Animal combined lactobacillus HR1 and application thereof

Abstract

The invention discloses animal combined lactobacillus HR1 and application thereof, and belongs to the technical field of microbial preparations. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 28, 2025, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.34037. The invention not only enriches a pet probiotic resource library, but also provides a non-drug and side-effect-free new way for treating cat milk allergic diarrhea, meets the urgent demand of the market on efficient and safe pet health management products, and has important practical value and scientific research significance. The implementation of the invention is expected to lead a new trend in the field of pet nutrition and health management, and contributes to sustainable development of the pet health industry.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial agents, and particularly to a Ligilactobacillus animalis HR1 and its application. Background Art

[0002] In recent years, with the rapid development of the pet economy, the health management of pet cats has become the focus of common concern for pet owners and researchers. Allergic diarrhea, as a common intestinal dysfunction symptom in felines, has a complex pathogenesis, involving multiple factors such as food intolerance, environmental allergen exposure, and intestinal microbial ecological imbalance. Although there are a variety of probiotic products on the current market, specific products for feline allergic diarrhea are still lacking, mainly due to the non-specificity of probiotic species, insufficient activity, and limitations in preparation technology. Therefore, the development of a probiotic preparation with high-efficiency targeting, high activity, and long-term stability has great scientific significance and market value for improving the quality of life of pet cats and reducing disease risks. Summary of the Invention

[0003] The object of the present invention is to provide a Ligilactobacillus animalis HR1 and its application to solve the problems existing in the above-mentioned prior art. The present invention involves a deep exploration in the field of pet nutrition and health, particularly focusing on the precise regulation of the intestinal microecology of felines and innovative improvement strategies for allergic diseases caused by common cow's milk. The present invention aims to provide an efficient solution for feline allergic diarrhea through a highly specialized and scientifically formulated probiotic combination and its fine preparation process, thus opening a new chapter in pet health management.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is a Ligilactobacillus animalis HR1, characterized in that the strain was deposited with the China General Microbiological Culture Collection Center on March 28, 2025, at the address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number of CGMCC NO. 34037.

[0006] Another technical solution of the present invention is a microbial agent, including the Ligilactobacillus animalis HR1.

[0007] A further technical solution of the present invention is the application of the Ligilactobacillus animalis HR1 or the microbial agent in the preparation of a product for treating feline allergic diarrhea.

[0008] Another technical solution of the present invention is a product for treating feline allergic diarrhea, including the Ligilactobacillus animalis HR1 or the microbial agent.

[0009] Based on the above technical solutions, the present invention has the following technical effects:

[0010] By comprehensively applying the 16S quantitative analysis technology in combination with the streak plate method and anaerobic culture, the present invention efficiently screens and identifies the core probiotic strain in the cat intestine - Lactobacillus animalis HR1. Through a comprehensive evaluation of the acid resistance, bile salt resistance, hydrophobicity, auto - and co - aggregation ability, and antibacterial ability of this strain, as well as the scientific verification of hemolytic safety and fermentation function, the safety and functionality of the probiotic are ensured. Further, the freeze - drying technology is used to effectively combine the probiotic with skim milk powder, which not only significantly improves the stability and portability of the probiotic, but also extends the shelf life of the product. Through clinical trials on healthy cats, the present invention successfully verifies the positive effect of the probiotic on improving cat intestinal health, including optimizing fecal quality and maintaining normal levels of blood routine and blood biochemical indexes. In summary, the present invention not only provides a scientific basis and technical support for the development of pet probiotic products, but also makes an important contribution to promoting the innovative development of the pet health industry.

[0011] Compared with the prior art, the present invention not only enriches the pet probiotic resource library, but also provides a new non - drug and non - side - effect approach for the treatment of cat cow milk allergic diarrhea, meeting the urgent market demand for highly efficient and safe pet health management products, and having important practical value and scientific research significance. The implementation of the present invention is expected to lead a new trend in the field of pet nutrition and health management, and contribute to the sustainable development of the pet health industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0013] Figure 1 Effects of probiotic supplements on cat diet intervention. Among them, A is the experimental procedure for constructing a cat allergic diarrhea model and evaluating the effect of probiotic intervention (n = 5), B is the representative fecal status observed during the experiment, C is the fecal score on the 7th day of the experiment (n = 5), and D is the fecal score on the 14th day of the experiment (n = 5). Data are expressed as the mean ± standard deviation of three independent experiments, and statistical significance is determined by ANOVA analysis (p < 0.05, p < 0.01, p < 0.001).

[0014] Figure 2Results of intestinal 16S sequencing. Among them, A shows the composition of the top 10 species in terms of abundance at the phylum level, B shows the composition of the top 20 species in terms of abundance at the genus level, C-D show the Chao-1 index analysis of intestinal α-diversity, G-H show the PCoA analysis of intestinal β-diversity, and I shows the results of Lefse analysis.

[0015] Figure 3 Results of serum metabolomics and multi-omics combined analysis. Among them, A-B are the principal component analysis (PCA) of serum metabolites in positive and negative ion modes respectively (comparison between the diarrhea group and the treatment group), C is the differential metabolites identified by serum metabolomics, D is the hierarchical clustering analysis of differential metabolites, E is the enrichment analysis of up-regulated differential metabolites, F is the combined analysis of key differential microbiota (from LEfSe) and serum metabolites, and G-I are the KEGG pathway analysis of the top 5 metabolites with the highest correlation. Detailed implementation manners

[0016] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0017] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0018] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0019] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are only exemplary.

[0020] The terms "comprising", "including", "having", "containing", etc. used in this text are all open-ended terms, meaning including but not limited to.

[0021] The technical solutions described in the present invention are all conventional solutions in the art unless otherwise specified. The reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0022] An embodiment of the present invention provides a strain of Ligilactobacillus animalis HR1, which was deposited on March 28, 2025 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit address being No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 34037.

[0023] An embodiment of the present invention also provides a microbial agent, including the Ligilactobacillus animalis HR1.

[0024] An embodiment of the present invention also provides the use of the Ligilactobacillus animalis HR1 or the microbial agent in the preparation of a product for treating feline allergic diarrhea.

[0025] An embodiment of the present invention also provides a product for treating feline allergic diarrhea, including the Ligilactobacillus animalis HR1 or the microbial agent.

[0026] An embodiment of the present invention also provides an animal feed for alleviating feline allergic diarrhea, including the Ligilactobacillus animalis HR1 or the microbial agent.

[0027] The present invention aims to solve the common problem of feline cow milk allergic diarrhea in the current pet health field and proposes an innovative probiotic solution. In the present invention, a strain of Ligilactobacillus animalis HR1 with a significant effect of alleviating feline cow milk allergic diarrhea was successfully isolated and identified from the fecal samples of young healthy cats. This strain showed a positive regulatory effect on the feline intestinal microecological environment in vitro and in vivo experiments, especially in alleviating the intestinal inflammatory response caused by cow milk protein.

[0028] The present invention further provides an optimized preparation method. The screened Ligilactobacillus animalis HR1 is processed through carefully designed fermentation culture, concentration, and freeze-drying processes to obtain a highly efficient and stable freeze-dried bacterial powder preparation. Through a 14-day feeding experiment on the test cats, the results showed that this probiotic preparation could significantly reduce the diarrhea frequency, improve the fecal characteristics, and at the same time improve the diversity of intestinal microorganisms and the abundance of beneficial bacteria, promoting the recovery of intestinal barrier function, verifying its effectiveness and safety in alleviating feline cow milk allergic diarrhea.

[0029] Efficient screening technology: By adopting a high-precision screening method, the Lactobacillus animalis HR1 strain, which can significantly relieve cow milk allergic diarrhea in cats, is specifically screened from the feces of young and healthy cats. This is one of the core technologies of the present invention, ensuring the specificity and effectiveness of the probiotics.

[0030] Optimized fermentation and freeze-drying process: Specific fermentation and culture conditions for the Lactobacillus animalis HR1, as well as subsequent concentration and freeze-drying processes, are invented to ensure the high activity, high stability, and long-term preservation of the probiotics, laying a solid foundation for the commercial application of probiotic preparations.

[0031] Clear verification of feeding effects: Through a strict animal experiment design, a feeding experiment is conducted on the test cats for 14 consecutive days to scientifically evaluate the improvement effects of the probiotic preparation on cow milk allergic diarrhea in cats, including the reduction of diarrhea frequency, the improvement of fecal characteristics, and the enhancement of intestinal health indicators, providing strong evidence support for the efficacy of the product.

[0032] Example 1

[0033] Test subjects and sample collection: Four young and healthy Maine cats are selected as test subjects. 0.2 g of feces is collected from each cat for quantitative analysis of microbial 16S.

[0034] Screening of probiotics: Prepare 2 ml sterile and enzyme-free centrifuge tubes and add 1 ml of PBS solution sterilized by high temperature and high pressure.

[0035] Sample treatment: Weigh 0.1 g of samples from the front, middle, and rear positions of each cat's feces and add them to the prepared centrifuge tubes respectively. Add magnetic beads with a diameter of 2 mm to each centrifuge tube and grind for 3 minutes at 70 Hz using a high-throughput tissue grinder. Centrifuge at 8000 r / min for 15 minutes at 4°C and repeat 3 times. Transfer the supernatant to a new 1.5 ml sterile and enzyme-free centrifuge tube to obtain a bacterial suspension.

[0036] Preparation and streaking of culture medium: Weigh 63.3 g of MRS solid agar medium (Beijing Solarbio Science & Technology Co., Ltd. 20240228), dissolve it in 1000 ml of distilled water, sterilize it at 121°C under high pressure for 15 minutes, and then dispense it into sterile bacterial culture dishes. After the culture medium solidifies, use the four-zone plate streaking method to streak the bacterial suspension in gradients, and make 2 replicates for each bacterial suspension.

[0037] Cultivation and selection: Place the petri dish in a 2.5L anaerobic cultivation bag, add a 2.5L anaerobic gas-generating pack, and cultivate in a mold incubator (MJ-250B, Shanghai Jiasheng Experimental Equipment Co., Ltd.) for 24 hours. Then, use the eight-zone streaking method to select single colonies with different morphological sizes and inoculate them into sterilized and dispensed MRS liquid medium (49.3g / 1000ml distilled water, autoclaved at 121°C for 15 minutes), and shake in a shaker at 37°C and 180r / min for 24 hours to obtain the bacterial liquid.

[0038] DNA extraction and sequencing: Use the Aikerui Biological Bacterial Genome DNA Extraction Kit (AG21007) to extract the DNA of the bacterial liquid and sequence it. Compare the sequencing results for homology in NCBI. The similarity of the comparison results is 99.13%, and the main probiotic strain is determined to be Ligilactobacillus animalis.

[0039] A strain of Ligilactobacillus animalis HR1 was isolated in the present invention. This strain was deposited at the General Microbiological Center of the China Microbial Culture Collection Center on March 28, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 34037.

[0040] Example 2

[0041] Detection of the characteristics of Ligilactobacillus

[0042] 1 Acid tolerance detection: Prepare MRS liquid medium with pH = 3. Inoculate the Ligilactobacillus bacterial liquid cultured to the logarithmic phase into normal and pH = 3 MRS liquid medium at 1% volume. After anaerobic incubation at 37°C for 2 hours, perform gradient dilution and plating, and count the colonies by the plate counting method after culturing for 24 hours.

[0043] The detection results show that the acid tolerance of this Ligilactobacillus is 78.38%.

[0044] 2 Bile salt tolerance detection: Prepare MRS liquid medium with bile salt concentrations of 0.3% (w / v) and 0.5% (w / v) (pH adjusted to 8). Similarly, inoculate the bacterial liquid, culture it, and then count.

[0045] The detection results show that the ability of this Ligilactobacillus to tolerate low-concentration bile salt 0.3% (w / v) is 9.82%, and the ability to tolerate high-concentration bile salt 0.5% (w / v) is 2.07%.

[0046] 3 Hydrophobicity detection: Take 1mL of the bacterial liquid cultured to the logarithmic phase, centrifuge and discard the supernatant, resuspend with PBS and adjust the absorbance. Add xylene, shake well, let it stand for stratification, measure the absorbance of the upper aqueous phase, and calculate the hydrophobicity.

[0047] The hydrophobicity test result of the combined Lactobacillus showed that it was 54.7%.

[0048] Self-aggregation ability test: Take 1 mL of the bacterial liquid, centrifuge and resuspend it, adjust the absorbance and record the initial value. After anaerobic static incubation at 37 °C for 2 hours, measure the absorbance of the supernatant and calculate the self-aggregation ability.

[0049] The self-aggregation ability test result of the combined Lactobacillus showed that it was 24.33%.

[0050] Co-aggregation ability test: Take 1 mL of the probiotic bacterial liquid and mix it with three pathogenic bacteria, namely Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium, respectively. After centrifugation, resuspend and adjust the absorbance. After anaerobic static incubation at 37 °C for 18 hours, measure the absorbance and calculate the co-aggregation ability.

[0051] The co-aggregation ability test results showed that the co-aggregation ability of the combined Lactobacillus with Salmonella was 53.79%, with Staphylococcus aureus was 85.39%, and with Escherichia coli was 58.88%.

[0052] Antibacterial ability test: Uniformly coat the three pathogenic bacteria on the MRS solid culture dish, place Oxford cups and add the probiotic bacterial liquid. After cultivation, measure the diameter of the antibacterial zone.

[0053] The antibacterial ability test results showed that the average diameter of the antibacterial zone produced when the combined Lactobacillus was co-cultured with Escherichia coli was 10.13 mm, with Salmonella was 11.16 mm, and with Staphylococcus aureus was 14.18 mm

[0054] Hemolysis ability test: Streak culture on Columbia CAN blood agar plate to judge the hemolysis type.

[0055] The hemolysis ability test result showed that the combined Lactobacillus did not produce a hemolysis zone on the plate.

[0056] Determination of short-chain fatty acid content: Use a gas chromatograph (7890B) to determine the content of short-chain fatty acids in the fresh bacterial liquid.

[0057] The short-chain fatty acid content test results showed that the concentration of acetic acid produced by the combined Lactobacillus was 50.38835 mmol / L, propionic acid was 8.784102 mmol / L, isobutyric acid was 4.280312 mmol / L, butyric acid was 2.247236 mmol / L, isovaleric acid was 1.430446, and valeric acid was 9.170651 mmol / L.

[0058] Example 3

[0059] Freeze-drying and preservation of probiotics: The purified Lactobacillus conjugatus was inoculated into MRS liquid medium and cultured for 16 hours, and then the bacterial sludge was obtained by centrifugation. Skim milk powder sterilized at 105°C for 30 minutes was added in a 1:1 ratio, and it was continuously freeze-dried in a freeze dryer for 24 hours and stored at 4°C for later use.

[0060] Cats from the Experimental Animal Center of Northwest A&F University were divided into a control group, a diarrhea group and a treatment group, with 5 cats in each group.

[0061] Treatment group: Healthy cats were fed 0.1 g of bacterial powder protectant or 0.1 g of freeze-dried bacterial powder every day. The feces of the cats were observed and fecal scores were recorded on days 0, 7, and 14. Serum and fecal samples were collected on days 7 and 14 for blood routine, blood biochemical examination, serum metabolomics, fecal microbiota analysis, etc. to evaluate the efficacy of probiotics.

[0062] Control group: Except for not feeding the freeze-dried bacterial powder, other feeding and experiments were exactly the same as those in the experimental group.

[0063] The results showed that the intervention of this Lactobacillus conjugatus strain could significantly improve diarrhea symptoms (the fecal scores of the experimental group returned to a healthy state), reduce the levels of serum total bilirubin (TBIL) and blood urea nitrogen (BUN), and showed a regulatory trend in cholesterol and triglyceride metabolism. The preparation process of this strain was stable (the resuscitation activity was 1.24×108 CFU / g), and it did not cause abnormalities in blood routine and most biochemical indexes, and had good safety, which was suitable for the relief of feline allergic diarrhea and the regulation of metabolic function.

[0064] Obviously, the above-mentioned embodiments of the present invention are only examples for clearly explaining the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A strain of Ligilactobacillus animalis HR1, characterized in that, This strain was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on March 28, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 34037.

2. Microbial inoculant, characterized in that, Including Lactobacillus animalis HR1 described in claim 1.

3. Use of Lactobacillus animalis HR1 described in claim 1 or the microbial agent described in claim 2 in the preparation of a product for treating feline allergic diarrhea.

4. A product for treating feline allergic diarrhea, characterized in that, Including Lactobacillus animalis HR1 described in claim 1 or the microbial agent described in claim 2.

5. An animal feed for relieving allergic diarrhea in cats, characterized in that, Including Lactobacillus animalis HR1 described in claim 1 or the microbial agent described in claim 2.

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