge test, it showed the effect of reducing weight suppression and inflammatory response, indicating that it helps to relieve acute stress and enhance the body's immune regulation ability.

[0024] (7) Diverse application methods: It can be applied through atomization, drinking water, feed addition and various dosage forms (such as powder, freeze-dried powder, granules, etc.) to adapt to poultry farming of different scales and models.

[0025] (8) Meets the requirements of antibiotic-free and green farming: It can improve the health and production performance of poultry without relying on antibiotics, reduce the risk of drug-resistant bacteria and drug residue problems, and is in line with the direction of sustainable development.

[0026] (9) High industrialization and promotion value: The strain has the characteristics of strong stability and easy large-scale preparation, and is suitable for widespread application in broiler, laying hen and breeding poultry farming, with broad market prospects.

[0027] In summary, the lung-derived lactic acid bacteria described in this invention are not only reliable in origin and highly safe, but also exhibit superior performance in antibacterial activity, immune regulation, and microecological optimization. Through flexible application methods, this strain can significantly improve the health and production efficiency of poultry in the context of antibiotic-free farming, thus providing a practical technical solution for modern poultry industry and possessing high potential for industrialization. Attached Figure Description

[0028] Figure 1 A schematic diagram showing the results of L. salivarius GHL02 isolation, purification, and Gram staining;

[0029] Figure 2 The image shows the drug susceptibility test results for L. salivarius GHL02, where labels 1-6 represent: 1: penicillin; 2: chloramphenicol; 3: erythromycin; 4: clindamycin hydrochloride; 5: cephalexin; 6: ciprofloxacin.

[0030] Figure 3 The graph shows the antibacterial effects of L. salivarius GHL02 supernatant and bacterial suspension on pathogens. The pathogens are, in order: Escherichia coli control (con), pathogenic Escherichia coli, Pseudomonas aeruginosa, Salmonella, and Staphylococcus aureus. Inhibition zones 1, 2, and 3 represent the antibacterial effect of the supernatant, while inhibition zones 4, 5, and 6 represent the antibacterial effect of the bacterial suspension.

[0031] Figure 4 A complete genome sequencing diagram of L. salivarius GHL02;

[0032] Figure 5 COG annotation diagram of the gene encoding the functional protein of L. salivarius GHL02;

[0033] Figure 6 KEGG annotation diagram of the gene encoding a functional protein in L. salivarius GHL02;

[0034] Figure 7 GO annotation diagram of the gene encoding the functional protein of L. salivarius GHL02;

[0035] Figure 8 Annotation diagram of the NR gene of L. salivarius GHL02;

[0036] Figure 9 Annotation diagram of the CAZymes gene encoding a functional protein in L. salivarius GHL02;

[0037] Figure 10Annotation results for L. salivarius GHL02 based on the Pfam structural domain;

[0038] Figure 11 The average body weight of broilers at 7 days (A), 14 days (B), 21 days (C), 28 days (D), 35 days (E) and 42 days (F) in the L. salivarius GHL02 atomization test;

[0039] Figure 12 The average daily weight gain of broilers aged 1-7 days (A), 7-14 days (B), 14-21 days (C), 21-28 days (D), 28-35 days (E) and 35-42 days (F) in the L. salivarius GHL02 atomization test;

[0040] Figure 13 The average daily feed intake of broilers aged 1-7 days (A), 7-14 days (B), 14-21 days (C), 21-28 days (D), 28-35 days (E) and 35-42 days (F) in the L. salivarius GHL02 atomization test;

[0041] Figure 14 Feed-to-meat ratio in L. salivarius GHL02 atomization test;

[0042] Figure 15 Body weight changes during acute LPS stress in L. salivarius GHL02 nebulization test. (A) Body weight before LPS injection at 36 days of age; (B) Body weight 24 hours after LPS injection at 37 days of age; (C) Body weight changes at 36-37 days of age. Detailed Implementation

[0043] The technical solutions involved in this application will be described in detail below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments that can be obtained by those skilled in the art without departing from the present invention are also within the scope of protection of this application.

[0044] Example 1: Isolation and purification of L. salivarius GHL02 lung-derived from high-yield broiler chickens

[0045] 1.1 Sample Collection

[0046] In this embodiment of the invention, 10 43-day-old roosters weighing 3.3–3.6 kg were obtained from a large-scale broiler farm, and their lung tissue was aseptically obtained and homogenized using a tissue grinding instrument as the original material for strain isolation.

[0047] 1.2 Preparation of MRS culture medium

[0048] (1) MRS liquid culture medium (1L): 10g peptone, 5g yeast powder, 20g glucose, 10g beef extract, 5g NaAc, 1 mL Tween 80, 1L distilled water, adjust pH to 6.2, autoclave at 121℃ for 15 min, and cool for later use.

[0049] (2) MRS solid medium (1L): Add 15g of agar powder to the above liquid medium, autoclave at 121℃ for 15min, cool to about 50℃, pour the plate, and invert it after solidification for later use.

[0050] 1.3 Enrichment Culture

[0051] The lung tissue homogenate mixture was inoculated into MRS liquid medium for enrichment culture and incubated at 37°C for 24 hours.

[0052] 1.4 Plate Separation

[0053] The enriched lung tissue homogenate mixture was serially diluted and directly spread onto MRS solid medium plates, and incubated at 37°C for 24 hours.

[0054] 1.5 Colony selection and preliminary observation

[0055] Observe the colony morphology in the petri dish, and record single colonies based on their color, state, transparency, smoothness, and size. Lactic acid bacteria colonies are mostly round or oval, with neat edges, 1-3 mm in size, pale white in color, and smooth on the surface.

[0056] 1.6 Monoclonal Purification

[0057] Single colonies with distinct morphological differences were selected and inoculated into the corresponding liquid culture medium, and the colonies were purified by single cloning for three generations.

[0058] 1.7 Morphological and Biochemical Identification

[0059] (1) When picking colonies, select colonies with different shapes, sizes or colors to ensure strain diversity.

[0060] (2) The purified strain was Gram stained and observed to be purple under a microscope, indicating that it was Gram positive.

[0061] 1.8 Strain identification

[0062] By observing the morphology and performing Gram staining tests, randomly selected colonies in the culture dish were identified, and strains that showed positive staining were tentatively classified as lactic acid bacteria. The lactic acid bacteria finally screened by this method were named Ligilactobacillus salivarius GHL02 (abbreviated as L. salivarius GHL02).

[0063] 1.9 Strain Characterization

[0064] The morphological characteristics of L. salivarius GHL02 are shown in Table 1, and colony photographs are shown in […]. Figure 1 .

[0065] Table 1. Colony morphology observation and Gram staining results of L. salivarius GHL02

[0066]

[0067] This strain was subsequently used for drug susceptibility testing, antibacterial experiments, acid and salt tolerance and acid production capacity testing, as well as 16S rRNA gene sequencing and whole genome analysis.

[0068] Example 2: Drug susceptibility assay of L. salivarius GHL02

[0069] To assess the drug susceptibility of the strain L. salivarius GHL02 of this invention, six commonly used antimicrobial agents—penicillin, chloramphenicol, erythromycin, ciprofloxacin, cephalexin, and clindamycin hydrochloride—were selected, and drug susceptibility testing was conducted using the disk agar diffusion method. The concentrations of each drug were set according to the NCCLS (CLSI) antimicrobial susceptibility standards.

[0070] 2.1 Preparation of main materials and culture media

[0071] (1) MRS medium: consistent with Example 1.2;

[0072] (2) Filter paper discs: 6mm diameter circles, sterilized at 121℃ for 15 min, then dried overnight in a 65℃ oven for later use;

[0073] (3) Activation of strain: L. salivarius GHL02 was inoculated in MRS liquid medium and cultured anaerobically at 37℃ for 24h, and activated for 2 generations;

[0074] (4) Preparation of test bacterial solution: Dilute the bacterial solution to a growth concentration of approximately 9 × 10⁻⁶. 8 CFU / mL, used for drug sensitivity testing.

[0075] 2.2 Drug sensitivity test procedure

[0076] (1) Take 100 μL of the test bacterial solution and spread it evenly on the surface of the MRS solid plate to ensure uniform distribution;

[0077] (2) Place the filter paper that has been soaked in the medicine solution and dried onto the surface of the plate with sterile tweezers and gently flatten it;

[0078] (3) The spacing between each sheet of paper is ≥24 mm, and the center of each sheet of paper is ≥15 mm from the edge of the flat plate;

[0079] (4) After incubation at 37℃ for 8-18 hours, remove the plate and measure the diameter of the inhibition zone using calipers. The inhibition zone should be perfectly circular and include the diameter of the paper. The edge of the inhibition zone should be such that no visible bacterial growth is visible to the naked eye.

[0080] 2.3 Test Results

[0081] The drug sensitivity results for L. salivarius GHL02 are shown in Table 2 and... Figure 2 According to the CLSI standards (Table 3) as translated by Sun Changgui, L. salivarius GHL02 is sensitive to clindamycin hydrochloride (Table 4).

[0082] Table 2. Antimicrobial diameter of L. salivarius GHL02

[0083]

[0084] Note: 1: Penicillin; 2: Chloramphenicol; 3: Erythromycin; 4: Clindamycin Hydrochloride; 5: Cephalexin; 6: Ciprofloxacin

[0085] Table 3 Antimicrobial susceptibility criteria

[0086]

[0087] Table 4. Sensitivity results of L. salivarius GHL02 to six antibacterial agents.

[0088]

[0089] Note: R indicates drug resistance; I indicates moderate drug resistance; S indicates susceptibility; 1: penicillin; 2: chloramphenicol; 3: erythromycin; 4: clindamycin hydrochloride; 5: cephalexin; 6: ciprofloxacin

[0090] Example 3: Antibacterial activity detection of L. salivarius GHL02

[0091] 3.1 Test Procedure

[0092] The inhibitory effect of L. salivarius GHL02 on common poultry pathogens was determined using the Oxford cup method.

[0093] 3.1.1 Activation and preparation of strains

[0094] (1) L. salivarius GHL02, which was frozen at -80℃, was inoculated into MRS liquid medium and cultured anaerobically at 37℃;

[0095] (2) Place the activated bacterial solution in a refrigerated centrifuge at 4℃, centrifuge at 8000 r / min for 5 min, take the supernatant and store it at 4℃;

[0096] (3) Dilute the precipitate to 1×10⁻⁶ with MRS liquid medium. 9 CFU / mL was used as a sample of lactic acid bacteria cells.

[0097] 3.1.2 Pathogen activation

[0098] (1) Select and store Escherichia coli, pathogenic Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus at -80℃;

[0099] (2) The pathogens were inoculated into LB broth and cultured at 37 °C. The bacterial suspension concentration was adjusted to 1 × 10⁻⁶. 9 CFU / mL.

[0100] 3.1.3 Preparation of indicator bacteria plates

[0101] (1) Pour LB solid medium into plates, let it solidify, and then dilute it to 1×10⁻⁶. 6 100 μL of CFU / mL bacterial suspension was evenly spread on the surface of a plate.

[0102] (2) After the surface is dry, place the Oxford cup in the center of the plate.

[0103] 3.1.4 Antibacterial test

[0104] (1) Add 200 μL of lactic acid bacteria supernatant to each Oxford cup (supernatant group);

[0105] (2) Negative control: MRS liquid culture medium;

[0106] (3) Positive controls: penicillin G (0.12 μg / mL) and streptomycin (10 μg / mL);

[0107] (4) Microbial cell group: The lactic acid bacteria precipitate was diluted to 1×10⁻⁶. 9 After reaching CFU / mL, add 200 μL to an Oxford cup;

[0108] (5) Incubate at 37℃ for 16-24h, observe and measure the diameter of the inhibition zone.

[0109] 3.2 Experimental Results

[0110] The experimental results showed that L. salivarius GHL02, both in its cell form and fermentation supernatant, exhibited significant inhibitory effects against common poultry pathogens such as pathogenic Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus, indicating that this strain has good potential for resisting pathogenic microorganisms. Figure 3 (Table 5-6).

[0111] Table 5. Antibacterial activity of lactic acid bacteria supernatant against pathogens.

[0112]

[0113] Table 6. Antibacterial activity of lactic acid bacteria culture against pathogens

[0114]

[0115] Example 4: Testing of the acid resistance, salt resistance, and acid production capacity of L. salivarius GHL02

[0116] 4.1 Test Procedure

[0117] 4.1.1 Preparation of bacterial culture

[0118] 100 μL of L. salivarius GHL02 strain was inoculated into MRS liquid medium with pH adjusted to neutral and incubated at 37 ℃ for 24 h to enrich and activate the culture, which was then used as the test bacterial solution.

[0119] 4.1.2 Acid resistance test

[0120] (1) Adjust the pH of MRS liquid culture medium to 1.0, 2.0, 3.0, and 4.0 using 3% hydrochloric acid or 4% sodium hydroxide;

[0121] (2) Sterilize at 121℃ for 30 min, cool to room temperature, and store at 4℃ for later use;

[0122] (3) In a clean bench, add 100 μL of activated bacterial solution to 2 mL of culture medium with different pH values;

[0123] (4) The culture medium was incubated at a constant temperature of 37℃, and the OD of the culture medium was measured at 12h, 24h and 48h. 600 Absorbance values ​​were used to compare the growth of strains under different acidity levels.

[0124] 4.1.3 Salt Tolerance Test

[0125] (1) Adjust the NaCl concentration in the MRS liquid culture medium to 2%, 4%, 6%, and 8%, respectively;

[0126] (2) Sterilize at 121 °C for 30 min, cool to room temperature, and store at 4 °C for later use;

[0127] (3) Under sterile conditions in a laminar flow hood, add 100 μL of activated bacterial solution to 5 mL of culture medium with different NaCl concentrations;

[0128] (4) Set up a standard MRS culture medium as the control group;

[0129] (5) Incubate at 37 ℃, and measure OD at 2h, 4h, 6h, 8h, 10h and 12h respectively. 600 Absorbance values ​​were used to analyze the growth of the strains under different salt concentrations.

[0130] 4.1.4 Acid production capacity test

[0131] (1) Under sterile conditions in a laminar flow hood, 50 μL of activated bacterial solution was inoculated into 2 mL of neutral MRS liquid culture medium;

[0132] (2) The culture was kept at 37 ℃ and the pH value of the culture medium was measured at 12h, 24h and 48h to analyze the acid production capacity.

[0133] 4.2 Test Results

[0134] 4.2.1 Results of acid resistance test

[0135] The acid resistance test results of L. salivarius GHL02 are shown in Table 7. The results indicate that in MRS medium with pH values ​​of 1.0, 2.0, and 3.0, the bacterial culture showed increasing OD values ​​with prolonged culture time. 600 The absorbance values ​​generally showed a decreasing trend, with the decrease being greater at lower pH levels; this decreasing trend slowed down as the pH increased. At pH 4.0, the OD... 600 The absorbance value actually increased over time. These results indicate that a strongly acidic environment inhibits the growth of L. salivarius GHL02, but the strain still exhibits strong tolerance to acidic environments, especially maintaining good growth activity in the pH range of 3.0–4.0.

[0136] Table 7. Determination of acid resistance of L. salivarius GHL02

[0137]

[0138] 4.2.2 Salt tolerance test results

[0139] The salt tolerance test results of L. salivarius GHL02 are shown in Table 8. The results showed that the strain could grow normally in MRS medium containing 2% NaCl; under 4% NaCl conditions, the growth phase of the strain was significantly prolonged, the time to enter the logarithmic growth phase was delayed, and the cell concentration was slightly lower than the control group; under 6% NaCl conditions, the OD... 600 The absorbance increased only slowly with prolonged culture time, failing to exhibit typical logarithmic growth phase characteristics; when the NaCl concentration reached 8%, the growth of the strain was almost completely inhibited. These results indicate that *L. salivarius* GHL02 has strong tolerance to low to medium salinity environments, but its growth is significantly inhibited under high salinity conditions, suggesting its potential for application under certain salinity conditions.

[0140] Table 8. Determination of salt tolerance of L. salivarius GHL02

[0141]

[0142] 4.2.3 Results of acid production experiment

[0143] The acid production performance of L. salivarius GHL02 is shown in Table 9. Lactic acid bacteria can directly lower the pH of the environment by producing acid through metabolism, thereby inhibiting the growth and reproduction of harmful microorganisms. They can also enhance their antibacterial effect through multiple mechanisms, such as nutrient competition, secretion of antibacterial substances, and regulation of the host's immune response. In this example, L. salivarius GHL02 produced a large amount of organic acid in the early stage of cultivation (12h), causing a significant decrease in the pH of the culture medium. Subsequently, in measurements at 24h and 48h, the pH value tended to stabilize without a significant decrease, indicating that this strain has a moderate acid production capacity, effectively inhibiting bacteria while avoiding the potential adverse effects of excessive acidification on the host or application environment.

[0144] Table 9. Determination of the acid-producing properties of L. salivarius GHL02

[0145]

[0146] Example 5: Detection of 16S rRNA gene in strain

[0147] The purified *L. salivarius* GHL02 strain was sent to Qingke Biotechnology Co., Ltd. for bidirectional sequencing of its 16S rRNA gene. The raw sequences obtained from sequencing were assembled using SeqMan software to obtain the complete 16S rRNA gene sequence. Subsequently, BLAST analysis was performed in the NCBI database, and the sequences were compared with those of known type strains. The results showed that the 16S rRNA gene sequence of this strain shared more than 99% homology with *Ligilactobacillus salivarius*, thus confirming its taxonomic position as *Ligilactobacillus salivarius*.

[0148] The 16S rRNA gene sequence of L. salivarius GHL02 is as follows:

[0149]

[0150] Table 10 Results of BLAST homology comparison of strains

[0151]

[0152] Example 6: Whole genome sequencing of L. salivarius GHL02 strain

[0153] The *L. salivarius* GHL02 strain was activated by inoculation in MRS liquid medium and cultured at 37°C in a shaker for 24 h. 50 mL of the bacterial culture was transferred to a centrifuge tube and centrifuged at 4500 r / min for 15 min at 4°C. The supernatant was discarded, and the bacterial pellet was retained. The bacterial cells were then sent to Hangzhou Lianchuan Biotechnology Co., Ltd. for library construction and sequencing.

[0154] 6.1 Sequencing Process

[0155] (1) Perform raw data quality control on the strains to remove low-quality and excessively short reads;

[0156] (2) Perform genome assembly on the filtered high-quality reads and correct errors in the preliminary assembly results (draft);

[0157] (3) Complete the analysis and functional annotation of the genome components.

[0158] 6.2 Sequencing Results

[0159] (1) The whole genome of L. salivarius GHL02 strain is 1.88 Mb, the genome type is circular, and the average GC content is 33.06%. It encodes 2172 genes, including 78 tRNA genes and 22 rRNA genes. Figure 4 );

[0160] (2) COG functional analysis results showed that the genome of this strain contains multiple key functional genes, which can enhance the synthesis and supply of metabolites, thereby improving the host's nutritional and metabolic status; at the same time, it has the function of inhibiting pathogens and maintaining the homeostasis of the lung microecology; its strong adhesion and colonization ability helps to form a protective barrier in the respiratory tract; in addition, this strain exhibits outstanding antioxidant and anti-stress properties, which can alleviate the inflammatory environment; and it can activate the host's immune response and promote the maintenance of immune system homeostasis. Figure 5 );

[0161] (3) KEGG functional analysis results showed that this strain improved local acidification levels through carbohydrate metabolism, lactic acid synthesis, and membrane transport processes, thereby effectively inhibiting respiratory pathogens such as mycoplasma and Pseudomonas, and improving the lung microecological environment; its specific signaling molecules and bacterial components could activate the host immune response, enhance immune alertness, and improve mucosal barrier function; it also had the ability to synthesize vitamins and amino acid derivatives, which helped to supplement host nutrition, regulate inflammation, and alleviate oxidative stress; in addition, it could express molecules such as flagella, adhesion proteins, and extracellular polysaccharides, enhancing its colonization ability in lung tissue and maintaining a stable symbiotic relationship with the host. Figure 6 );

[0162] (4) GO enrichment analysis showed that this strain can maintain its own stability and evolutionary potential, improve its stress resistance, inhibit virulence factors, and ensure its probiotic properties; it can stably express functional proteins and maintain structural integrity, which helps to stably colonize the host respiratory tract and enhance the antigen barrier effect; it can synthesize beneficial metabolites such as lactic acid, short-chain fatty acids and amino acids, thereby maintaining the host's nutritional and metabolic homeostasis; through the transport of antimicrobial peptides, lactic acid and vitamins, it can effectively inhibit pathogens and build a lung microbial barrier; it can rapidly express homeostasis-related proteins, enhance its adaptability and survival ability to the host environment; and it can ensure the activity of key enzymes and activation of signaling pathways, achieving a comprehensive effect of antioxidation, immune regulation and stable functional expression. Figure 7 );

[0163] (5) Comparison with the NR database showed that this strain had the highest homology with Ligilactobacillus salivarius, accounting for 68.426%. Figure 8 );

[0164] (6) CAZys strains exhibit diverse carbohydrate metabolic functions: glycosyltransferases promote colonization and immune regulation; carbohydrate esterases provide energy and inhibit pathogens; glycoside hydrolases improve carbohydrate utilization; and carbohydrate binding modules enhance substrate recognition and promote interaction with the host mucosa, thereby supporting strain colonization and host health. Figure 9 );

[0165] (7) Pfam domain analysis showed that this strain has significant functions in material transport and energy metabolism, which can promote nutrient absorption, maintain microbial homeostasis, and enhance probiotic colonization and anti-infection ability; its adhesion and host interaction characteristics help Lactobacillus form a stable symbiotic community in the lungs, inhibit pathogen colonization, and maintain immune balance; genomic stability and stress response capabilities enable probiotics to survive stably and continue to function under lung environmental stress; at the same time, its metabolic-related characteristics help stabilize the microecological community, enhance host immune tolerance, and optimize energy metabolism interactions. Figure 10 );

[0166] The L. salivarius GHL02 genome is complete and stable, possessing multiple probiotic properties such as enhancing metabolite synthesis, inhibiting pathogens, maintaining lung microecology, promoting colonization barrier formation, anti-stress, and immune regulation. These properties provide a molecular basis for improving host nutrition, enhancing immunity, and improving poultry health and production performance.

[0167] Example 7: Evaluation of the effects of atomized L. salivarius GHL02 on broiler production performance and immune function

[0168] 7.1 Test Procedure

[0169] 7.1.1 Experimental animals and their rearing conditions

[0170] This experiment used 520 newly hatched AA broiler chickens (1 day old, average weight 47.0±0.5 g), all healthy and with consistent immunization programs. The chickens were raised in a three-tiered cage system at the Northwest A&F University Animal Husbandry Experimental Base, with free access to feed and water throughout the experiment. The entire rearing and management process strictly followed the Aviagen UK's "AA Broiler Management Manual." Operations and handling complied with the regulations of the Northwest A&F University Laboratory Animal Management and Ethics Committee, and were approved (Approval No.: 2023-DK-015).

[0171] 7.1.2 Preparation of bacterial culture

[0172] (1) L. salivarius GHL02, which was frozen at −80℃, was incubated statically at 37℃ for 18h in MRS liquid medium;

[0173] (2) Collect the bacterial cells by centrifugation at 8000×g for 10 min, wash three times with PBS and resuspend;

[0174] (3) Use spectrophotometry combined with plate counting to calibrate the concentration to 1×10⁻⁶. 9 CFU / mL, used as a bacterial suspension for nebulization.

[0175] 7.1.3 Grouping and Atomization Processing

[0176] (1) Random grouping: control group (PBS group): 200 animals, 25 replicates, 8 animals per replicate; treatment group (nebulization group): 320 animals, 40 replicates, 8 animals per replicate.

[0177] (2) Nebulization device and parameters: Medical ultrasonic nebulizer (median droplet diameter MMAD about 2-5µm), the nebulization chamber is relatively closed and well ventilated; the nebulization head is 0.8-1.2 m away from the flock of chickens and the height is slightly higher than the chicken back line.

[0178] (3) Dosing schedule: Nebulization once every 3 days, 20 min each time; the treatment group was given 1×10 9 The aerosol was administered CFU / mL of L. salivarius GHL02 suspension, with the control group receiving PBS, at the same frequency and duration. Regular ventilation was maintained during nebulization to ensure uniform aerosol diffusion.

[0179] (4) Safety protection: Operators should wear masks, gloves and protective clothing, and can only enter the operation 20 minutes after atomization.

[0180] 7.1.4 Indicator Measurement and Recording

[0181] (1) Growth performance: Weigh and record the feed consumption of each group at 7, 14, 21, 28, 35, and 42 days of age, and calculate:

[0182] Average daily weight gain = (final weight - initial weight) / number of days;

[0183] Average daily feed intake = (feed consumption per bird during the period) / number of days;

[0184] Feed conversion ratio = feed consumption (kg) / body weight gain (kg).

[0185] 7.1.5 LPS Acute Stress Model (35 Days Old)

[0186] After 35 days of continuous nebulization, 14 animals were randomly selected from each group and injected intraperitoneally with lipopolysaccharide (LPS) at a dose of 1 mg / kg of body weight. The weight change was recorded 6 hours later.

[0187] 7.2 Experimental Results

[0188] 7.2.1 Growth performance

[0189] This study systematically evaluated the effects of atomized L. salivarius GHL02 on the entire production cycle of AA broilers. Results showed that in the early growth stages, atomization significantly promoted body weight gain: compared with the control group (PBS group), the atomized group showed significantly increased mean body weight at 7, 14, and 21 days of age, while the difference was no longer significant at 28 days of age and thereafter. Figure 11 Regarding average daily weight gain, the nebulized group showed significantly higher gains than the control group during the 1–7 day and 14–21 day age stages, with no significant differences in other stages. Figure 12 The results of feed intake and feed conversion efficiency showed that the average daily feed intake of the atomized group was significantly higher than that of the control group from 1 to 7 days of age, but significantly lower than that of the control group from 28 to 35 days of age. Figure 13 It is worth noting that the feed conversion ratio of the atomizing group decreased significantly throughout the entire production cycle, indicating a continuous improvement in feed utilization efficiency. Figure 14 ).

[0190] Atomized L. salivarius GHL02 can significantly promote growth and feed intake in broilers in the early stages, while improving feed utilization efficiency throughout the entire cycle, indicating its good application potential as a means of improving production performance.

[0191] 7.2.2 LPS Acute Stress Model

[0192] In the immune stress experiment, after LPS stimulation, the body weight of broilers in the nebulized group was significantly higher than that in the PBS+LPS group, and the degree of inhibition of body weight gain during LPS injection was significantly less than that in the PBS+LPS group. Figure 15 This result suggests that nebulized L. salivarius GHL02 not only promotes growth performance in the early stages, but also enhances immune function and alleviates LPS-induced acute stress response.

[0193] 7.2.3 Clinical observation and safety

[0194] Throughout the experiment, no significant adverse reactions related to nebulization (such as persistent tearing, sneezing, or significant wheezing) were observed, and the mortality and attrition rates did not increase abnormally. Based on the drug susceptibility testing and in vitro safety evaluation results of Examples 2–4, it can be concluded that this strain and its nebulization application have good safety and feasibility under the experimental conditions.

[0195] 7.3 Conclusion

[0196] Nebulized L. salivarius GHL02 significantly promoted growth in broilers during the early stages and improved feed utilization throughout the production cycle. It also demonstrated enhanced immunity and reduced stress damage in the LPS acute stress model. No adverse reactions were observed during the experiment. Combined with drug sensitivity and safety results, this indicates that the nebulized application of this strain is safe and reliable, and has the potential for application in broiler production.

Claims

1. A strain of lactic acid bacteria, Ligilactobacillus salivarius, SunLab-GHL02, with accession number CCTCCNO: M 20251726.

2. Use of the strain according to claim 1 in the preparation of formulations for the prevention, mitigation or treatment of respiratory diseases, digestive diseases or systemic infections in poultry.

3. Use of the strain according to claim 1 in the preparation of a formulation for improving poultry production performance, wherein the production performance includes feed conversion ratio, daily weight gain, survival rate and stress resistance.

4. Use of the strain according to claim 1 in the preparation of formulations for inhibiting pathogenic Escherichia coli, Salmonella, Pseudomonas aeruginosa or Staphylococcus aureus.

5. The application of the strain according to claim 1 in feed additives, drinking water additives, aerosol preparations, spray preparations, capsule preparations, powders, freeze-dried powders, granules or gels.

6. A method for applying the strain of claim 1, characterized in that, The live bacterial preparation containing the strain is applied to poultry via atomization, drinking water, or feed to achieve the use described in claims 2 to 5.

7. The method according to claim 6, characterized in that, The atomization method involves atomizing the live bacterial preparation containing the strain into an aerosol with a particle size of 1-10 µm and spraying it into the poultry rearing environment so that the poultry can inhale it through their respiratory tract.

8. The method according to claim 6, characterized in that, The method of administration is to add a live bacterial preparation containing the aforementioned strain to the drinking water, with a live bacterial count of 10. 6 -10 9 CFU / mL.

9. The method according to claim 6, characterized in that, The feeding method involves uniformly mixing the preparation containing the strain with the feed at a ratio of 0.05-2% (by mass).

10. A composition containing the strain of claim 1, characterized in that, The composition further comprises a carrier or excipient selected from lactose, glucose, maltodextrin, starch, skim milk powder, or combinations thereof.

11. A composition, characterized in that, The product comprises the strain of claim 1 or its metabolites used in combination with at least one other probiotic, wherein the other probiotic is selected from Lactobacillus plantarum, Bifidobacterium animalis, Bacillus subtilis or Enterococcus faecium.

12. Use of the strain according to claim 1, its metabolites or inactivated cell lysates in the preparation of formulations for improving poultry health or production performance, wherein the metabolites or lysates can enhance the immune function, stress resistance or inhibit the growth of pathogenic bacteria in poultry.

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