Lactobacillus gallinaceus and probiotic bacterial agent, and application of freeze-dried bacterial powder

Abstract

The application discloses a kind of Lactobacillus gallinarum and probiotic bacterial agent and freeze-dried bacterial powder and application thereof.The Latin name of the Lactobacillus gallinarum is Lactobacillus gallinarum, named HNAU2301, and the preservation number of China General Microbiological Culture Collection Center is CGMCC NO.29242;The nucleotide sequence of 16SrDNA is SEQ ID NO.1.The Lactobacillus gallinarum provided in the application has weak acid resistance and bile salt resistance, good self-aggregation and biofilm formation capacity, and good inhibition capacity for chicken white dysentery salmonella;Therefore, the use of antibiotics can be replaced, and the damage of antibiotics to the body is reduced.The Lactobacillus gallinarum provided in the application can be added to feed, which can significantly increase the serum IgY level of breeding hen and the egg yolk IgY level, improve the fertilization rate and hatching rate of breeding hen, reduce the dead embryo rate, and improve the production performance of breeding hen.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a hen lactobacillus and probiotic inoculant and freeze-dried bacterial powder and their applications. Background Technology

[0002] High mortality rates in chicks are a significant factor limiting the level of poultry farming and the rapid development of the industry. Many factors contribute to high chick mortality, with early pathogen infection being a key factor. For example, pullorum disease caused by Salmonella pullorum can lead to fowl cholera. Bacterial infections further slow chick growth, and in hens, they can cause decreased egg production. In the past, antibiotics were widely used to prevent and treat bacterial infections like pullorum disease. However, the overuse of antibiotics leads to drug resistance, ultimately resulting in antibiotic treatment failure. Furthermore, the accumulation of antibiotics in farmed animals not only harms poultry health, but excessive antibiotic excretion into the environment also causes pollution, which in turn has a series of impacts on human health.

[0003] Microbial preparations, with their unique non-specific antibacterial and bactericidal effects, environmental friendliness, and residue-free characteristics, are gradually becoming alternatives to antibiotics.

[0004] Chinese patent document CN102031235A discloses a strain of Enterococcus faecalis ANSE228, which exhibits significant prebiotic properties. By adding it to the drinking water and / or feed of farmed animals, it can significantly inhibit Salmonella pullorum. The strain also demonstrates strong resistance, tolerating simulated gastric acid, simulated bile salts, and high-temperature environments, and maintaining a high viable bacterial survival rate of over 80%.

[0005] Chinese patent document CN115820482A discloses a strain of Bacillus amyloliquefaciens YN-BA2. This strain has good heat, acid and bile salt resistance and exhibits high inhibitory effects against Salmonella pullorum and Salmonella enteritidis in vitro. This strain can be used to prepare biological control microecological preparations, which can be administered orally to chickens and are suitable for the prevention and control of Salmonella pullorum disease in the livestock and poultry industry.

[0006] However, these microorganisms and their preparations are primarily intended for direct consumption by farmed animals. Since chicks are highly susceptible to pathogenic microorganisms and their immature immune systems have a limited ability to produce adaptive immune effectors such as antibodies, the aforementioned microorganisms are not particularly effective for chicks. However, maternal / yolk antibodies (IgY) can provide passive immune protection for newly hatched chicks and play an important role in preventing pathogenic infections in chicks.

[0007] Furthermore, studies have found that maternal gut microbiota can shape an antibody repertoire. The microbiota plays a crucial role in triggering protective antibodies against specific neonatal pathogens. For example, maternal symbiotic microbiota can induce antibodies that recognize antigens expressed by enterotoxigenic Escherichia coli and other Enterobacteriaceae species, protecting newborns from enterotoxigenic Escherichia coli infection. Gut symbiotic Gram-negative bacteria can spread systemically to induce immunoglobulin G (IgG) responses, targeting Gram-negative bacterial antigens and promoting phagocytic killing through direct bacterial coating, thereby providing protection against systemic infections by Escherichia coli and Salmonella. These studies suggest that gut microbiota may play an important role in the production of maternal antibodies.

[0008] However, probiotic products that can increase maternal antibody levels are rare in poultry farming, so there is an urgent need to develop a strain and product that can increase maternal antibody levels. Summary of the Invention

[0009] The technical problem to be solved by this invention is to provide a hen lactobacillus and probiotic agent and freeze-dried bacterial powder and their application, which can improve the level of maternal antibody IgY in breeding hens and the offspring's resistance to Salmonella pullorum infection, improve the fertilization rate and hatchability of hen eggs, and reduce the embryo mortality rate.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a *Lactobacillus gallinarum*, whose Latin name is *Lactobacillus gallinarum*, named HNAU2301, and whose accession number at the China General Microbiological Culture Collection Center is CGMCC NO.29242; the nucleotide sequence of its 16S rDNA is SEQ ID NO.1.

[0012] Secondly, the present invention provides a probiotic preparation containing the aforementioned *Lactobacillus henii*.

[0013] Furthermore, the probiotic agent also includes excipients. The excipients are preferably maltodextrin.

[0014] Furthermore, the formulation of the microbial agent is a liquid, powder, or granules. Freeze-dried microbial powder is preferred.

[0015] Thirdly, the present invention also provides a freeze-dried Lactobacillus heniorum powder, which is prepared from the aforementioned Lactobacillus heniorum, specifically including the following steps:

[0016] Take the frozen Lactobacillus heniella, revive and propagate it, with a volume ratio of culture medium to bacterial solution of 500:1;

[0017] After culturing for 24 hours, centrifuge the expanded Lactobacillus heniol, discard the supernatant, and wash 3 times with sterile 1×PBS.

[0018] After washing, centrifugation was used to obtain bacterial sludge. A 10% maltodextrin PBS solution was added to the centrifuged bacterial sludge at a volume ratio of 1:1. After mixing, the mixture was frozen at -80°C.

[0019] The frozen bacteria are freeze-dried and then ground into powder.

[0020] Fourthly, the present invention also provides the application of the above-mentioned Lactobacillus heniorum or bacterial agent or freeze-dried Lactobacillus heniorum powder in poultry breeding.

[0021] Furthermore, the application includes the use of the *Lactobacillus henilis* or its agent or freeze-dried *Lactobacillus henilis* powder in improving the level of maternal antibody IgY in breeder chickens.

[0022] Furthermore, the application includes the use of the *Lactobacillus henilis* or its agent or freeze-dried *Lactobacillus henilis* powder in enhancing the ability of breeding chicken offspring to inhibit *Salmonella pullorum* infection.

[0023] Furthermore, the application includes the use of the *Lactobacillus henilis* or its agent or freeze-dried *Lactobacillus henilis* powder in improving the fertilization rate and hatchability of hatching eggs.

[0024] The present invention has the following beneficial effects:

[0025] 1. The *Lactobacillus henii* HNAU2301 provided by this invention has weak acid and bile salt resistance, good self-aggregation and biofilm formation capabilities, and good inhibitory ability against *Salmonella pullorum*. Therefore, it can replace the use of antibiotics and reduce the damage of antibiotics to the body.

[0026] 2. Adding the hen lactobacillus provided by this invention to the feed can significantly increase the serum IgY level and egg yolk IgY level of breeder hens, and can also improve the fertilization rate and hatchability of hatching eggs from breeder hens, reduce the embryo mortality rate, and thus improve the production performance of breeder hens. Attached Figure Description

[0027] Figure 1 The image shows the screening results of the *Lactobacillus* strains from hens in this invention, wherein: Figure 1 Figure A shows the screening results of 26 strains of Lactobacillus heniorum in acid resistance test (pH=3); Figure 1 B shows the screening results of 26 strains of Lactobacillus heniorum in acid resistance test (pH=4); Figure 1 C represents the screening results of 26 strains of Lactobacillus heniorum in the bile salt tolerance test (bile salt concentration of 0.15%). Figure 1 D is the screening results of 26 strains of Lactobacillus heniorum in the bile salt tolerance test (bile salt concentration of 0.3%). Figure 1E represents the screening results of the self-aggregation test of 26 strains of Lactobacillus heniella; Figure 1 F shows the screening results of the biofilm formation test for 26 strains of Lactobacillus heniella; Figure 1 G represents the screening results of the antibacterial test of 26 strains of Lactobacillus heniella.

[0028] Figure 2 This is a colony morphology diagram of Lactobacillus henii HNAU2301 of the present invention.

[0029] Figure 3 This is a Gram staining image of Lactobacillus henii HNAU2301 of the present invention.

[0030] Figure 4 The diagram shows the effect of Lactobacillus henii HNAU2301 of the present invention on the production performance of breeding chickens. Among them: 4A is the weekly egg production rate control curve of each experimental group; 4B is the egg production rate control curve of each experimental group throughout the experimental period; 4C is the comparison diagram of fertilization rate, hatching rate and embryo mortality rate of each experimental group.

[0031] Figure 5 The figure shows the effect of Lactobacillus henii HNAU2301 on egg yolk and chick serum IgY, as described in this invention. Figure 5 A shows the comparison chart of IgY concentration in egg yolk of each experimental group; Figure 5 B is a comparison chart of IgY concentration in the serum of chicks in each experimental group.

[0032] Figure 6 The figure shows the effect of Lactobacillus henii HNAU2301 of this invention on organ indices in chicks challenged with Salmonella pullorum after infection, wherein: Figure 6 A is a comparison chart of liver indices in chicks from each experimental group 1 day after being challenged with Salmonella pullorum. Figure 6 B is a comparison chart of spleen indices in chicks from each experimental group 1 day after being challenged with Salmonella pullorum. Figure 6 C is a comparison chart of the thymus index of chicks in each experimental group 1 day after being challenged with Salmonella pullorum. Figure 6 D is a comparison chart of the bursa of Fabricius index of chicks in each group after 1 day of infection with Salmonella pullorum. Figure 6 E is a comparison chart of liver indices in chicks of each group after 7 days of exposure to Salmonella pullorum. Figure 6 F is a comparison chart of spleen indices in chicks of each group after 7 days of exposure to Salmonella pullorum. Figure 6 G is a comparison chart of thymus index of chicks in each group after 7 days of exposure to Salmonella pullorum. Figure 6 H is a comparison chart of the bursal index of chicks in each group 7 days after being challenged with Salmonella pullorum.

[0033] Figure 7 The graph shows the effect of *Lactobacillus henryi* HNAU2301 on the bacterial load in organ tissues of chicks after challenge with *Salmonella pullorum*. Figure 7A is a comparison chart of the bacterial load in the spleen of chicks in each group 1 day after being challenged with Salmonella pullorum. Figure 7 B is a comparison chart of the bacterial load in the livers of chicks in each group 1 day after being challenged with Salmonella pullorum. Figure 7 C is a comparison chart of bacterial load in the cecal contents of chicks in each group 1 day after being challenged with Salmonella pullorum. Figure 7 D is a comparison chart of the bacterial load in the spleen of chicks in each group after 7 days of exposure to Salmonella pullorum. Figure 7 E is a comparison chart of the bacterial load in the livers of chicks in each group after 7 days of exposure to Salmonella pullorum. Figure 7 F is a comparison chart of bacterial load in the cecal contents of chicks in each group 7 days after being challenged with Salmonella pullorum.

[0034] Figure 8 The figure shows the effect of Lactobacillus henii HNAU2301 on the expression of inflammatory factors mRNA in the immune organs of chicks after challenge with Salmonella pullorum. Figure 8 A shows the expression of inflammatory factor mRNA in the spleen of chicks in each group 1 day after being challenged with Salmonella pullorum. Figure 8 B is a control diagram showing the expression of cecal tonsil inflammatory factor mRNA in chicks 1 day after being challenged with Salmonella pullorum. Figure 8 C is a control diagram showing the expression of inflammatory factor mRNA in the spleen of chicks in each group 7 days after being challenged with Salmonella pullorum. Figure 8 D is a comparison diagram showing the expression of inflammatory factor mRNA in the cecal tonsils of chicks in each group 7 days after being challenged with Salmonella pullorum.

[0035] Figure 9 The figure shows the effect of Lactobacillus henryi HNAU2301 on the mRNA expression of jejunal barrier-related genes in chicks after challenge with Salmonella pullorum. Figure 9 A shows the control of Occludin gene expression levels in the jejunum of chicks 1 day after being challenged with Salmonella pullorum. Figure 9 B is a comparison chart of the expression level of ZO-1 gene in the jejunum of chicks in each group 1 day after being challenged with Salmonella pullorum. Figure 9 C is a control diagram showing the expression level of the MUC2 gene in the jejunum of chicks 1 day after being challenged with Salmonella pullorum. Figure 9 D is a comparison diagram of the expression level of Claudin gene in the jejunum of chicks in each group 1 day after being challenged with Salmonella pullorum. Figure 9 E is a control diagram showing the expression level of the Occludin gene in the jejunum of chicks in each group 7 days after being challenged with Salmonella pullorum. Figure 9 F is a control diagram showing the expression level of ZO-1 gene in the jejunum of chicks after 7 days of infection with Salmonella pullorum. Figure 9 G is a control diagram showing the expression level of the MUC2 gene in the jejunum of chicks after 7 days of infection with Salmonella pullorum. Figure 9H is a comparison diagram of the expression level of the Claudin gene in the jejunum of chicks in each group 7 days after being challenged with Salmonella pullorum. Detailed Implementation

[0036] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0038] The MRS culture medium used in this embodiment of the invention has the following formulation: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diamine hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1 g / L Tween 80, pH 5.8–6.2, sterilized at 121°C for 15 min.

[0039] The present invention will be further described below with reference to the embodiments.

[0040] Example 1: Isolation and Screening of Strains

[0041] 1.1 Sample Source

[0042] Feces of healthy 400-day-old Shimen Black Chickens at the Xiangjia Shimen Black Chicken Breed Testing Station in Shimen County, Changde City, Hunan Province.

[0043] 1.2 Strains Isolation

[0044] Place 0.5g of fecal sample into 50ml of sterile PBS or physiological saline to prepare a sample suspension. After vortexing and mixing, transfer the suspension to a clean bench and dilute it to different concentration gradients (generally 10). -5 and 10 -6After vortexing, 50-100 μl of the culture medium was spread onto an agar plate and then placed in an anaerobic bag (containing anaerobic indicator and anaerobic gas generator) for inverted culture in a constant temperature incubator (37℃). After 24-48 hours of culture, the culture dishes were removed, and colonies were picked up with a sterile pipette tip and placed into centrifuge tubes pre-filled with 1 ml of broth. These tubes were then placed in a constant temperature shaker for further culture and propagation. The cultured bacterial solution was removed, purified twice, and then sent for sequencing. The sequencing company was Beijing Qingke Biotechnology Co., Ltd. (Beijing, China). Bidirectional sequencing was performed using universal primers 27F / 1492R. The base sequences were then copied to BLAST for alignment. Sequences with over 97% similarity were considered to be of the same species, over 95% to the same genus, and over 80% to the same phylum.

[0045] The applicant isolated a total of 26 strains of Lactobacillus heniella, numbered LG1 to LG26.

[0046] 1.3 Strain screening

[0047] The acid tolerance of third-generation bacterial suspensions was evaluated by adding 2% (v / v) to MRS broth medium at pH 2. An equal volume of bacterial suspension was added to broth medium (pH 6) as a control group. All experimental bacterial suspensions were incubated at 37°C with a shaking incubator at 100 rpm for 4 hours. Samples were taken at 0, 2, and 4 hours, with three replicates at each time point, using 10... 4 10 5 10 6 Three gradients were streaked on agar plates, and the viable counts for each gradient were calculated after incubation at 37°C for 24 hours.

[0048] Third-generation bacterial suspensions were added at 2% (v / v) to broth medium containing 0.15% and 0.3% (w / v) bile salts, respectively, to evaluate bile salt tolerance. An equal volume of third-generation bacterial suspension was added to MRS medium as a control group. All experimental bacterial suspensions were incubated at 37°C with a shaking incubator at 100 rpm for 4 h. Samples were taken at 2 and 4 h, with three replicates at each time point, using 10... 4 10 5 10 6 Three gradients were streaked on agar plates, and the viable counts for each gradient were calculated after incubation at 37°C for 24 hours.

[0049] Survival rate (%) = (Number of viable bacteria in experimental group / Number of viable bacteria in control group) × 100%

[0050] Lactobacillus heniella, Escherichia coli, and Salmonella pullorum were revived and enriched in enrichment broth (100 μl of bacterial suspension was added to 100 ml of culture medium) for antibacterial tests. Salmonella pullorum (CVCC519) and avian pathogenic Escherichia coli (CVCC1490) were purchased from the China Veterinary Microbiological Culture Collection Center. 100 μl of the revived and amplified Escherichia coli was added to a 10 ml petri dish and immediately spread evenly on an agar plate. Using the Oxford cup method, 200 μl of Lactobacillus heniella was added to an Oxford cup and incubated overnight at 37°C. After complete absorption of the sample in the Oxford cup, the diameter of the inhibition zone for each group was measured with calipers, and the mean value was recorded.

[0051] Biofilm formation was evaluated using a 96-well plate-crystal violet staining method. *Lactobacillus heniella* was incubated overnight in MRS broth, and the bacterial concentration was adjusted to OD600nm = 0.5. The bacterial solution was then diluted 1000-fold (10⁻⁶ oz). 5 (CFU / mL) After thorough mixing, 200 μl of the bacterial suspension was added to each well of a 96-well plate. After incubation at 37°C for 48 h and 72 h, the culture medium was discarded, and the plate was washed twice with sterile PBS. The plate was then fixed with methanol and dried. 200 μl of 0.1% crystal violet solution was added to each well, followed by washing with running water and drying. 200 μl of 33% glacial acetic acid solution was added to each well, and the plate was placed on a decolorizing shaker for 5 min to dissolve. The absorbance at OD590 nm was measured using a microplate reader.

[0052] The method for evaluating self-aggregation was as follows: A suspension of *Lactobacillus henilis* was collected, centrifuged at 4000 rpm for 10 min, washed twice with sterile physiological saline, and then centrifuged at 5000 rpm for 10 min to collect the bacterial cells. The concentration of *Lactobacillus henilis* was adjusted with physiological saline so that the absorbance of the bacterial suspension at 600 nm was approximately 1.0 (OD600nm = 1.00). 4 mL of the prepared bacterial suspension was placed in a 10 mL centrifuge tube and allowed to stand at room temperature for stratification. After standing for 1 and 4 hours, 200 μL of the supernatant suspension was aspirated to measure the OD600nm value, with three replicates for each time point. The cell self-aggregation rate (C) was calculated as: C = [1 - (At / A0)] × 100%. Where A0 and At are the OD values ​​of the upper suspension measured at 600 nm before and after self-aggregation, respectively.

[0053] The results are as follows Figure 1 As shown, Figure 1 The x-coordinates of A-1G correspond to LG1-LG26 respectively. Figure 1It is known that *Lactobacillus henilis* has weak acid and bile salt tolerance. At pH 3, the survival rate after 2 hours of culture is about 5%, and drops rapidly to about 0.05% after 4 hours; at pH 4, the survival rate after 2 hours is about 6%, and drops rapidly to about 0.5% after 4 hours. At a bile salt concentration of 0.15%, the survival rate after 2 hours is about 0.7%, and drops rapidly to about 0.03% after 4 hours; at a bile salt concentration of 0.3%, the survival rate after 2 hours is about 0.06%, and drops rapidly to about 0.0005% after 4 hours. The self-aggregation and biofilm formation abilities of probiotics can reflect their survival and adhesion abilities. The self-aggregation ability of *Lactobacillus henilis* varies, with LG1, LG4, LG5, LG9, LG15, and LG21 showing the worst self-aggregation effects. Biofilm formation ability was assessed, with LG1, LG5, LG10, LG14, LG18, LG22, LG24, LG25, and LG26 showing relatively poor effects. Antibacterial activity was not significantly different among the various *Lactobacillus* strains found in hens, with LG17, LG21, LG23, LG24, and LG26 exhibiting better antibacterial effects. Based on the comprehensive evaluation of the probiotic effects of *Lactobacillus* in hens, LG23 was ultimately selected as the strain for the subsequent validation experiments.

[0054] Example 2 Identification of LG23 strain

[0055] 2.1 Colony morphology identification

[0056] After activation, the bacterial strain was inoculated into liquid culture medium at a rate of 2%, with an initial pH of 6.5. The medium was then placed in a 37°C incubator and cultured at 120 rpm for 30 hours. The strain was then inoculated onto agar plates and cultured anaerobically at 37°C for 24-48 hours. Colony morphology was then observed, and colonies were picked for Gram staining. Bacterial morphology was observed under a microscope and recorded.

[0057] like Figure 2 and Figure 3 It can be seen that the colonies of strain LG23 are round with irregular edges, opaque, grayish-white on the front, and raised in the middle. Under a microscope, Gram staining is purple, and the shape is short rod-shaped.

[0058] 2.216S rRNA identification

[0059] The purified LG23 strain was activated, and 1 ml of bacterial culture was prepared and sent to a sequencing company for sequencing. The results showed that the sequence of strain LG23 was SEQ ID NO.1, with a length of 1481 bp. The specific sequence is as follows:

[0060] ACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCAGAACCAGCAGATTTAC

[0061] TTCGGTAATGACGCTGGGGACGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCT

[0062] GCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGAT

[0063] CGCATGATCAGCTTATAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGC

[0064] ATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAG

[0065] ACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTA

[0066] GGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGG

[0067] TTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTA

[0068] TTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATAC

[0069] GTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAAAATA

[0070] AGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTG

[0071] AGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAA

[0072] GAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCA

[0073] TGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAG

[0074] TGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGG

[0075] AGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGG

[0076] AGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGTGC

[0077] CATCCTAAGAGATTAGGAGTTCCCTTCGGGGACGCTAAGACAGGTGGTGCATGGCTGTC

[0078] GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTATT

[0079] AGTTGCCAGCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAG

[0080] GTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATG

[0081] GGCAGTACAACGAGAAGCGAGCCTGCGAAGGCAAGCGAATCTCTGAAAGCTGTTCTCA

[0082] GTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGAT

[0083] CAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGA

[0084] AGTCTGCAATGCCCAAAGCCGGTGGCCTAACCTTCGGGAAGGAGCCGTCTAAGGCAGG

[0085] CAGATGACTGGGGTGA.

[0086] A BLAST comparison of SEQ ID NO.1 in the NCBI database revealed a 99.80% similarity to Lactobacillus gallinarum.

[0087] Based on the above analysis, and combining the colony morphology, microscopic characteristics, and 16S rRNA identification of strain LG23, the applicant identified the strain as *Lactobacillus gallinarum* and named it *Lactobacillus gallinarum* HNAU2301. The applicant deposited *Lactobacillus gallinarum* HNAU2301 at the China General Microbiological Culture Collection Center in Beijing, China on December 6, 2023, with accession number CGMCCNO.29242.

[0088] Example 3: Effects of Lactobacillus heniella HNAU2301 on maternal antibody IgY levels and offspring's resistance to Salmonella pullorum infection.

[0089] 3.1 Preparation of freeze-dried bacterial powder

[0090] Take the frozen *Lactobacillus henii* HNAU2301 and perform resuscitation and propagation (50ml centrifuge tube, 50ml culture medium + 0.1ml bacterial suspension; for large-scale production of bacterial powder, use a 500ml or 1L Erlenmeyer flask). After 24 hours, centrifuge the amplified *Lactobacillus henii* (5000rpm 5min), discard the supernatant, and wash three times with sterile 1×PBS. After washing, add 10% maltodextrin (1g maltodextrin + 9ml PBS) at a 1:1 volume ratio to the centrifuged bacterial slurry, mix well, and freeze at -80℃ (for at least 3 hours). Open the centrifuge tube caps and freeze-dry the frozen bacteria for at least 24 hours. Then grind it into bacterial powder, aliquot it (centrifuge tubes, 1g or 5g / tube), and store at -80℃ for long-term use.

[0091] 3.2 Animal Experiment Design

[0092] Experimental design for breeding hens: 450 38-week-old Ephedra sinica breeding hens were selected. Three eggs were collected from each hen for IgY level testing. The hens were then randomly divided into 5 groups based on IgY levels, with 10 replicates per group and 8 hens per replicate. The control group was fed a basal diet, while the experimental groups were fed a basal diet supplemented with 2.5 × 10⁻⁶ IgY. 8 5.0×10 8 7.5×10 8 10×10 8 Diets containing CFU / kg of Lactobacillus HNAU2301 in hens were designated as 2.5 HNAU2301, 5.0 HNAU2301, 7.5 HNAU2301, and 10 HNAU2301, respectively, with an experimental period of 6 weeks. The experiment was conducted at Jitai Agricultural and Animal Husbandry Co., Ltd. in Liling County, Zhuzhou City, Hunan Province.

[0093] 3.3 Sample Collection and Index Measurement

[0094] 3.3.1 Egg production rate and hatching performance of breeding hens

[0095] Egg counts were collected daily, and repeated weekly. Egg production rate was calculated using the formula below after the experiment. Hatching eggs were collected continuously for 7 days starting one week before the end of the experiment. Feces and other contaminants were removed from the surface of the eggs, and they were fumigated with formalin for 30 minutes before incubation. The number of hatched eggs in each treatment was counted. Candling was performed on day 17 of incubation to count infertile eggs and dead embryos. The number of hatched chicks was counted after hatching. The initial weight of the remaining chicks was used for the next stage of the experiment. The relevant indicators were calculated as follows:

[0096] Egg production rate (%) = Total eggs produced / (Number of hens × Number of days of laying) × 100%

[0097] Fertilization rate (%) = (Number of eggs put into incubation - Number of infertile eggs) / Number of eggs put into incubation × 100%

[0098] Infant mortality rate (%) = (Number of dead embryos / Number of fertilized eggs) × 100%

[0099] Hatching rate (%) = (Number of hatched chicks / Number of fertilized eggs) × 100%

[0100] The results are as follows Figure 4 The results showed that adding Lactobacillus henii HNAU2301 to the diet of each experimental group had no significant effect on the weekly egg production rate of hens. Figure 4 A), but throughout the entire experimental period, the group with the highest dose of Lactobacillus lactis HNAU2301 showed a significant trend in total egg production rate compared to the control group (0.1). <P<0.05)( Figure 4B). Furthermore, adding Lactobacillus henii HNAU2301 to the diet can improve the fertilization rate and hatchability of hatching eggs, and reduce the mortality rate of embryos, with the highest dose (10 HNAU2301 group) showing the most significant effect (P<0.05).

[0101] 3.3.2 Egg yolk and serum IgY levels

[0102] Eggs were collected consecutively for 3 days in each replicate. After mixing each egg, 200 μL of yolk was extracted into a 1.5 ml centrifuge tube and vortexed to mix. Then, 200 μL of yolk was transferred into another 1.5 ml centrifuge tube, and PBS was added at a volume ratio of 1:4. The mixture was vortexed and mixed, then incubated overnight at 4°C. The supernatant was then collected by centrifugation at 1000×g for IgY level detection. The results are as follows: Figure 5 As shown in Figure A.

[0103] Blood was collected from the jugular vein of chicks, allowed to stand for 15-30 minutes, then centrifuged at 3000 rpm for 15 minutes. The supernatant serum was aliquoted into centrifuge tubes and quickly transferred to a -80°C freezer for storage. Serum IgY levels were detected using an ELISA kit purchased from Jiangsu Enzyme-Label Biotechnology Co., Ltd. Results are as follows: Figure 5 As shown in B.

[0104] like Figure 5 As shown, compared with the control group, the experimental groups of Lactobacillus henii HNAU2301 of the present invention can significantly increase the serum IgY level of breeder chickens (P<0.05) and significantly increase the yolk IgY level of hatching eggs (P<0.05).

[0105] 3.3.3 Salmonella challenge in chicks

[0106] Salmonella pullorum strain was cultured overnight at 37°C with shaking at 180 rpm in a sterile laminar flow hood using an appropriate amount of liquid-free culture medium. The resulting bacterial suspension was centrifuged at 8000 rpm for 10 min in a high-speed benchtop centrifuge, and the supernatant was discarded. The bacterial pellet was washed twice with sterile PBS, and finally resuspended in sterile PBS and diluted to the specified bacterial concentration. It was then stored on ice for use in chick challenge. When chicks reached 8 days of age, 0.5 mL of the bacterial suspension (bacterial concentration: 2 × 10⁻⁶) was administered via crop gavage. 9 (cfu / ml).

[0107] 3.3.4 Organ Index

[0108] Chicks were weighed on days 1 and 7 after viral infection, then euthanized by bleeding from the neck. The thymus, spleen, bursa of Fabricius, and liver were removed, weighed, and organ indices were calculated.

[0109] Organ index (g / kg) = Organ weight (g) / Broiler live weight (kg).

[0110] like Figure 6 As shown, compared with the control group, the *Lactobacillus henii* HNAU2301 experimental groups of the present invention significantly increased the serum IgY levels of 1-day-old chicks and chicks on days 1 and 7 after challenge with *Salmonella pullorum* (P<0.05).

[0111] 3.3.5 Tissue bacterial load

[0112] On days 1 and 7 post-infection in chicks, small amounts of liver, spleen, and cecal contents were aseptically collected, weighed, and added to sterile PBS at a 1:9 ratio. The mixture was then homogenized at 60 Hz for 240 seconds using a high-throughput homogenizer (SCIENTZ-48, Ningbo Xinzhi Biotechnology Co., Ltd.) to prepare a 10% homogenate. The homogenate was subsequently diluted 10% with sterile PBS. 4 10 5 10 6 Take 100 μl and spread it on a DHL (bile salt sulfur milk agar) plate. After incubating in an incubator for 24 hours, calculate the number of colonies and the bacterial load per gram of tissue and contents, and perform logarithmic processing.

[0113] like Figure 7 As shown, on day 7 of challenge with Salmonella pullorum in chicks, compared with the control group, the experimental groups of Lactobacillus HNAU2301 significantly reduced the bacterial load in the spleen and liver (P<0.05).

[0114] 3.3.5 Real-time quantitative PCR

[0115] Cecal tonsils, liver, spleen, and jejunum tissues were aseptically collected from chicks on days 1 and 7 after viral challenge. After flash freezing in liquid nitrogen, the tissues were stored at -80°C for the detection of mRNA expression levels of inflammatory factors and intestinal barrier-related genes. Total RNA was extracted using the SteadyPure Universal RNA Extraction Kit (Hunan Aikerui Biotechnology Co., Ltd.), following the kit's instructions. The concentration and purity of the RNA were determined using a BioPhotometer nucleic acid and protein analyzer. The qualified RNA was then used to synthesize cDNA using the Evo M-MLV reverse transcription kit (Hunan Aikerui Biotechnology Co., Ltd.) for mRNA expression level detection, following the kit's instructions. cDNA synthesis conditions were 37°C for 15 min and 85°C for 5 s. qPCR reactions were strictly performed according to the instructions for the SYBR Green Pro Taq HS premixed qPCR kit (Hunan Aikerui Biotechnology Co., Ltd.). Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. β-actin was used as an internal reference gene, and 2... -ΔΔCt The mRNA expression level of the target gene relative to the internal reference gene β-actin was calculated using the method described in Table 1 below. The gene primer sequences are shown in Table 1.

[0116] Table 1 Primer sequences of related genes

[0117]

[0118]

[0119] The results are as follows Figure 8 , 9 As shown. Figure 8 As shown, the addition of *Lactobacillus henii* HNAU2301 to the diet significantly reduced the mRNA expression of pro-inflammatory factors IFN-G, TNF-α, and IL-6 in the spleen of chicks challenged with *Salmonella pullorum* on day 1 (P<0.05), and increased the mRNA expression of the anti-inflammatory factor IL-10 (P>0.05). Figure 9 As shown, on day 1 of challenge with Salmonella pullorum in chicks, compared with the control group, the addition of Lactobacillus HNAU2301 in each experimental group significantly increased the mRNA expression of MUC2 in the jejunum (P<0.05).

[0120] In summary, the *Lactobacillus henii* HNAU2301 provided by this invention can significantly increase the level of yolk IgY, and can improve the fertilization rate and hatchability of hatching eggs, thereby improving the production performance of hens. Analysis of hatched offspring revealed that adding a certain dose of *Lactobacillus henii* HNAU2301 to the hen's diet significantly reduced the bacterial load in the spleen, liver, and cecal contents of chicks, decreased the expression of pro-inflammatory factors (IFN-G, TNF-α, IL-6) and the anti-inflammatory factor IL-10 mRNA in the spleen and cecal tonsils of chicks challenged with *Salmonella pullorum*, and increased the expression of jejunal mucin MUC2 mRNA in chicks challenged with *Salmonella pullorum*. This indicates that the *Lactobacillus henii* of this invention can improve the production performance of breeder hens and protect chicks from pathogen infection by increasing maternal antibody levels, also demonstrating the important role of yolk antibodies in protecting chicks from pathogen infection.

[0121] The above description is only a part of the preferred embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of *Lactobacillus henii* that can increase the level of maternal antibody IgY in breeder hens, characterized in that, The Latin name of the *Lactobacillus gallinarum* is HNAU2301, and its accession number at the China General Microbiological Culture Collection Center is CGMCC NO.29242.

2. A probiotic preparation, characterized in that, The probiotic agent contains *Lactobacillus henii* as described in claim 1.

3. The probiotic agent according to claim 2, characterized in that, The probiotic agent also includes excipients, namely maltodextrin.

4. The probiotic agent according to claim 2 or 3, characterized in that, The formulation of the microbial agent is liquid, powder, or granules.

5. The probiotic agent according to claim 4, characterized in that, The bacterial agent is freeze-dried bacterial powder.

6. A freeze-dried Lactobacillus heniorum powder, characterized in that, The freeze-dried Lactobacillus henii powder is prepared from the Lactobacillus henii described in claim 1, specifically including the following steps: Take the frozen Lactobacillus heniella, revive and propagate it, with a volume ratio of culture medium to bacterial solution of 500:1; After culturing for 24 hours, the expanded Lactobacillus hen was centrifuged, the supernatant was discarded, and the sample was washed three times with sterile 1 × PBS. After washing, centrifugation was used to obtain bacterial sludge. A 10% maltodextrin PBS solution was added to the centrifuged bacterial sludge at a volume ratio of 1:

1. After mixing, the mixture was frozen at -80°C. The frozen bacteria are freeze-dried and then ground into powder.

7. The application of the *Lactobacillus henensis* as described in claim 1, or the bacterial agent as described in any one of claims 2-5, or the freeze-dried *Lactobacillus henensis* powder as described in claim 6, in the preparation of feed for breeding chickens; the feed has at least one of the following functions: (1) Increase the level of maternal antibody IgY in breeder chickens; (2) Enhance the ability of breeding chicken offspring to inhibit Salmonella pullorum infection; (3) Improve the fertilization rate and hatching rate of hatching eggs.

Images