Bifidobacterium nichini hc2 and use thereof

Abstract

The application discloses a lactobacillus plantarum HC2 and application thereof, the strain is preserved in the China General Microbiological Culture Collection Center on August 29, 2023, the preservation address is No. 3, Xibei Road, Chaoyang District, Beijing, the preservation number is CGMCC No.28288, and the Latin name is Lactobacillus kitasatonis The application further discloses application of the lactobacillus plantarum HC2.The lactobacillus plantarum HC2 provided by the application has certain tolerance to an acidic environment, can adhere to intestinal epithelial cells, remodels intestinal flora, and secretes metabolic products to inhibit adhesion and colonization of intestinal harmful bacteria, so that the lactobacillus plantarum HC2 can improve immune organ indexes of laying hens and be used for improving the immunity of chicken groups.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering and relates to a new strain of Lactobacillus Kitasato, specifically Lactobacillus Kitasato HC2 and its applications. Background Technology

[0002] The gut microbiota is closely related to the absorption and utilization of various nutrients in animals, and plays an important role in enhancing the host's disease resistance. When the gut microbiota is in balance, the native flora not only does not cause disease in the host, but also has important functions such as synthesizing vitamins and other nutrients and aiding digestion. It also has biological antagonistic and immune effects against pathogens and opportunistic pathogens from the intestinal flora. Therefore, the balance of the gut microbiota is of great significance to the host.

[0003] Lactobacillus Kitasato is a lactic acid bacterium found in the intestines of vertebrates and mammals. It has a strong ability to adhere to the intestinal mucosa, which can improve the distribution of intestinal flora. It also has biological antagonistic and immune effects against foreign pathogens and conditional pathogens, thereby enhancing the host's mucosa's resistance to infection.

[0004] There is currently relatively little research on Lactobacillus Kitasato. Summary of the Invention

[0005] The purpose of this invention is to provide a Kitasato Lactobacillus HC2, which can effectively inhibit a variety of pathogenic bacteria and increase the content of lactic acid bacteria, thereby improving immunity.

[0006] Another object of the present invention is to provide an application of the aforementioned Lactobacillus Kitasato HC2.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A strain of Lactobacillus kitasatonis, HC2, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is August 29, 2023, and the accession number is CGMCC No. 28288.

[0009] As a limitation, its 16S rRNA sequence is as follows:

[0010] TGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCGGAACCAACAGATTTGC

[0011] TTCGGTAATGACGTTGGGAAAGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCTAAGT

[0012] CTGGGATACCATTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTTTAAA

[0013] AGGCGGCGTAAGCTGTCGCTAAGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTA

[0014] CCAAGGCAACGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAA

[0015] CTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTG

[0016] AGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCT

[0017] TTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT

[0018] GGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAAAATAAGTCTGATGTGAAAG

[0019] CCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACT

[0020] CCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTG

[0021] CAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTA

[0022] AACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCG

[0023] CCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCA

[0024] TGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGTGCAATCTGTAGAGAT

[0025] ACGGAGTTCCCTTCGGGGACGCTAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGT

[0026] TGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTATTAGTTGCCAGCATTAAGTTGGGCACTCTAATG

[0027] AGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGG

[0028] CTACACACGTGCTACAATGGGCAGTACAACGAGAAGCAAGCCTGCGAAGGCAAGCGAATCTCTTAAAG

[0029] CTGTTCTCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGAT

[0030] CAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTCTGCAA.

[0031] The present invention also provides the application of the above-mentioned Lactobacillus Kitasato HC2, which is used to regulate intestinal flora and improve animal immunity.

[0032] By adopting the above technical solution, the technical progress achieved by this invention compared with the prior art is as follows:

[0033] The present invention provides a Kitasato Lactobacillus HC2 strain that exhibits a certain degree of tolerance to acidic environments. It adheres to intestinal epithelial cells and can effectively inhibit pathogenic bacteria such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, Serratia marcescens, Proteus mirabilis, Campylobacter jejuni, and Clostridium perfringens. At the same time, it increases the content of probiotics such as Lactobacillus johnsonii, Lactobacillus reuteri, and Lactobacillus salivarius. Its secreted metabolites inhibit the adhesion and colonization of harmful bacteria in the intestines, promote the growth and development of immune organs, and improve animal immunity. Attached Figure Description

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments:

[0035] Figure 1 This is an electrophoresis diagram of the 16S rRNA sequence of Lactobacillus Kitasato HC2 in Example 2 of the present invention. Detailed Implementation

[0036] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] In the following examples: tryptone soybean agar medium (TSA medium), MRS agar medium, MRS broth medium and nutrient agar medium (NA) were purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0040] Tryptone and yeast powder were purchased from OXOID.

[0041] Sodium chloride was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.

[0042] Biochemical test tubes and Gram staining solution were purchased from Hangzhou Binhe Biological Reagent Co., Ltd.

[0043] Calf serum, purchased from Thermo Fisher Scientific.

[0044] Example 1: A type of Lactobacillus Kitasato HC2

[0045] This embodiment provides a strain of Lactobacillus kitasatonis HC2, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is August 29, 2023, the accession number is CGMCC No. 28288, and the Latin name is Lactobacillus kitasatonis.

[0046] The 16S rRNA sequence of one of the Lactobacillus Kitasato HC2 strains is as follows:

[0047] TGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCGGAACCAACAGATTTGC

[0048] TTCGGTAATGACGTTGGGAAAGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCTAAGT

[0049] CTGGGATACCATTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTTAAA

[0050] AGGCGGCGTAAGCTGTCGCTAAGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTA

[0051] CCAAGGCAACGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAA

[0052] CTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTG

[0053] AGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCT

[0054] TTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT

[0055] GGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAAAATAAGTCTGATGTGAAAG

[0056] CCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACT

[0057] CCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTG

[0058] CAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTA

[0059] AACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCG

[0060] CCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCA

[0061] TGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGTGCAATCTGTAGAGAT

[0062] ACGGAGTTCCCTTCGGGGACGCTAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGT

[0063] TGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTATTAGTTGCCAGCATTAAGTTGGGCACTCTAATG

[0064] AGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGG

[0065] CTACACACGTGCTACAATGGGCAGTACAACGAGAAGCAAGCCTGCGAAGGCAAGCGAATCTCTTAAAG

[0066] CTGTTCTCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGAT

[0067] CAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTCTGCAA.

[0068] Example 2: A method for isolating and purifying Lactobacillus Kitasato HC2

[0069] This embodiment provides a method for isolating and purifying Lactobacillus Kitasato HC2, which is carried out according to the following steps:

[0070] S1. Sample Collection

[0071] Under aseptic conditions, the contents of the ileum of an adult healthy chicken were collected and placed in a sterile anaerobic incubator for later use.

[0072] S2. Isolation and purification of lactobacilli from the sample

[0073] Take 1g of the contents and inoculate it into casein peptone soybean agar medium (TSA medium) containing 1.5% (w / w) fetal bovine serum, and incubate it in an anaerobic incubator at 37°C for 48h.

[0074] The anaerobic environment in the anaerobic incubator consists of H2, CO2, and N2 in a volume ratio of 10:5:85.

[0075] Single colonies from TSA medium were inoculated onto MRS agar medium and incubated statically in a bacterial incubator at 37°C for 48 hours to obtain pure cultures.

[0076] Single colonies of the pure culture were picked and inoculated into MRS broth medium. The inoculated MRS broth medium was placed in a bacterial incubator at 37°C and incubated for 48 hours to obtain the purified strain, which was then stored.

[0077] S3. Identification of strains

[0078] The preserved strain was subjected to molecular biological identification. Through DNA extraction, PCR amplification, and 16S rDNA gene sequencing, the strain was finally identified as Lactobacillus Kitasato HC2 using the NCBI BLAST website.

[0079] Electrophoresis image of the 16S rRNA sequence of Lactobacillus Kitasato HC2 is shown below. Figure 1 As shown; the electrophoresis conditions were 3 μL sample + 1.0% agarose gel, and the marker band composition was: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp. The concentration of the 750bp band was 60 ng / 3 μL, and the concentration of the other bands was 30 ng / 3 μL. The electrophoresis direction was from top to bottom.

[0080] Example 3: Characterization of Lactobacillus Kitasato HC2

[0081] 1) Basic characteristics

[0082] This embodiment studies the basic bacteriological characteristics of Lactobacillus Kitasato HC2, and the results are shown in Table 1:

[0083] Table 1. Basic characteristics of Lactobacillus Kitasato HC2

[0084]

[0085]

[0086] 2) Biochemical characteristics

[0087] Lactobacillus Kitasato HC2 is a facultative anaerobic bacterium with good acid tolerance, high growth rate, and rapid acid production. Its optimal growth temperature is 36-40℃, and its culture has a certain acidic taste.

[0088] Lactobacillus Kitasato HC2 was inoculated into microbiochemical reaction tubes containing glucose, lactose, sucrose, fructose, maltose, ribose, salicin, sorbitol, xylose, and mannitol, respectively, and incubated in a 37°C bacterial incubator for 48 hours. The bacterial strain was observed to see if it could utilize these substances, and the biochemical results were recorded.

[0089] Lactobacillus HC2 from Kitasato was inoculated into a semi-solid agar biochemical test tube and cultured in a 37°C bacterial incubator for 48 hours. The motility of the strain was observed and the biochemical experimental results were recorded.

[0090] A single colony of *Lactobacillus Kitasato* HC2 was mixed with 20 μL of 3.0% hydrogen peroxide (a reagent for catalase biochemical tests). The presence or absence of bubbles was observed (bubble formation indicated a positive result), and the biochemical results were recorded. The biochemical characteristics are shown in Table 2.

[0091] Table 2 Biochemical characteristics of Lactobacillus Kitasato HC2

[0092] Experimental Project result Experimental Project result glucose + Salicylic acid - lactose + Sorbitol - sucrose + catalase - fructose + Xylose - maltose + Mannitol - Ribose + athleticism -

[0093] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0094] As shown in Table 2, Lactobacillus Kitasato HC2 utilizes most sugars such as glucose, lactose, sucrose, fructose, maltose, and ribose, does not produce catalase, and has no motility (in a semi-solid biochemical test tube, it grows along the inoculation line without diffusion, i.e., it has no motility).

[0095] 3) Acid resistance

[0096] Take 6 100 μL portions with a concentration of 1.0 × 10⁻⁶. 7 CFU / mL of *Lactobacillus Kitasato* HC2 bacterial suspension was inoculated into MRS broth medium at pH 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0, respectively, and incubated statically at 37℃ for 48 h. The viable count was calculated according to the bacterial plating counting method. The results are shown in Table 3.

[0097] Table 3. Viable bacterial count of Lactobacillus Kitasari HC2 cultured at different pH values.

[0098] pH value viable bacteria count (CFU / mL) 1.0 0 2.0 0 3.0 <![CDATA[1.70×10 7 ]]> 4.0 <![CDATA[2.10×10 8 ]]> 5.0 <![CDATA[5.80×10 8 ]]> 6.0 <![CDATA[8.00×10 8 ]]>

[0099] As shown in Table 3, Lactobacillus Kitasato HC2 has good acid resistance and can grow in an environment with a pH of 3.0.

[0100] Example 4: Inhibitory effect of Lactobacillus Kitasato HC2 on pathogenic bacteria

[0101] This embodiment studies the inhibitory effect of Lactobacillus Kitasato HC2 on pathogenic bacteria. The specific method is as follows:

[0102] S1. Streak Kitari Lactobacillus HC2 onto MRS agar medium and incubate at 37°C for 48 hours. Then, pick a single colony of Kitari Lactobacillus HC2 and inoculate it onto MRS broth medium. Incubate at 37°C for 48 hours to obtain Kitari Lactobacillus HC2 culture solution.

[0103] S2. Take pathogenic bacterial strains (including Escherichia coli, Salmonella, Pseudomonas aeruginosa, Serratia marcescens, Proteus mirabilis, Campylobacter jejuni, and Clostridium perfringens) preserved at -75℃, streak them onto suitable agar medium, incubate them at 37℃ for 24 hours, pick single colonies and inoculate them into suitable medium, incubate them in a shaker at 37℃ for 16 hours to obtain the pathogenic bacterial strain culture solution;

[0104] S3. After diluting the pathogenic bacterial strain culture 10 times, take 100 μL of the liquid and spread it onto a suitable agar medium. Make three circular wells on the plate, and add 100 μL of Lactobacillus Kitasato HC2 culture medium to each well. Transfer the agar medium to a 37℃ environment and incubate for 24-48 h. Observe the antibacterial results, as shown in Table 4:

[0105] Table 4. Inhibitory effect of Lactobacillus Kitasato HC2 on pathogenic bacteria.

[0106]

[0107]

[0108] As shown in Table 4, Lactobacillus Kitasato HC2 has an inhibitory effect on pathogenic bacteria such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, Serratia marcescens, Proteus mirabilis, Campylobacter jejuni, and Clostridium perfringens.

[0109] Example 5: Biosafety of Lactobacillus Kitasato HC2

[0110] In this embodiment, *Lactobacillus Kitasato* HC2 was inoculated onto chicken embryos to test its biosafety. The specific procedures are as follows:

[0111] S1. *Lactobacillus Kitari* HC2 was streaked onto MRS agar medium. After incubation at 37°C for 48 hours, a single colony of *Lactobacillus Kitari* HC2 was picked and inoculated onto MRS broth medium. The culture was then incubated at 37°C for 48 hours to obtain *Lactobacillus Kitari* HC2 bacterial culture. The viable count was calculated to be 8.00 × 10⁻⁶ cells / day using the plating count method. 8 Take four portions of Lactobacillus HC2 bacterial culture medium and dilute them to four concentrations: 100 CFU / 0.1 mL, 300 CFU / 0.1 mL, 1000 CFU / 0.1 mL, and 3000 CFU / 0.1 mL.

[0112] S2. Take 50 11-day-old SPF chicken embryos and divide them into 5 groups with 10 SPF chicken embryos in each group. Four groups are experimental groups and are inoculated with the same volume of the above four concentrations of Lactobacillus Kitasato HC2 bacterial culture medium. One group is the control group and is inoculated with the same volume of physiological saline. After inoculation, observe for 7 days, candling the eggs every day and counting the survival of chicken embryos.

[0113] Seven days later, all chicken embryos survived normally, proving that Lactobacillus Kitasato HC2 has biosafety.

[0114] Example 6: Application of Lactobacillus Kitasato HC2

[0115] In this embodiment, *Lactobacillus Kitasato* HC2 was applied to Hy-Line Brown chicks to study its effect on chicken immunity. The specific experimental method is as follows:

[0116] I. Laboratory Animals:

[0117] Four hundred healthy 1-day-old Hy-Line Brown chicks with no significant weight difference were randomly divided into two groups of 200 each and fed complete formulated pelleted feed.

[0118] Group I served as the control group, with free access to water; Group II served as the experimental group, with participants drinking 0.1 mL of *Lactobacillus Kitasato* HC2 bacterial solution (1×10⁻⁶) daily. 8 (CFU / mL), after which the animals were allowed free access to water; there were 5 replicates in each of the experimental and control groups, with 40 animals in each replicate.

[0119] II. Experimental Preparation:

[0120] Disinfect the chicken house, chicken cages and surrounding environment before the experiment, and maintain constant light and mechanical ventilation during the experiment.

[0121] The experiment used cage rearing, with automatic temperature control and heating devices in the chicken house. Hy-Line Brown chicks had free access to feed, and the experiment lasted for 42 days. The daily chicken house management plan and related immunization program were implemented in accordance with the chicken farm brooding farm standards.

[0122] III. Test Methods

[0123] At 43 days of age, four experimental chickens close to the average weight were selected from each replicate group for slaughter. Feed was withheld at 20:00 the day before slaughter, but water was allowed to flow freely.

[0124] Experiment 1.

[0125] After slaughter, 4 cm of the middle ileum and cecum were cut from each experimental chicken and placed into 10 mL sterile centrifuge tubes, which were then labeled. The contents were immediately squeezed out after sampling to detect the number of lactic acid bacteria and Escherichia coli. Lactic acid bacteria were counted using solid MRS medium, and Escherichia coli were counted using crystal violet neutral red bile agar (VRBA). The bacterial count was expressed as the logarithm of the number of colonies per 1 g of intestinal contents [lg(CFU / g)]. The results are shown in Table 5.

[0126] Table 5. Effects of Lactobacillus Kitasato HC2 on the intestinal flora of Hy-Line Brown chicks.

[0127] Bacterial count (lg(CFU / g)) Group I Group II ileal lactic acid bacteria count <![CDATA[7.14±0.12 a ]]> <![CDATA[9.13±0.11 b ]]> ileal Escherichia coli count <![CDATA[8.19±0.04 a ]]> <![CDATA[6.01±0.15 b ]]> Cecal lactobacillus count <![CDATA[7.42±0.08 a ]]> <![CDATA[9.28±0.10 b ]]> cecal E. coli count <![CDATA[7.96±0.08 a ]]> <![CDATA[5.75±0.10 b ]]>

[0128] Note: Different lowercase superscript letters in the same row indicate significant differences (P<0.05).

[0129] Table 5 shows that the content of lactic acid bacteria in the ileum and cecum of the experimental group was significantly higher than that of the control group, while the content of Escherichia coli was significantly lower. Lactic acid bacteria mainly protect the body's health by activating the intestinal autoimmunity, regulating the adhesion of intestinal cells, promoting the development of immune organs, or having metabolites with effects similar to immune adjuvants. This indicates that Lactobacillus Kitasato HC2 can effectively regulate the intestinal flora, increase the number of probiotics in chickens, reduce the number of pathogens, and improve immunity.

[0130] Experiment 2.

[0131] Lactic acid bacteria with different colony morphologies were isolated, purified, and cultured from the solid MRS medium in Experiment 1. The strains were preserved and sent to Beijing Liuhe Huada Genomics Co., Ltd. for 16S rDNA gene sequencing. The specific bacterial species were finally determined by NCBI BLAST.

[0132] The strains isolated in this experiment were identified as containing four types of lactic acid bacteria: *Lactobacillus johnsonii*, *Lactobacillus reuteri*, *Lactobacillus salivarius*, and *Lactobacillus kitasato*. Studies have shown that after entering the intestines of livestock and poultry, lactic acid bacteria can increase the relative abundance of beneficial intestinal flora through biological oxygen depletion and competitive site occupation, and produce metabolites such as lactic acid and lactic acid peptides to inhibit the proliferation of pathogenic microorganisms. For example, *Lactobacillus reuteri* secretes bacteriocins (reuterin), which have broad-spectrum antibacterial activity and can effectively inhibit pathogenic bacteria; furthermore, its extracellular polysaccharides have an inhibitory effect on the inflammatory response caused by enterotoxigenic *Escherichia coli* infection. *Lactobacillus johnsonii* exerts its antibacterial effect by producing hydrogen peroxide and lactic acid. *Lactobacillus salivarius* can alleviate inflammation caused by enterotoxigenic *Escherichia coli* infection through immune regulation pathways.

[0133] This experiment demonstrates that Lactobacillus Kitasato HC2 can significantly increase the total lactic acid bacteria content in the ileum and cecum, thereby increasing the number of probiotics, inhibiting the growth of pathogenic bacteria, and thus improving immunity.

[0134] Experiment 3.

[0135] The internal organs of the experimental chickens were completely removed after slaughter, weighed, and the organ index was calculated. The results are shown in Table 6.

[0136] Organ Index Calculation Formula: Organ Index = Fresh Organ Weight (g) / Live Body Weight (kg) × 100%

[0137] Table 6. Effects of Lactobacillus Kitasato HC2 on organs in Hy-Line Brown chicks.

[0138]

[0139]

[0140] Note: Different lowercase superscript letters in the same row indicate significant differences (P<0.05).

[0141] The growth and development of immune organs are important indicators of the strength of a chicken's immunity. Changes in their weight and organ indices can reflect the strength of the body's immune function and the degree of lymphocyte proliferation to a certain extent, and can roughly estimate the strength of immune function. The period from 1 to 42 days of age is an important period for the development of internal organs and immune organs in laying hens. As shown in Table 6, compared with the control group, the organ indices of organs closely related to immunity, such as the spleen, thymus, and bursa of Fabricius, in the experimental group were significantly increased. This shows that the experimental group that took Lactobacillus Kitasato HC2 had stronger immunity than the control group.

[0142] Lactobacillus Kitasato HC2 can promote the growth and development of immune organs and improve organ index, possibly because it can significantly increase the content of probiotics such as lactic acid bacteria. The ability of lactic acid bacteria to improve the immune organ index may be due to: 1. Lactic acid bacteria contain immune-active components (such as peptidoglycan), which can promote the development and maturation of immune organs; 2. Lactic acid bacteria can act as an antigen, stimulating the development of immune organs and thus promoting their maturation; 3. During intestinal reactions, lactic acid bacteria can produce nutrients that are beneficial to the development and maturation of immune organs.

[0143] In addition to direct administration of bacterial solutions, Lactobacillus Kitasato HC2 can also be used to prepare probiotic agents, which can be used as additives in food, medicine, and feed to regulate intestinal flora and improve animal immunity, thereby achieving probiotic and health benefits for animals.

Claims

1. A type of Lactobacillus Kitasato ( Lactobacillus kitasatonis HC2, characterized in that, The *Lactobacillus kiwi* HC2 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is August 29, 2023, and the accession number is CGMCC No. 28288.

2. The *Lactobacillus Kitasato* as described in claim 1 (… Lactobacillus kitasatonis The application of HC2 is characterized by, The aforementioned Lactobacillus Kitasato HC2 is used to regulate intestinal flora and improve animal immunity.

Images