Lactobacillus acidophilus for relieving chemotherapeutic diarrhea and improving constipation and application of lactobacillus acidophilus

Abstract

The invention discloses lactobacillus acidophilus for relieving chemotherapeutic diarrhea and improving constipation and application of the lactobacillus acidophilus, and relates to the technical field of microorganisms. According to the invention, a strain of Lactobacillus acidophilus LIHUO 1978 is screened out, and the preservation number of the Lactobacillus acidophilus is GDMCC No: 65514. The lactobacillus acidophilus LIHUO 1978 has good safety, in-vitro probiotic characteristics and in-vitro immunoregulation characteristics, has the effects of relieving chemotherapeutic diarrhea, improving constipation and regulating intestinal flora, and has good application prospects in preparation of drugs for relieving chemotherapeutic diarrhea and / or preparation of products for improving constipation and regulating intestinal flora.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a Lactobacillus acidophilus strain capable of relieving chemotherapy-induced diarrhea and improving constipation and an application thereof. Background Art

[0002] Chemotherapy remains the main treatment strategy for most cancer patients, especially those with advanced tumors, distant metastases, and resistance to molecular targeted therapy or immunotherapy. Gastrointestinal toxicity is a common complication induced by chemotherapy, including clinical symptoms such as diarrhea, constipation, nausea, and vomiting. Chemotherapy-induced diarrhea is diarrhea caused by chemotherapy, which seriously affects the quality of life of patients. The inducing factors of chemotherapy-induced diarrhea are related to intestinal epithelial damage and inflammation that cause damage to the absorption, secretion, and mucosal barrier of the small intestine. Opioid derivatives such as loperamide, deodorized opium tincture, and octreotide are often used to treat chemotherapy-induced diarrhea, but these drugs are usually accompanied by more side effects such as abdominal pain, rash, goiter, allergies, etc. Therefore, it is urgent to develop drugs with good efficacy and high safety for the prevention or treatment of chemotherapy-induced diarrhea.

[0003] Constipation is more common in women than in men, and more common in the elderly than in the young. There are many causes of constipation, which may be caused by intestinal obstruction, lack of exercise, low fiber intake or personal factors. Functional constipation is the most common type of constipation. Imbalance of intestinal flora is a potential predisposing factor for functional constipation, and further limits intestinal motility and immune barrier function. Functional constipation causes patients to lose productivity and affects their quality of life. Typical methods for preventing or treating constipation include improving the patient's diet, adjusting their lifestyle, and using various types of medications such as bulking agents, osmotic agents and stimulant laxatives. These treatments have limitations, such as inconsistent efficacy, different degrees of symptom relief and potential safety issues. Therefore, there is an urgent need to develop drugs or dietary supplements with good efficacy and high safety for relieving or treating constipation.

[0004] In recent years, the role of probiotics in regulating intestinal flora, alleviating chemotherapy-induced diarrhea, and improving constipation has been confirmed, and they have a wide history of human use and are relatively safe. Some probiotics, such as Lactobacillus, Bifidobacterium, and yeast, can prevent or improve chemotherapy-induced diarrhea. The potential mechanisms of action of probiotics in improving chemotherapy-induced diarrhea include regulating intestinal flora, regulating immunity, and reducing the levels of proinflammatory cytokines (Daylia T, Nutthada A, Tippawan S. Effects of Probiotics on Chemotherapy-induced Diarrhea. [J]. Nutrition and cancer, 2023, 75 (10): 11-11.). Probiotics such as Lactobacillus can relieve constipation by regulating intestinal flora, short-chain fatty acid levels, water channel protein expression levels, neurotransmitter and hormone levels, inflammation levels, antioxidant levels, etc. (Dimidi E, Scott MS, Whelan K. Probiotics and constipation: mechanisms of action, evidence for effectiveness and utilisation by patients and healthcare professionals [J]. Proceedings of the Nutrition Society, 2020, 79 (1): 147-157.).

[0005] The effects of probiotics in relieving chemotherapy-induced diarrhea and constipation still vary between species and strains (Linlin W, Lujun H, Qi X, et al. Bifidobacteria exert species-specific effects onconstipation in BALB / c mice. [J]. Food&function, 2017, 8 (10): 3587-3600.). Therefore, providing a Lactobacillus acidophilus strain with good safety, in vitro probiotic properties, in vitro immune regulation properties, and the ability to relieve chemotherapy-induced diarrhea, improve constipation, and regulate intestinal flora, and its application, is of great value and has considerable application prospects. Summary of the Invention

[0006] The present invention aims to provide a strain of Lactobacillus acidophilus for relieving chemotherapy-induced diarrhea and improving constipation, and its application. The strain has good safety, in vitro probiotic properties, and in vitro immune regulation properties. The strain has the effects of relieving chemotherapy-induced diarrhea, improving constipation, and regulating intestinal flora. The strain can be used to prepare medicines for relieving chemotherapy-induced diarrhea, and / or prepare health foods, animal nutrition products, and medicines for improving constipation and / or regulating intestinal flora, and has good application prospects.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides a strain of Lactobacillus acidophilus ( Lactobacillus acidophilus ) LIHUO1978, the preservation number of the Lactobacillus acidophilus LIHUO 1978 is GDMCC No:65514.

[0008] Specifically, the sequence of 16S rDNA of Lactobacillus acidophilus LIHUO 1978 is shown in SEQ ID NO: 1.

[0009] Furthermore, the Lactobacillus acidophilus LIHUO 1978 was isolated from farmyard cream in Bayannur City, Inner Mongolia Autonomous Region. Its colony morphology on MRS medium is white, round, with a moist, opaque surface and neat edges. Its bacterial cells are rod-shaped and arranged singly or in pairs. It is Gram-positive. It has excellent safety: negative hemolysis reaction, no detectable putrescine, cadaverine, histamine, or tyramine, and is non-pathogenic. It exhibits excellent in vitro probiotic properties: good growth, acid production, skim milk fermentation, and tolerance in simulated gastrointestinal fluid. It also exhibits excellent in vitro immunomodulatory properties: live bacteria of this strain can significantly promote the production of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) by normal macrophages. Heat-killed bacteria of this strain can significantly promote the production of TNF-α and IL-10 by normal and inflammatory macrophages, and can significantly promote TNF-α production in a Caco-2 cell-macrophage co-culture system.

[0010] In another aspect, the present invention provides a Lactobacillus acidophilus LIHUO 1978 preparation, which is prepared by culturing Lactobacillus acidophilus LIHUO 1978 in a culture medium.

[0011] Specifically, the Lactobacillus acidophilus LIHUO 1978 preparation includes any one or more of live bacteria, fermentation broth, fermentation broth precipitate, heat-inactivated bacteria, and freeze-dried powder.

[0012] Furthermore, the fermentation liquid refers to the liquid obtained after the bacteria are inoculated into the culture medium and cultured.

[0013] Furthermore, the fermentation liquid precipitate refers to the liquid precipitate after centrifugation, including free protein, residual bacteria, broken cells, and residues of the culture medium.

[0014] Furthermore, the method for preparing the heat-killed bacteria is to sterilize the test bacterial solution of Lactobacillus acidophilus LIHUO 1978 at 121° C. for 15 minutes to prepare the heat-killed bacteria.

[0015] Furthermore, the preparation method of the lyophilized powder can adopt the method currently commonly used in the art, or other methods that may appear in the future to obtain a solid form product of a microorganism or microbial culture. It should be clarified that the preparation method of the lyophilized powder should not become a condition that limits the scope of protection of the present invention.

[0016] Furthermore, the culture medium can be any one or more of a suitable solid culture medium, a semi-solid culture medium, and a liquid culture medium.

[0017] Furthermore, the culture medium includes but is not limited to MRS medium, M17 medium, RCM medium, MC medium, TTB medium, BS medium, SC medium, VRBA medium, and red Bengal medium.

[0018] In another aspect, the present invention provides a microbial agent, comprising the aforementioned Lactobacillus acidophilus LIHUO 1978 or the aforementioned Lactobacillus acidophilus LIHUO 1978 preparation.

[0019] Specifically, the bacterial agent is a powder or liquid preparation.

[0020] Specifically, the bacterial agent can be solid, liquid, semi-solid or any physical form that can be obtained by those skilled in the art through current or future technologies and contains the necessary active ingredients to achieve any application, function or effect of the present invention.

[0021] In another aspect, the present invention provides use of the aforementioned Lactobacillus acidophilus LIHUO 1978 or the aforementioned Lactobacillus acidophilus LIHUO 1978 preparation or the aforementioned microbial agent in the preparation of a medicament for treating diarrhea.

[0022] Furthermore, the diarrhea is chemotherapy-induced diarrhea.

[0023] Specifically, the viable count of Lactobacillus acidophilus LIHUO 1978 in the medicine is 1.00×10 6 -1.00×10 12 CFU / mL or 1.00×10 6 -1.00×1012 CFU / g.

[0024] Furthermore, the viable count of Lactobacillus acidophilus LIHUO 1978 in the medicine is 5.00×10 9 CFU / mL or 5.00×10 9 CFU / g.

[0025] Specifically, the drug has at least one of the following effects: (1) Significantly reduce diarrhea scores; (2) Significantly increase the length of the small intestine; (3) Significantly reduced jejunal tissue pathology score; (4) Significantly reduce serum interleukin-1β (IL-1β) concentration; (5) Significantly increase serum IL-10 concentration.

[0026] In another aspect, the present invention provides a drug for alleviating diarrhea, wherein the drug comprises the aforementioned Lactobacillus acidophilus LIHUO 1978 or the aforementioned Lactobacillus acidophilus LIHUO 1978 preparation or the aforementioned microbial agent.

[0027] Specifically, the diarrhea is chemotherapy-induced diarrhea.

[0028] Specifically, the medicine may further include pharmaceutical excipients.

[0029] Furthermore, the pharmaceutical excipients are selected from any one or more of adjuvants, stabilizers, protective agents, antibacterial agents, excipients, solubilizers, flavoring agents, diluents, and buffers.

[0030] Specifically, the dosage form of the drug is any one of tablets, capsules, granules, ophthalmic preparations, nasal preparations, suppositories, pills, ointments, creams, sprays, gels, powders, paints or lyophilized preparations.

[0031] In another aspect, the present invention provides use of the aforementioned Lactobacillus acidophilus LIHUO 1978 or the aforementioned Lactobacillus acidophilus LIHUO 1978 preparation or the aforementioned microbial agent in the preparation of a product for improving constipation.

[0032] In another aspect, the present invention provides a product for improving constipation, wherein the product comprises the aforementioned Lactobacillus acidophilus LIHUO 1978 or the aforementioned Lactobacillus acidophilus LIHUO 1978 preparation or the aforementioned microbial agent.

[0033] Specifically, the products include health foods, animal nutrition products or medicines.

[0034] Furthermore, the health food and animal nutrition products also include auxiliary materials.

[0035] Furthermore, the auxiliary materials include additives.

[0036] Furthermore, the additives are selected from any one or more of acidity regulators, anticaking agents, defoaming agents, antioxidants, bleaching agents, leavening agents, colorants, color protectants, emulsifiers, enzyme preparations, flavor enhancers, coating agents, moisture retaining agents, nutritional enhancers, preservatives, stabilizers and coagulants, sweeteners, thickeners, food flavorings, and processing aids for the food industry.

[0037] Furthermore, the animal nutrition product includes any one or more of animal feed and pet snacks.

[0038] Specifically, when the product is a medicine, the medicine may also include pharmaceutical excipients; Furthermore, the pharmaceutical excipients are selected from any one or more of adjuvants, stabilizers, protective agents, antibacterial agents, excipients, solubilizers, flavoring agents, diluents, and buffers.

[0039] Specifically, the dosage form of the drug is any one or more of tablets, capsules, granules, ophthalmic preparations, nasal preparations, suppositories, pills, ointments, creams, sprays, gels, powders, paints or lyophilized preparations.

[0040] Specifically, the viable count of Lactobacillus acidophilus LIHUO 1978 in the product is 1.00×10 6 -1.00×10 12 CFU / mL or 1.00×10 6 -1.00×10 12 CFU / g.

[0041] Furthermore, the viable count of Lactobacillus acidophilus LIHUO 1978 in the product is 1.00×10 8 CFU / mL or 1.00×10 8 CFU / g.

[0042] Specifically, the product has at least one of the following effects: (1) Significantly increase the number of fecal particles and fecal water content; (2) Significantly improve gastrointestinal propulsion rate; (3) Significantly reduced serum endothelin-1 (ET-1), serum vasoactive intestinal peptide (VIP), serum somatostatin (SS), and serum nitric oxide synthase (iNOS) concentrations; (4) Significantly increased serum motilin (MTL), serum substance P (SP), serum acetylcholine (Ach), colon tissue 5-hydroxytryptamine (5-HT), and colon tissue IL-10 concentrations; (5) Significantly increased the content of mucus-secreting goblet cells; (6) Significantly increase the concentration of aquaporin 9 (AQP9) in colon tissue; (7) Regulate intestinal flora; (8) Significantly increase the concentrations of isobutyric acid, isovaleric acid, and valeric acid in the colon contents.

[0043] In another aspect, the present invention provides use of the aforementioned Lactobacillus acidophilus LIHUO 1978 or the aforementioned Lactobacillus acidophilus LIHUO 1978 preparation or the aforementioned microbial agent in the preparation of a product for regulating intestinal flora.

[0044] In particular, the product is able to improve the beta diversity of the cecal content microbiome.

[0045] Furthermore, the product can significantly increase the relative abundance of Lactobacillus and Lactobacillus mucis in the intestine.

[0046] In another aspect, the present invention provides a product for regulating intestinal flora, wherein the product comprises the aforementioned Lactobacillus acidophilus LIHUO 1978 or the aforementioned Lactobacillus acidophilus LIHUO 1978 preparation or the aforementioned microbial agent.

[0047] Specifically, the products include health foods, animal nutrition products or medicines.

[0048] Furthermore, the health food and animal nutrition products also include auxiliary materials that can be added to food.

[0049] Furthermore, the additives are selected from any one or more of acidity regulators, anticaking agents, defoaming agents, antioxidants, bleaching agents, leavening agents, colorants, color protectants, emulsifiers, enzyme preparations, flavor enhancers, coating agents, moisture retaining agents, nutritional enhancers, preservatives, stabilizers and coagulants, sweeteners, thickeners, food flavorings, and processing aids for the food industry.

[0050] Furthermore, the health food includes probiotic products and nutritional health products, and the animal nutrition products include any one or more of animal feed and pet snacks.

[0051] Specifically, when the product is a medicine, the medicine may also include pharmaceutical excipients; Furthermore, the pharmaceutical excipients are selected from any one or more of adjuvants, stabilizers, protective agents, antibacterial agents, excipients, solubilizers, flavoring agents, diluents, and buffers.

[0052] Furthermore, the dosage form of the drug is any one or more of tablets, capsules, granules, ophthalmic preparations, nasal preparations, suppositories, pills, ointments, creams, sprays, gels, powders, paints or lyophilized preparations.

[0053] Furthermore, the viable count of Lactobacillus acidophilus LIHUO 1978 in the product is 1.00×10 6 -1.00×10 12 CFU / mL or 1.00×10 6 -1.00×10 12 CFU / g.

[0054] Furthermore, the viable count of Lactobacillus acidophilus LIHUO 1978 in the product is 1.00×10 8 CFU / mL or 1.00×10 8 CFU / g.

[0055] The beneficial effects of the present invention are: (1) The Lactobacillus acidophilus LIHUO 1978 of the present invention has good safety, in vitro probiotic properties, and good in vitro immune regulation properties.

[0056] (2) The effect of Lactobacillus acidophilus LIHUO 1978 of the present invention on alleviating chemotherapy-induced diarrhea is specifically manifested as follows: the live bacteria of Lactobacillus acidophilus LIHUO 1978 can significantly reduce the diarrhea score of mice with chemotherapy-induced diarrhea, significantly increase the small intestine length of mice with chemotherapy-induced diarrhea, significantly reduce the jejunum tissue pathology score of mice with chemotherapy-induced diarrhea, significantly reduce the serum IL-1β concentration of mice with chemotherapy-induced diarrhea, and significantly increase the serum IL-10 concentration of mice with chemotherapy-induced diarrhea.

[0057] (3) The effects of the Lactobacillus acidophilus LIHUO 1978 of the present invention on improving constipation are specifically manifested as follows: both the live and heat-killed bacteria of Lactobacillus acidophilus LIHUO 1978 can significantly increase the number of fecal particles, fecal water content, and gastrointestinal propulsion rate of constipated rats; significantly regulate the serum hormone levels of constipated rats: significantly reduce the concentrations of ET-1, VIP, and SS, and significantly increase the concentrations of MTL and SP; significantly increase the neurotransmitter levels of constipated rats: significantly increase the serum Ach concentration and the 5-HT concentration in colon tissue; increase the content of secretory mucus goblet cells and the AQP9 concentration in colon tissue of constipated rats; reduce the serum iNOS concentration in constipated rats, and significantly increase the IL-10 concentration in colon tissue; and regulate the intestinal flora. The live bacteria of Lactobacillus acidophilus LIHUO 1978 can increase the concentrations of isobutyric acid, isovaleric acid, and valeric acid in constipated rats, and improve the level of short-chain fatty acids to relieve constipation.

[0058] Preservation Instructions Deposit number: GDMCC No: 65514; Category naming: Lactobacillus acidophilus LIHUO 1978; Chinese name of the strain: Lactobacillus acidophilus LIHUO 1978; Deposit date: November 20, 2024; Depository: Guangdong Provincial Microbial Culture Collection Center; Abbreviation of depository unit: GDMCC; Collection address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a phylogenetic tree constructed based on the 16S rDNA sequence of Lactobacillus acidophilus LIHUO 1978 in Example 1. The scale bar in the figure represents 0.005, and the superscript “T” represents the model strain.

[0060] Figure 2 The colony morphology (left) and bacterial morphology (right) of Lactobacillus acidophilus LIHUO 1978 in Example 1 are shown. The scale bar in the bacterial morphology diagram represents 5 μm.

[0061] Figure 3 The growth curves of Lactobacillus acidophilus LIHUO 1978 and Lactobacillus acidophilus NCFM in MRS liquid medium in Example 3 (top: OD 600 value curve, bottom: viable cell count curve).

[0062] Figure 4 This is the pH curve of Lactobacillus acidophilus LIHUO 1978 and Lactobacillus acidophilus NCFM cultured in MRS liquid culture medium in Example 3.

[0063] Figure 5 The acidity value of Lactobacillus acidophilus LIHUO 1978 and Lactobacillus acidophilus NCFM fermented in skim milk in Example 3.

[0064] Figure 6 The concentrations of TNF-α and IL-10 produced by macrophages in each group under normal conditions in Example 5 are shown in Table 5. P <0.001.

[0065] Figure 7 is the concentration of TNF-α and IL-10 produced by macrophages in each group under inflammatory conditions in Example 5, ** indicates P <0.01, *** indicates P <0.001.

[0066] Figure 8 is the TNF-α concentration of each group of Caco-2 cell-macrophage co-culture system in Example 5, * indicates P <0.05.

[0067] Figure 9 is the diarrhea score of each group of mice in Example 6, ** indicates P <0.01, *** indicates P <0.001.

[0068] Figure 10 is the small intestine length of each group of mice in Example 6, ns represents P >0.05, ** indicates P <0.01, *** indicates P <0.001.

[0069] Figure 11 These are the HE staining results of the jejunum tissues of each group of mice in Example 7. The scale bars all represent 200 μm.

[0070] Figure 12 In Example 7, the pathological scores of the jejunum of each group of mice are as follows: P <0.001.

[0071] Figure 13 is the serum IL-1β and IL-10 concentration of each group of mice in Example 8, ns represents P >0.05, ** indicates P <0.01, *** indicates P <0.001.

[0072] Figure 14 is the number of feces pellets of rats in each group in Example 9, ns represents P >0.05, * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0073] Figure 15 is the feces water content of each group of rats in Example 9, *** indicates P <0.001.

[0074] Figure 16 is the gastrointestinal propulsion rate of each group of rats in Example 9, ** indicates P <0.01, *** indicates P <0.001.

[0075] Figure 17is the concentration of ET-1, VIP, SS, MTL and SP in the serum of each group of rats in Example 10, ns represents P >0.05, * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0076] Figure 18 The serum Ach concentration and colon tissue 5-HT concentration of each group of rats in Example 11 are as follows: P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0077] Figure 19 These are the AB staining results of the colon tissues of rats in each group in Example 12. The scale bars represent 500 μm.

[0078] Figure 20 is the concentration of AQP9 in the colon of each group of rats in Example 12, ** indicates P <0.01, *** indicates P <0.001.

[0079] Figure 21 is the serum iNOS concentration of rats in each group in Example 13, *** indicates P <0.001.

[0080] Figure 22 is the IL-10 concentration of each group of rat colon in Example 13, ns represents P >0.05, * indicates P <0.05, *** indicates P <0.001.

[0081] Figure 23 are the Ace index and Chao index of the cecal contents microbiome of each group of rats in Example 14, and ns represents P >0.05, * indicates P <0.05.

[0082] Figure 24 This is the PCA analysis and PCoA analysis of the cecal contents microbiome at the genus level of each group of rats in Example 14.

[0083] Figure 25 is the relative abundance of the microbial genus level in the cecal contents of each group of rats in Example 14.

[0084] Figure 26 This is a differential analysis of the microbial genus levels in the cecal contents of each group of rats in Example 14.

[0085] Figure 27 is the concentration of isobutyric acid, isovaleric acid, and valeric acid in the colon contents of each group of rats in Example 15, ns represents P >0.05, * indicates P <0.05, ** indicates P <0.01. DETAILED DESCRIPTION

[0086] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, and the equipment used are all conventional equipment.

[0087] The strain Lactobacillus acidophilus NCFM in the following examples has been disclosed in patent CN108102960A.

[0088] Example 1 Isolation, purification, identification and preservation of Lactobacillus acidophilus LIHUO 1978 Experimental methods: The Lactobacillus acidophilus LIHUO 1978 of this embodiment was isolated, purified, and identified in the following manner: (1) Isolation and purification of Lactobacillus acidophilus LIHUO 1978: Collected the farmer's cream samples in Bayannur City, Inner Mongolia Autonomous Region, and quickly transported them back to the laboratory at low temperature to carry out the strain isolation experiment. The collected cream samples were diluted with sterile saline and 10 -4 , 10 -5 Flow dilutions were plated onto MRS solid medium and incubated anaerobically at 37°C for 48 hours. Based on morphological characteristics, dominant colonies were selected and placed on MRS liquid medium. Incubated anaerobically at 37°C for 24 hours, the colonies were streaked onto MRS solid medium, and incubated anaerobically at 37°C for 48 hours. Repeat the streak inoculation process five times until a single colony appeared on the medium.

[0089] (2) Identification of Lactobacillus acidophilus LIHUO 1978: 1.00 mL of the culture medium of the isolated strain was cultured anaerobically at 37°C for 24 h. After centrifugation and washing with sterile PBS solution, the genomic DNA of the strain was extracted using a DNA extraction kit (manufacturer: Beijing Tiangen Biochemical, Cat.# DP302-02). The 16S rDNA sequence of the isolated strain was amplified by PCR using the universal primers for bacterial 16S rDNA (27F, 1492R). The successfully amplified PCR product was sent to Shanghai Saiheng Biotechnology Co., Ltd. for sequencing. The sequence homology was obtained by BLAST comparison using the NCBI GeneBank database and identified as Lactobacillus acidophilus. The strain was named: Lactobacillus acidophilus LIHUO 1978. The phylogenetic tree of the strain was constructed based on the 16S rDNA of Lactobacillus acidophilus LIHUO 1978 using MEGA software and the neighbor-joining method.

[0090] (3) The colony morphology and bacterial morphology observation, as well as the physiological and biochemical characteristics of Lactobacillus acidophilus LIHUO 1978 were commissioned to CICC for testing. The testing methods were based on the FMIC-QO01-001-2015 Microbiological Detection of Bacteria Multiple Identification Test Methods.

[0091] (4) Deposit of Lactobacillus acidophilus LIHUO 1978: The identified Lactobacillus acidophilus LIHUO 1978 was deposited in Guangdong Provincial Microbial Culture Collection Center (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou).

[0092] Experimental results: A BLAST comparison of the sequencing results of the 16S rDNA PCR product of Lactobacillus acidophilus LIHUO 1978 against the NCBI GeneBank database showed that the 16S rDNA sequence of this strain was 99.73% similar to that of Lactobacillus acidophilus. The 16S rDNA sequence of Lactobacillus acidophilus LIHUO 1978 is shown in SEQ ID NO. 1:

[0093] The phylogenetic tree based on the 16S rDNA sequence of Lactobacillus acidophilus LIHUO 1978 is shown in Figure 1 .

[0094] The colony morphology of Lactobacillus acidophilus LIHUO 1978 on MRS medium is white, round, moist, opaque, and with neat edges (see Figure 2 The bacterial morphology of this strain on MRS medium is rod-shaped, arranged singly or in pairs (see Figure 2 The strain is a Gram-positive strain.

[0095] The physiological and biochemical characteristics of Lactobacillus acidophilus LIHUO 1978 are shown in Table 1 (“+” indicates positive, and “-” indicates negative).

[0096] Table 1 Physiological and biochemical characteristics of Lactobacillus acidophilus LIHUO 1978

[0097] Example 2 Safety Evaluation of Lactobacillus acidophilus LIHUO 1978 Experimental methods: (1) Hemolytic test of Lactobacillus acidophilus LIHUO 1978: The hemolytic test of Lactobacillus acidophilus LIHUO 1978 was commissioned to CICC for testing. The test method was based on FMIC-QO01-043-2024 Microbiological Detection of Hemolytic Microbial Species.

[0098] (2) Detection of biogenic amines in Lactobacillus acidophilus LIHUO 1978: CICC was commissioned to conduct the detection of biogenic amines in Lactobacillus acidophilus LIHUO 1978. The detection method was based on GB 5009.208-2016 National Food Safety Standard - Determination of biogenic amines in foods (Method 1).

[0099] (3) Bacterial animal pathogenicity detection of Lactobacillus acidophilus LIHUO 1978: The bacterial animal pathogenicity detection of Lactobacillus acidophilus LIHUO 1978 was commissioned to CICC for testing. The detection method refers to Appendix A "Technical Guidelines for Safety Testing and Evaluation of Bacteria for Health Food Ingredients" (2020 Edition) - Bacterial Pathogenicity Detection Methods for Health Food Ingredients.

[0100] The activated strain Lactobacillus acidophilus LIHUO 1978 was inoculated on an MRS agar plate containing 500 μg / mL cysteine ​​hydrochloride. The colonies on the agar plate were scraped and suspended in sterile saline to prepare a 5.8×10 7CFU / mL bacterial suspension was used for intraperitoneal injection in mice. Each test mouse was injected with 0.2 mL of bacterial suspension at one time and observed continuously for 21 days after intraperitoneal injection. The broth culture was prepared with a concentration of 3.3×10 8 CFU / mL of bacterial suspension and the concentration was 1.7×10 9 The mice in the corresponding groups were gavaged with a 5-fold concentrated bacterial suspension at a dose of 20.0 mL / kg・BW of CFU / mL, once a day for 3 consecutive days, and the mice were observed for 21 consecutive days starting from the first day of gavage.

[0101] Experimental results: The hemolytic reaction of Lactobacillus acidophilus LIHUO 1978 was negative.

[0102] No putrescine, cadaverine, histamine, or tyramine was detected in Lactobacillus acidophilus LIHUO 1978, indicating that it did not produce biogenic amines.

[0103] There was no significant difference in the initial and final body weights of the mice in each experimental group of Lactobacillus acidophilus LIHUO 1978 compared with the corresponding control group ( P＞ 0.05). No adverse effects of the Lactobacillus acidophilus LIHUO 1978 culture on the general health of the animals were observed, and no toxic reactions or deaths were observed in the test mice. Therefore, the strain Lactobacillus acidophilus LIHUO 1978 is non-pathogenic.

[0104] A strain with good safety is a prerequisite for becoming a probiotic. The experimental results of this example show that Lactobacillus acidophilus LIHUO 1978 has a negative hemolytic reaction, no putrescine, cadaverine, histamine, and tyramine were detected, and it is non-pathogenic, indicating that the strain has good safety and can be used for further development.

[0105] Example 3 Determination of Growth Characteristics, Acid Production Characteristics, and Skim Milk Fermentation Characteristics of Lactobacillus acidophilus LIHUO 1978 Experimental methods: (1) Activation of Lactobacillus acidophilus LIHUO 1978 and control strain Lactobacillus acidophilus NCFM The culture fluids of Lactobacillus acidophilus LIHUO 1978 and the control strain Lactobacillus acidophilus NCFM were inoculated into MRS liquid culture medium respectively and cultured anaerobically at 37°C for 16 h (this was the first strain activation). The strains were activated three times in sequence.

[0106] (2) Determination of growth curve and pH curve of Lactobacillus acidophilus LIHUO 1978 and control strain Lactobacillus acidophilus NCFM: After activation three times, Lactobacillus acidophilus LIHUO 1978 and control strain Lactobacillus acidophilus NCFM were inoculated into MRS liquid medium (pH 6.70) at a 3% inoculum volume and cultured anaerobically at 37°C for 24 h. During this period, samples were taken every 2 h (repeated 3 times) to measure the OD value of the culture medium. 600 The viable bacteria count of the culture medium was determined by plate counting method at 4-h intervals (repeated 3 times). The growth curve and pH value curve of Lactobacillus acidophilus LIHUO 1978 and the control strain Lactobacillus acidophilus NCFM were drawn.

[0107] (3) Determination of the fermentation characteristics of skim milk by Lactobacillus acidophilus LIHUO 1978 and the control strain Lactobacillus acidophilus NCFM: After activation three times, Lactobacillus acidophilus LIHUO 1978 and the control strain Lactobacillus acidophilus NCFM were inoculated into 15% skim milk culture medium at a 3% inoculum size and cultured anaerobically at 37°C for 24 h. During this period, samples were taken every 2 h (repeated three times) and the total acid content of the samples was determined according to the national standard method for the determination of food acidity (GB5009.239-2016).

[0108] Experimental results: 0h-24h, with the increase of culture time, the OD value of Lactobacillus acidophilus LIHUO 1978 and the control strain Lactobacillus acidophilus NCFM in MRS liquid medium 600 The values ​​and viable bacteria counts showed an upward trend, and OD 600 The trend of the change of the value is generally consistent with that of the viable bacteria count (see Figure 3 When the culture time was 24 h, the viable count of Lactobacillus acidophilus LIHUO 1978 was (2.12±0.08)×10 9 The CFU / mL was higher than that of the control strain Lactobacillus acidophilus NCFM (the number of viable bacteria was (1.98±0.03)×10 9 CFU / mL.

[0109] From 0h to 24h, the pH values ​​of the bacterial cultures of Lactobacillus acidophilus LIHUO 1978 and the control strain Lactobacillus acidophilus NCFM in MRS liquid medium showed a downward trend as the culture time increased (see Figure 4 ), after culturing for 12 hours, the pH value of the culture liquid of Lactobacillus acidophilus LIHUO 1978 dropped to 4.42±0.01, which was lower than that of the control strain Lactobacillus acidophilus NCFM (pH value was 4.57±0.00); after culturing for 24 hours, the pH value of the culture liquid of Lactobacillus acidophilus LIHUO 1978 dropped to 3.94±0.01, which was lower than that of the control strain Lactobacillus acidophilus NCFM (pH value was 4.00±0.02).

[0110] From 0h to 24h, the acidity of Lactobacillus acidophilus LIHUO 1978 and the control strain Lactobacillus acidophilus NCFM in skim milk increased with the increase of culture time; during the fermentation period (0h-24h), the acidity of Lactobacillus acidophilus LIHUO 1978 was always higher than that of the control strain Lactobacillus acidophilus NCFM (see Figure 5 After culturing for 24 h, the acidity of the skim milk system of Lactobacillus acidophilus LIHUO1978 was (151.63±0.93)°T, which was higher than that of the control strain Lactobacillus acidophilus NCFM (acidity was (129.13±0.38)°T).

[0111] The experimental results of this example show that Lactobacillus acidophilus LIHUO 1978 has good growth characteristics, acid production characteristics, and skim milk fermentation characteristics. The good growth characteristics and skim milk fermentation characteristics are conducive to its industrialization and save its production energy consumption. Organic acids can inhibit the growth of pathogenic bacteria. The good acid production characteristics are conducive to its inhibition of intestinal pathogens in the host, thereby reducing the probability of intestinal diseases such as diarrhea and constipation.

[0112] Example 4 Determination of tolerance of Lactobacillus acidophilus LIHUO 1978 and control strain Lactobacillus acidophilus NCFM in simulated gastrointestinal fluid Experimental methods: (1) Preparation of test bacterial suspensions of Lactobacillus acidophilus LIHUO 1978 and control strain Lactobacillus acidophilus NCFM The activation of Lactobacillus acidophilus LIHUO 1978 and the control strain Lactobacillus acidophilus NCFM was the same as in Example 3. After the strains were activated three times, they were washed three times with sterile saline and an appropriate amount of sterile saline was added to resuspend the bacteria to prepare a bacterial suspension.

[0113] (2) Determination of tolerance of Lactobacillus acidophilus LIHUO 1978 and control strain Lactobacillus acidophilus NCFM in simulated gastrointestinal fluid 1.00 mL of bacterial suspension of Lactobacillus acidophilus LIHUO 1978 and control strain Lactobacillus acidophilus NCFM were inoculated into 9.00 mL of simulated gastric fluid (pH 2.50, containing 1.00% pepsin), respectively. The cells were anaerobically treated at 37°C for 3 h. Samples were collected at 0 h and 3 h, and the number of viable bacteria (N0 and N1, respectively) was determined by the plate count method. The survival rate was calculated (the survival rate in simulated gastric fluid was N1 / N0×100%).

[0114] 1.00 mL of Lactobacillus acidophilus LIHUO 1978 and the control strain Lactobacillus acidophilus NCFM culture fluid treated with simulated gastric fluid for 3 h were transferred to 9.00 mL of simulated intestinal fluid (pH 8.00, containing 1.00% trypsin and 1.80% bile salts). After treatment at 37°C for 8 h, samples were collected and the number of viable cells (N2) was determined by the plate count method, and the survival rate was calculated (the survival rate in simulated intestinal fluid was N2 / N1×100%, and the survival rate in simulated gastrointestinal fluid was N2 / N0×100%).

[0115] Experimental results: The survival rates of Lactobacillus acidophilus LIHUO 1978 and the control strain Lactobacillus acidophilus NCFM after treatment in simulated gastric fluid for 3 h, simulated intestinal fluid for 8 h, and simulated gastrointestinal fluid for 11 h were 95.18%, 84.71%, and 80.63%, respectively, which were all higher than the survival rates of the control strain Lactobacillus acidophilus NCFM (84.94%, 82.53%, and 70.10%, see Table 2).

[0116] The experimental results of this example show that Lactobacillus acidophilus LIHUO 1978 has good tolerance in simulated gastrointestinal fluid, which helps the strain resist the low pH value of gastric acid and the intestinal environment with high bile salt concentration, and helps it play a probiotic role in the host intestine.

[0117] Table 2 Survival rates of Lactobacillus acidophilus LIHUO 1978 and Lactobacillus acidophilus NCFM in simulated gastrointestinal fluid

[0118] Example 5 Evaluation of the immune-regulating properties of Lactobacillus acidophilus LIHUO 1978 in vitro Experimental methods: (1) Preparation of test solution and heat-killed cells of Lactobacillus acidophilus LIHUO 1978 The activation of Lactobacillus acidophilus LIHUO 1978 was the same as in Example 3. After the strain was activated three times, it was washed three times with sterile PBS solution and resuspended in an appropriate amount of sterile PBS solution to prepare a viable cell count of 1.00×10 7 CFU / mL test bacterial solution; the test bacterial solution of Lactobacillus acidophilus LIHUO 1978 was sterilized at 121℃ for 15min to prepare heat-killed bacteria.

[0119] (2) Effects of live and heat-killed Lactobacillus acidophilus LIHUO 1978 on normal macrophages (experiments were conducted with reference to the method described in patent CN119570698A): After activation, macrophages were injected with 1.00×10 5Cells were inoculated into 48-well plates at a density of 100 cells / well and incubated overnight at 37°C in a 5% CO2 incubator. After cell attachment, the supernatant was discarded and 900 μL of DMEM culture medium was added to each well. For the blank group, 100 μL of sterile PBS solution was added to each well; for the LIHUO 1978 group, 100 μL of the test Lactobacillus acidophilus LIHUO 1978 bacteria were added to each well; and for the heat-killed LIHUO 1978 group, 100 μL of heat-killed Lactobacillus acidophilus LIHUO 1978 bacteria were added to each well. Each group was repeated three times. After incubation at 37°C in a 5% CO2 incubator for 2 h, the cell culture supernatant was collected from each well and the concentrations of TNF-α (manufacturer: Shanghai ELISA, catalog number: ml002095A) and IL-10 (manufacturer: Shanghai ELISA, catalog number: ml037873A) in the cell culture supernatant were determined using enzyme-linked immunosorbent assay kits.

[0120] (3) Effect of heat-killed Lactobacillus acidophilus LIHUO 1978 on macrophages in an inflammatory state (experiments were conducted with reference to the method described in patent CN119570698A): Macrophages were activated and 1.00×10 5 Cells were inoculated into 48-well plates at a density of 100 cells / well and incubated overnight at 37°C in a 5% CO2 incubator. After cell attachment, the supernatant was discarded and 900 μL of DMEM culture medium was added to each well. For the blank group, 100 μL of sterile PBS solution was added to each well; for the model group, 100 μL of sterile PBS solution was added to each well; for the heat-killed LIHUO1978 group, 100 μL of heat-killed Lactobacillus acidophilus LIHUO 1978 cells were added to each well. Each group was repeated three times. After incubation at 37°C in a 5% CO2 incubator for 2 h, the supernatant was aspirated, the cells were washed three times with sterile PBS, and 900 μL of DMEM culture medium was added to each well. For the blank group, 100 μL of sterile PBS solution was added to each well; for the model group, 100 μL of LPS solution (10 μg / mL) was added to each well; for the heat-killed LIHUO1978 group, 100 μL of LPS solution (10 μg / mL) was added to each well. The cells were incubated in a 37°C, 5% CO2 incubator for 2 h, and the cell culture supernatant was collected from each well. The concentrations of TNF-α and IL-10 in the cell culture supernatant of each group were determined using enzyme-linked immunosorbent assay kits (TNF-α (manufacturer: Shanghai ELISA, catalog number: ml002095A) and IL-10 (manufacturer: Shanghai ELISA, catalog number: ml037873A).

[0121] (4) Effect of heat-killed Lactobacillus acidophilus LIHUO 1978 on Caco-2 cell-macrophage co-culture system (refer to the method described in patent CN119570698A for the experiment): Caco-2 cells were activated and 1.00×10 5The inoculum volume was inoculated into 24-well plates, and 1.00×10 5 A co-culture system was established by inserting the reverse side of the chamber into a 24-well plate. 500 μL of DMEM medium was added and the cells were incubated at 37°C with 5% CO₂. Once a dense monolayer of Caco-2 cells had formed in the chamber, 200 μL of DMEM was added to each well. For the blank control group, 100 μL of sterile PBS was added to each well. For the heat-killed LIHUO1978 group, 100 μL of heat-killed Lactobacillus acidophilus LIHUO 1978 cells were added to each well. Each group was replicated three times. The cells were incubated at 37°C with 5% CO₂ for 2 hours. After completion of the incubation, the lower chamber and cell culture supernatants were aspirated and the TNF-α concentration in the cell culture supernatants of each group was determined using an enzyme-linked immunosorbent assay (ELISA, Shanghai ELISA, Cat. No. ml002095A).

[0122] Experimental results: Compared with the blank group, the concentrations of TNF-α and IL-10 produced by macrophages in the normal state of the LIHUO 1978 group and the heat-inactivated LIHUO 1978 group were significantly increased (see Figure 6 ).

[0123] Compared with the blank group, the concentrations of TNF-α and IL-10 produced by macrophages in the inflammatory state of the model group were significantly increased; compared with the model group, the concentrations of TNF-α and IL-10 produced by macrophages in the inflammatory state of the heat-killed LIHUO1978 group were significantly increased (see Figure 7 ).

[0124] Compared with the blank group, the TNF-α concentration in the Caco-2 cell-macrophage co-culture system in the heat-inactivated LIHUO1978 group was significantly increased (see Figure 8 ).

[0125] The experimental results of this example demonstrate that live Lactobacillus acidophilus LIHUO 1978 significantly promotes the production of TNF-α and IL-10 by normal macrophages; heat-killed Lactobacillus acidophilus LIHUO 1978 significantly promotes the production of TNF-α and IL-10 by normal and inflammatory macrophages, and significantly promotes TNF-α production in a Caco-2 cell-macrophage co-culture system. Both live and heat-killed Lactobacillus acidophilus LIHUO 1978 exhibit excellent in vitro immunomodulatory properties, which facilitates their immunomodulatory effects in the host intestine, thereby exerting their probiotic properties.

[0126] Example 6 Effect of Live Lactobacillus acidophilus LIHUO 1978 on Symptoms of Chemotherapy-Induced Diarrhea in Mice Experimental methods: (1) Preparation of Lactobacillus acidophilus LIHUO 1978 bacterial solution The activation of Lactobacillus acidophilus LIHUO 1978 was the same as in Example 3. After the strain was activated three times, it was washed three times with sterile PBS solution and resuspended in an appropriate amount of sterile PBS solution to prepare a viable cell count of 5.00×10 9 CFU / mL bacterial solution.

[0127] (2) Animal experiment design SPF-grade BALB / c male mice (weighing 18-20 g), provided by Changsha Tianqin Biotechnology Co., Ltd., were used as research subjects. All mice were housed in a stable environment with a temperature of (23 ± 2)°C and a humidity of (50 ± 5)%. Throughout the experiment, mice were housed under a 12-hour light-dark cycle and had free access to SPF-grade feed and sterile water. After acclimating to the diet for 7 days, all mice were grouped using a weight-based serpentine grouping method (10 per group) into the control group, model group, drug group, and LIHUO 1978 experimental group. Animal experiments were conducted according to Table 3.

[0128] Table 3 Animal experimental design of Lactobacillus acidophilus LIHUO 1978 in relieving chemotherapy-induced diarrhea

[0129] (3) Diarrhea score of mice After gavage and intraperitoneal injection on the 16th day, each mouse was placed in a clean container with a clean filter paper at the bottom of the container. The mice were allowed to defecate and the feces were observed. The diarrhea score of the mice was calculated according to the scoring rules in Table 4.

[0130] Table 4 Diarrhea score table

[0131] (4) Dissection of mice and measurement of small intestine length All mice were deprived of food and water for 12 hours after gavage, intraperitoneal injection, and diarrhea scoring on day 16. They were anesthetized with isoflurane and euthanized. Eyeballs were removed from all mice for blood sampling. The abdomens of the mice were disinfected with 75% ethanol and dissected. The entire small intestine was removed and its total length was measured with a ruler.

[0132] The jejunal tissues of mice in each group were washed with ice-cold saline, and about 0.5 cm of the distal jejunal tissue was cut and fixed in 4% paraformaldehyde solution.

[0133] After the blood samples of each group of mice were allowed to stand and stratified, they were centrifuged at 4°C and 3000 r / min for 15 min to obtain serum, which was then packaged and stored at -80°C for testing.

[0134] Experimental results: Compared with the blank group, the diarrhea scores of mice in the model group increased significantly; compared with the model group, the diarrhea scores of mice in the drug group and LIHUO1978 experimental group decreased significantly by 29.17% and 37.50%, respectively (see Figure 9 ).

[0135] Compared with the blank group, the small intestine length of the model group mice was significantly shortened by 16.80%; compared with the model group, the small intestine length of the drug group mice was not significantly different, while the small intestine length of the LIHUO 1978 experimental group mice was significantly increased by 9.29% (see Figure 10 ).

[0136] The experimental results of this example show that Lactobacillus acidophilus LIHUO 1978 can significantly reduce diarrhea scores and significantly increase small intestine length, thereby alleviating the symptoms of chemotherapy-induced diarrhea in mice.

[0137] Example 7 Effects of Lactobacillus acidophilus LIHUO 1978 on jejunal histological damage in mice with chemotherapy-induced diarrhea Experimental methods: In Example 6, all mouse jejunal tissues fixed in 4% paraformaldehyde solution were dehydrated, paraffin-embedded, 5 μm sliced, dewaxed, and stained with hematoxylin-eosin (H&E). The damage of the mouse jejunal tissue was observed under a microscope, and the pathological score of the jejunal tissue was performed according to the following criteria: (1) mononuclear cell infiltration or mixed neutrophil infiltration in the mucosa; (2) thinning of the mucosa or glands, reduction of goblet cells, and crypts and glands away from the basement membrane; (3) disappearance of crypts and dominance of neutrophils; (4) epithelial destruction, increase of basal plasma cells, and aggregation of lymphocytes. The pathological scoring standard is that each of the above pathological manifestations is scored cumulatively with 1 point, and if more than three of the above pathological manifestations are present, the cumulative score is 4 points.

[0138] The experimental results are as follows: The HE staining results of the jejunum tissues of mice in each group are shown in Figure 11The results showed that the jejunum tissue of the blank group mice had abundant villi, which were wide and neatly arranged, regular intestinal crypts, and abundant goblet cells, with no obvious histopathological changes. The jejunum tissue structure of the model group mice was severely abnormal, with significant atrophy and disappearance of the intestinal villi, edema of the epithelial cells, a significant decrease in the number of goblet cells, and a large number of intestinal crypts destroyed and disappeared, with disordered arrangement, accompanied by a large number of inflammatory cell infiltrations. The jejunum tissue structure of the drug group mice was mildly abnormal, with mild atrophy and slightly disordered intestinal villi, a decrease in the number of goblet cells, disordered crypt arrangement, moderate edema of the mucosal layer, and moderate inflammatory cell infiltration. The jejunum tissue structure of the LIHUO 1978 experimental group mice was mildly abnormal, with mild atrophy and slightly disordered intestinal villi, abundant goblet cells, and mild inflammatory cell infiltration of the mucosal layer.

[0139] Compared with the blank group, the pathological scores of the jejunum of the model group mice were significantly increased; compared with the model group, the pathological scores of the intestinal tissue of the drug group and the LIHUO 1978 experimental group mice were significantly decreased by 30.77% and 53.85%, respectively (see Figure 12 ).

[0140] The experimental results of this example show that Lactobacillus acidophilus LIHUO 1978 can significantly improve the degree of jejunal tissue lesions in mice with chemotherapy-induced diarrhea, thereby alleviating chemotherapy-induced diarrhea.

[0141] Example 8 Effects of Lactobacillus acidophilus LIHUO 1978 on serum inflammatory-related factors in mice with chemotherapy-induced diarrhea Experimental methods: The IL-1β (manufacturer: Shanghai ELISA, catalog number: ml098416A) and IL-10 (manufacturer: Shanghai ELISA, catalog number: ml037873A) enzyme-linked immunosorbent assay kits were used and the IL-1β and IL-10 concentrations in the serum of each group of mice in Example 6 were determined strictly according to the instructions.

[0142] The experimental results are as follows: Compared with the blank group, the serum IL-1β concentration of mice in the model group increased significantly by 98.41%; compared with the model group, the serum IL-1β concentration of mice in the drug group and LIHUO 1978 experimental group decreased significantly by 27.39% and 35.19%, respectively (see Figure 13 ).

[0143] Compared with the blank group, the serum IL-10 concentration of mice in the model group decreased significantly by 27.69%; compared with the model group, the serum IL-10 concentration of mice in the drug group increased by 19.70%, but the difference was not significant. However, the serum IL-10 concentration of mice in the LIHUO 1978 experimental group increased significantly by 55.82% ( Figure 13 ).

[0144] The experimental results of this example show that Lactobacillus acidophilus LIHUO 1978 can significantly reduce the concentration of the pro-inflammatory factor IL-1β in the serum of mice with chemotherapy-induced diarrhea, significantly increase the concentration of the serum anti-inflammatory factor IL-10, regulate the concentration of serum inflammatory factors, and relieve chemotherapy-induced diarrhea.

[0145] Example 9 Effects of live and heat-killed Lactobacillus acidophilus LIHUO 1978 on symptoms of constipation in rats Experimental methods: (1) Preparation of Lactobacillus acidophilus LIHUO 1978 bacterial suspension and heat-killed bacteria The activation of Lactobacillus acidophilus LIHUO 1978 was the same as in Example 3. After the strain was activated three times, it was washed three times with sterile PBS solution and resuspended in an appropriate amount of sterile PBS solution to prepare a viable cell count of 1.00×10 8 CFU / mL bacterial suspension; the bacterial suspension of Lactobacillus acidophilus LIHUO 1978 was sterilized at 121℃ for 15min to prepare heat-killed bacteria.

[0146] (2) Animal experiment design SPF-free male Sprague-Dawley rats (weighing 180-220 g), 6-8 weeks old and provided by Changsha Tianqin Biotechnology Co., Ltd., were used as research subjects. All rats were housed in a stable environment with a temperature of (23 ± 2)°C and a humidity of (50 ± 5)%. Throughout the experiment, the rats were housed under a 12-hour light-dark cycle and had free access to SPF-free feed and sterile water. After acclimating to the diet for 7 days, all rats were divided into groups (8 per group) using a weight-based serpentine grouping method: a normal group, a constipation model group, a polyethylene glycol 4000 powder group, a LIHUO 1978 group, and a heat-inactivated LIHUO 1978 group. Animal experiments were conducted according to Table 5.

[0147] Table 5 Animal experimental design of Lactobacillus acidophilus LIHUO 1978 live and heat-killed bacteria in relieving constipation

[0148] (3) Determination of rat fecal pellet number and water content On the 21st day, feces excreted within 1 h from each rat in all the above groups were collected, the number of fecal particles was recorded, the feces were weighed (m1), and then dried in a 60°C oven to a constant weight (m2). The fecal moisture content of each rat was calculated according to the formula: fecal moisture content (%) = (m1-m2) / m1×100%.

[0149] (4) Rat anatomy and determination of rat gastrointestinal propulsion rate All rats were deprived of food and water for 12 hours after gavage and intraperitoneal injection on day 21. Each rat was gavaged with 2 mL of Evans blue paste (prepared according to the method described in patent CN119331785B: 2.5 g carboxymethyl cellulose, 8 g milk powder, 4 g sucrose, 4 g starch, and 3.5 mL of 1% Evans blue solution were dissolved and sterilely mixed using a vortex mixer to prepare 150 mL of Evans blue semisolid paste). Blood samples were collected by orbital puncture at 15 minutes, and the rats were sacrificed. Dissections were performed on the rats, and intact gastrointestinal tissue was removed. The distance from the pyloric sphincter to the end of the Evans blue paste and the total length of the small intestine were measured and recorded using a ruler. The gastrointestinal propulsion rate (%) was calculated for each rat using the formula: distance from the end of the Evans blue paste (cm) / total length of the small intestine (cm) × 100%.

[0150] The colon tissues of rats in each group were washed with ice-cold saline, and about 1 cm of distal colon tissue was cut and fixed in 4% paraformaldehyde solution; the remaining colon tissues were collected into sterile enzyme-free cryopreservation tubes, quickly frozen in liquid nitrogen, and stored at -80°C for testing.

[0151] After the blood samples of each group of rats were allowed to stand and stratified, they were centrifuged at 4°C and 3000 r / min for 15 min to obtain serum, which was then packaged and stored at -80°C until testing.

[0152] Under sterile conditions, the cecal contents and colonic contents of rats in each group were collected, quickly frozen in liquid nitrogen, and stored at -80°C for testing.

[0153] The experimental results are as follows: Compared with the normal group, the number of fecal particles in the constipation model group decreased significantly by 53.85%. Compared with the constipation model group, the number of fecal particles in the polyethylene glycol 4000 powder group increased by 79.33%, but the difference was not significant. However, the number of fecal particles in the LIHUO 1978 group and the heat-inactivated LIHUO 1978 group increased significantly by 137.67% and 100.00%, respectively (see Figure 14 ).

[0154] Compared with the normal group, the fecal water content of the rats in the constipation model group decreased significantly by 29.08%; while compared with the constipation model group, the fecal water content of the rats in the polyethylene glycol 4000 powder group, LIHUO 1978 group, and heat-inactivated LIHUO 1978 group increased significantly by 32.75%, 34.27%, and 33.18%, respectively (see Figure 15 ).

[0155] Compared with the normal group, the gastrointestinal propulsion rate of rats in the constipation model group decreased significantly by 42.44%; while compared with the constipation model group, the gastrointestinal propulsion rate of rats in the polyethylene glycol 4000 powder group, LIHUO 1978 group, and heat-inactivated LIHUO 1978 group increased significantly by 22.73%, 65.19%, and 61.48%, respectively (see Figure 16 ).

[0156] The experimental results of this example showed that the number of fecal particles, fecal water content, and gastrointestinal propulsion rate in the constipation model group were significantly lower than those in the normal group, indicating that the constipation rat model was successfully established. Both live and heat-killed Lactobacillus acidophilus LIHUO 1978 bacteria alleviated constipation symptoms in rats by significantly increasing the number of fecal particles, fecal water content, and gastrointestinal propulsion rate.

[0157] Example 10 Effects of live and heat-killed Lactobacillus acidophilus LIHUO 1978 on serum ET-1, VIP, SS, MTL, and SP concentrations in constipated rats Experimental methods: The ET-1 (manufacturer: Shanghai ELISA, catalog number: ml002890V), VIP (manufacturer: Shanghai ELISA, catalog number: ml936589V), SS (manufacturer: Shanghai ELISA, catalog number: ml003102V), MTL (manufacturer: Shanghai ELISA, catalog number: ml003079V), and SP (manufacturer: Shanghai ELISA, catalog number: ml003119V) enzyme-linked immunosorbent assay kits were used to determine the ET-1, VIP, SS, MTL, and SP concentrations in the serum of the rats in each group in Example 9 in strict accordance with the instructions.

[0158] Experimental results: Compared with the normal group, the serum ET-1 concentration of rats in the constipation model group increased significantly by 56.82%; while compared with the constipation model group, the serum ET-1 concentration of rats in the polyethylene glycol 4000 powder group, LIHUO 1978 group, and heat-inactivated LIHUO 1978 group decreased significantly by 12.35%, 16.60%, 16.21%, and 10.81%, respectively (see Table 2). Figure 17 ).

[0159] Compared with the normal group, the serum VIP concentration of rats in the constipation model group increased significantly by 150.29%; while compared with the constipation model group, the serum VIP concentration of rats in the polyethylene glycol 4000 powder group, LIHUO 1978 group, and heat-inactivated LIHUO 1978 group decreased significantly by 20.32%, 31.18%, and 26.55%, respectively (see Figure 17 ).

[0160] Compared with the normal group, the serum SS concentration of rats in the constipation model group increased significantly by 133.43%; while compared with the constipation model group, the serum SS concentration of rats in the polyethylene glycol 4000 powder group, LIHUO 1978 group, and heat-inactivated LIHUO 1978 group decreased significantly by 27.28%, 33.78%, and 38.87%, respectively (see Figure 17 ).

[0161] Compared with the normal group, the MTL concentration in the serum of rats in the constipation model group decreased significantly by 40.46%; while compared with the constipation model group, the MTL concentration in the serum of rats in the polyethylene glycol 4000 powder group, LIHUO 1978 group, and heat-inactivated LIHUO 1978 group increased significantly by 27.97%, 39.48%, and 28.92%, respectively (see Figure 17 ).

[0162] Compared with the normal group, the SP concentration of rats in the constipation model group decreased significantly by 44.00%. Compared with the constipation model group, the SP concentration of rats in the polyethylene glycol 4000 powder group increased by 16.15%, but the difference was not significant. The SP concentration of rats in the LIHUO 1978 group and the heat-inactivated LIHUO 1978 group increased significantly by 56.32% and 48.95%, respectively (see Figure 17 ).

[0163] ET-1 can inhibit smooth muscle contraction, VIP can dilate blood vessels, and SS can inhibit gastrointestinal fluid secretion, thereby inhibiting intestinal motility; MTL can enhance gastrointestinal motility and emptying capacity, and SP can stimulate intestinal contraction by activating intestinal smooth muscle, thereby promoting intestinal motility. The experimental results of this example show that both live and heat-killed Lactobacillus acidophilus LIHUO 1978 bacteria can significantly reduce the serum ET-1, VIP, and SS concentrations of constipated rats, significantly increase the serum MTL and SP concentrations of constipated rats, and regulate the hormone levels in the serum of constipated rats, thereby alleviating constipation.

[0164] Example 11 Effects of live and heat-killed Lactobacillus acidophilus LIHUO 1978 on serum acetylcholine concentration and colon tissue 5-hydroxytryptamine concentration in constipated rats Experimental methods: (1) Determination of ACh concentration in rat serum The Ach concentration in the serum of the rats in each group in Example 9 was determined using an Ach enzyme-linked immunosorbent assay kit (manufacturer: Shanghai ELISA, catalog number: ml003048V) and in strict accordance with the instructions.

[0165] (2) Determination of protein concentration in rat colon tissue The colon tissues of the rats in each group in Example 9 were homogenized and centrifuged to obtain the supernatant, and the protein concentration was determined using the BCA protein concentration assay kit (enhanced) (manufacturer: Biyuntian, product number: Cat No. P0010) in strict accordance with the instructions.

[0166] (3) Determination of 5-HT concentration in rat colon tissue The concentration of 5-HT in the colon tissue of rats in each treatment group in Example 9 was determined using a 5-HT enzyme-linked immunosorbent assay kit (manufacturer: Shanghai ELISA, catalog number: ml059511V) in strict accordance with the instructions.

[0167] Experimental results: Compared with the normal group, the serum ACh concentration of rats in the constipation model group decreased significantly by 44.60%; while compared with the constipation model group, the serum ACh concentration of rats in the polyethylene glycol 4000 powder group, LIHUO 1978 group, and heat-inactivated LIHUO 1978 group increased significantly by 36.80%, 55.53%, and 47.07%, respectively (see Figure 18 ).

[0168] Compared with the normal group, the 5-HT concentration in the colon tissue of rats in the constipation model group decreased significantly by 21.05%; while compared with the constipation model group, the 5-HT concentration in the colon tissue of rats in the polyethylene glycol 4000 powder group, LIHUO 1978 group, and heat-inactivated LIHUO 1978 group increased significantly by 10.00%, 13.33%, and 10.00%, respectively (see Figure 18 ).

[0169] ACh promotes the secretion of gastric and intestinal juices, thereby promoting intestinal motility. 5-HT is an important neurotransmitter that reduces the transit time of feces in the colon. The experimental results of this example show that both live and heat-killed Lactobacillus acidophilus LIHUO 1978 bacteria can significantly increase the serum ACh concentration and colon tissue 5-HT concentration in constipated rats, thereby regulating neurotransmitter levels in constipated rats and alleviating constipation.

[0170] Example 12 Effects of live and heat-killed Lactobacillus acidophilus LIHUO 1978 on the content of mucus goblet cells and the level of aquaporin 9 in colonic tissue of constipated rats Experimental methods: (1) AB staining of rat colon tissue All rat colon tissues fixed in 4% paraformaldehyde solution in Example 9 were sequentially dehydrated, paraffin-embedded, 5 μm sliced, dewaxed, stained with Alcian blue (AB), and scanned to observe the distribution and content of mucus-secreting goblet cells in the rat colon tissues.

[0171] (2) Determination of AQP9 concentration in rat colon tissue The colon tissues of the rats in each group in Example 9 were homogenized and centrifuged to obtain supernatants. The protein concentrations of the colon tissues of the rats in each group were determined in the same manner as in Example 11. AQP9 (manufacturer: Shanghai ELISA, catalog number: ml059561V) was used to determine the AQP9 concentrations in the colon tissues of the rats in each treatment group in Example 9 in strict accordance with the instructions.

[0172] Experimental results: AB staining was used to detect the distribution and content of mucus-secreting goblet cells in the colon tissue of rats in each group (see Figure 19 ), the results showed that compared with the normal group, the content of secretory mucus goblet cells in the colon tissue of rats in the constipation model group was decreased; compared with the constipation model group, the content of secretory mucus goblet cells in the colon tissue of rats in the polyethylene glycol 4000 powder group, LIHUO 1978 group, and heat-inactivated LIHUO 1978 group were increased.

[0173] Compared with the normal group, the AQP9 concentration in the colon tissue of rats in the constipation model group decreased significantly by 49.84%; while compared with the constipation model group, the AQP9 concentration in the colon tissue of rats in the polyethylene glycol 4000 powder group, LIHUO 1978 group, and heat-inactivated LIHUO 1978 group increased significantly by 26.56%, 53.36%, and 60.24%, respectively (see Figure 20 ).

[0174] Mucus goblet cells in colonic tissue secrete mucins, which protect the colonic mucosa and promote defecation. AQP9 in colonic tissue can promote defecation by participating in the secretion of colonic mucins. The experimental results of this example demonstrate that both live and heat-killed Lactobacillus acidophilus LIHUO 1978 bacteria significantly increase AQP9 concentrations in colonic tissue by increasing the content of mucus-secreting goblet cells in the colonic tissue of constipated rats, thereby alleviating constipation.

[0175] Example 13 Effects of live and heat-killed Lactobacillus acidophilus LIHUO 1978 on serum nitric oxide synthase levels and colon tissue IL-10 levels in constipated rats Experimental methods: (1) Determination of rat serum iNOS concentration The iNOS concentration in the serum of the rats in each group in Example 9 was determined using an iNOS enzyme-linked immunosorbent assay kit (manufacturer: Shanghai ELISA, catalog number: ml059045V) and in strict accordance with the instructions.

[0176] (2) Determination of IL-10 concentration in rat colon tissue The colon tissues of the rats in each group in Example 9 were homogenized and centrifuged to obtain supernatants. The protein concentration of the colon tissues of the rats in each group was determined in the same manner as in Example 11. The IL-10 concentration in the colon tissues of the rats in each treatment group in Example 9 was determined using an ELISA kit (manufacturer: Shanghai ELISA, catalog number: ml002813V) and strictly in accordance with the instructions.

[0177] The experimental results are as follows: Compared with the normal group, the serum iNOS concentration of rats in the constipation model group increased significantly by 521.21%; while compared with the constipation model group, the serum iNOS concentration of rats in the polyethylene glycol 4000 powder group, LIHUO 1978 group, and heat-inactivated LIHUO 1978 group decreased significantly by 50.49%, 59.76%, and 54.63%, respectively (see Figure 21 ).

[0178] Compared with the normal group, the IL-10 concentration in the colon tissue of rats in the constipation model group decreased significantly by 22.07%; compared with the constipation model group, the IL-10 concentration in the colon tissue of rats in the polyethylene glycol 4000 powder group increased by 4.42%, but the difference was not significant. However, the IL-10 concentration in the colon tissue of rats in the LIHUO 1978 group and the heat-inactivated LIHUO 1978 group increased significantly by 29.20% and 17.70%, respectively (see Figure 22 ).

[0179] iNOS is associated with intestinal inflammation and can cause intestinal smooth muscle relaxation by producing excessive NO, thereby aggravating constipation symptoms. IL-10 is an anti-inflammatory factor that can alleviate intestinal inflammation caused by constipation. The experimental results of this example show that both live and heat-killed Lactobacillus acidophilus LIHUO 1978 bacteria can significantly reduce serum iNOS concentrations in constipated rats, significantly increase IL-10 concentrations in colon tissue, and regulate the levels of inflammation-related factors, thereby alleviating constipation.

[0180] Example 14 Effects of live and heat-killed Lactobacillus acidophilus LIHUO 1978 on the intestinal flora of constipated rats Experimental methods: In Example 9, cecal contents of six rats were randomly selected from each group, and microbial diversity was analyzed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. (the experimental method was based on the method described in patent CN119331785B). Total DNA was extracted from the rat cecal contents, and the V3-V4 region of the 16S rDNA was amplified using 338F and 806R. All sequences were divided into OTUs based on different similarity levels, and bioinformatics statistical analysis was performed on OTUs at the 97% similarity level. Alpha and beta diversity, as well as the proportions of different species in samples from different groups and species differences, were analyzed. Plots were performed using the R language.

[0181] The experimental results are as follows: Compared with the normal group, the Ace index of the cecal contents microbiome of the rats in the constipation model group at the genus level was not significantly different; compared with the constipation model group, the Ace index of the cecal contents microbiome of the rats in the polyethylene glycol 4000 powder group, the LIHUO 1978 group, and the heat-inactivated LIHUO 1978 group at the genus level was not significantly different (see Figure 23 ).

[0182] Compared with the normal group, the Chao index of the cecal contents microbiome of rats in the constipation model group at the genus level was not significantly different; compared with the constipation model group, the Chao index of the cecal contents microbiome of rats in the polyethylene glycol 4000 powder group at the genus level was significantly increased, while the Chao index of the cecal contents microbiome of rats in the LIHUO 1978 group and the heat-inactivated LIHUO 1978 group at the genus level was not significantly different (see Figure 23 ).

[0183] Principal component analysis (PCA) and principal coordinate analysis (PCoA) of the cecal microbiome at the genus level in each group of rats (see Figure 24 ) It can be seen that the normal group is far away from the constipation model group, indicating that the composition of the microbiome of the cecal contents of rats in the normal group and the constipation model group is different; the LIHUO 1978 group and the heat-killed LIHUO 1978 group are closer to the normal group and farther away from the constipation model group, indicating that the composition of the microbiome of the cecal contents of rats in the LIHUO 1978 group, the heat-killed LIHUO 1978 group and the normal group is closer. Clostridia _UCG-014, Rombutsiella spp. Romboutsia ), Lactobacillus ( Lactobacillus ) in the cecal contents of rats in each group had a high relative abundance and was the dominant bacterial species (see Figure 25 The relative abundance of microorganisms at the genus level in the cecal contents of rats in the LIHUO 1978 group and the heat-killed LIHUO 1978 group was closer to that in the normal group.

[0184] Compared with the normal group, the cecal contents of rats in the constipation model group contained more Rombutsiella ( Romboutsia ), Staphylococcus spp. ( Staphylococcus ), Mammalian cocci ( Mammaliicoccus ), Saltwater Sea Coccus ( Jeotgalicoccus ), host-associated Lactobacillus spp. ( Ligilactobacillus ), Zurichobacterium spp. ( Turicibacter ) decreased significantly, norank_f_ Muribaculaceae 、 Lachnospiraceae _NK4A136_group, Bacteroides spp. Bacteroides )、[ Eubacterium ]_ siraeum Compared with the constipation model group, the relative abundance of Lactobacillus genus in the cecal contents microbiome of the rats in the polyethylene glycol 4000 powder group was significantly increased. Lactobacillus ), Rombutsiella ( Romboutsia ), Corynebacterium spp. ( Corynebacterium ), Faecalibacterium ( Faecalibaculum ), Mammalian cocci ( Mammaliicoccus ), Zurichobacterium spp. ( Turicibacter ), Clostridium ( Clostridium ) increased significantly. Lachnospiraceae _NK4A136_group、norank_f_ Oscillospiraceae , Colonobacter spp. ( Colidextribacter ) decreased significantly. Compared with the constipation model group, the relative abundance of Rombutsella ( Romboutsia ), Lactobacillus spp. ( Lactobacillus ), Corynebacterium spp. ( Corynebacterium ), Staphylococcus spp. ( Staphylococcus ), Lactobacillus mucilaginosus ( Limosilactobacillus ), Zurichobacterium spp. ( Turicibacter ), Saltwater Sea Coccus ( Jeotgalicoccus ) increased significantly, norank_f_ Muribaculaceae 、 Christensenellaceae _R-7_group, Colonobacter spp. ( Colidextribacter ) decreased significantly. Compared with the constipation model group, the relative abundance of Lactobacillus genus ( Lactobacillus ), Rombutsiella ( Romboutsia ), Staphylococcus spp. ( Staphylococcus ), Mammalian cocci ( Mammaliicoccus ), Zurichobacterium spp. ( Turicibacter ) increased significantly, norank_f_ Muribaculaceae 、 Lachnospiraceae _NK4A136_group、norank_f_ Oscillospiraceae , Ruminococcus spp. ( Ruminococcus ), Colonobacter spp. ( Colidextribacter ) decreased significantly (see Figure 26 ).

[0185] The experimental results of this example show that both live and heat-killed Lactobacillus acidophilus LIHUO 1978 bacteria can improve the β diversity of the cecal contents of constipated rats. Live Lactobacillus acidophilus LIHUO 1978 bacteria can significantly increase the Lactobacillus genus ( Lactobacillus ), Lactobacillus mucilaginosus ( Limosilactobacillus ) relative abundance, Lactobacillus acidophilus LIHUO 1978 heat-killed bacteria can significantly increase the Lactobacillus genus ( Lactobacillus ) and thus regulate the intestinal flora.

[0186] Example 15 Effects of live and heat-killed Lactobacillus acidophilus LIHUO 1978 on the content of short-chain fatty acids in the intestinal contents of constipated rats Experimental methods: In Example 9, the colon contents of 5 rats were randomly selected from each group, and the contents of short-chain fatty acids, including isobutyric acid, isovaleric acid, and valeric acid, were detected by GC-MC method by Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0187] The specific results are as follows: Compared with the normal group, the isobutyric acid concentration in the colon contents of rats in the constipation model group decreased significantly by 54.84%. Compared with the constipation model group, the isobutyric acid concentration in the colon contents of rats in the polyethylene glycol 4000 powder group and the heat-inactivated LIHUO 1978 group increased by 35.71% and 42.86%, respectively, but the differences were not significant. However, the isobutyric acid concentration in the colon contents of rats in the LIHUO 1978 group increased significantly by 92.86% (see Figure 27 ).

[0188] Compared with the normal group, the isovaleric acid concentration in the colon contents of rats in the constipation model group decreased significantly by 63.04%. Compared with the constipation model group, the isovaleric acid concentration in the colon contents of rats in the polyethylene glycol 4000 powder group and the heat-inactivated LIHUO 1978 group increased by 82.35% and 82.35%, respectively, but the differences were not significant. The isovaleric acid concentration in the colon contents of rats in the LIHUO 1978 group increased significantly by 129.41% (see Figure 27 ).

[0189] Compared with the normal group, the valeric acid concentration in the colon contents of rats in the constipation model group decreased significantly by 43.14%. Compared with the constipation model group, the valeric acid concentration in the colon contents of rats in the polyethylene glycol 4000 powder group and the heat-inactivated LIHUO 1978 group increased by 31.03% and 48.28%, respectively, but the differences were not significant. However, the valeric acid concentration in the colon contents of rats in the LIHUO 1978 group increased significantly by 82.76% (see Figure 27 ).

[0190] Isobutyric acid, isovaleric acid, and valeric acid are short-chain fatty acids that serve as energy for intestinal epithelial cells and promote mucosal growth. In this example, the live Lactobacillus acidophilus LIHUO 1978 bacteria increased the concentrations of isobutyric acid, isovaleric acid, and valeric acid in constipated rats, promoting colonic mucosal growth and reducing stool transit time, thereby alleviating constipation.

[0191] The above description is only a preferred embodiment 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 in the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus acidophilus ( Lactobacillus acidophilus ) LIHUO 1978, characterized by, The deposit number of the Lactobacillus acidophilus LIHUO 1978 is GDMCC No: 65514.

2. A Lactobacillus acidophilus LIHUO 1978 preparation, characterized in that: The Lactobacillus acidophilus LIHUO 1978 preparation is prepared by culturing Lactobacillus acidophilus LIHUO 1978 in a culture medium.

3. The Lactobacillus acidophilus LIHUO 1978 preparation according to claim 2, characterized in that The Lactobacillus acidophilus LIHUO 1978 preparation comprises live bacteria, fermentation liquid, fermentation liquid precipitation, heat-inactivated bacteria and freeze-dried powder.

4. A microbial agent, characterized in that: The microbial agent comprises the Lactobacillus acidophilus LIHUO 1978 according to claim 1 or the Lactobacillus acidophilus LIHUO 1978 preparation according to any one of claims 2-3.

5. Use of the Lactobacillus acidophilus LIHUO 1978 according to claim 1, the Lactobacillus acidophilus LIHUO 1978 preparation according to any one of claims 2 to 3, or the microbial agent according to claim 4 in the preparation of a medicament for treating diarrhea.

6. The use according to claim 5, characterized in that The diarrhea is chemotherapy-induced diarrhea.

7. The use according to claim 5, characterized in that The viable count of Lactobacillus acidophilus LIHUO 1978 in the medicine is 1.00×10 6 -1.00×10 12 CFU / mL or 1.00×10 6 -1.00×10 12 CFU / g.

8. The use according to claim 7, characterized in that The viable count of Lactobacillus acidophilus LIHUO 1978 in the medicine is 5.00×10 9 CFU / mL or 5.00×10 9 CFU / g.

9. The use according to any one of claims 5 to 8, characterized in that: The drug has at least one of the following effects: (1) Significantly reduce diarrhea scores; (2) Significantly increase the length of the small intestine; (3) Significantly reduced jejunal tissue pathology score; (4) Significantly reduce serum interleukin-1β concentration; (5) Significantly increase serum interleukin-10 concentration.

10. A drug for relieving diarrhea, characterized in that: The medicine comprises the Lactobacillus acidophilus LIHUO 1978 according to claim 1 or the Lactobacillus acidophilus LIHUO according to any one of claims 2-3 1978 preparation or the microbial agent according to claim 4.

11. Use of the Lactobacillus acidophilus LIHUO 1978 according to claim 1, the Lactobacillus acidophilus LIHUO 1978 preparation according to any one of claims 2 to 3, or the microbial agent according to claim 4 in preparing a product for improving constipation.

12. The use according to claim 11, characterized in that The product is a health food, an animal nutrition product or a medicine.

13. The use according to claim 12, characterized in that When the product is a medicine, the medicine also includes pharmaceutically acceptable excipients; When the product is a health food, the health food also includes auxiliary materials acceptable in food.

14. The use according to claim 11, characterized in that The viable count of Lactobacillus acidophilus LIHUO 1978 in the product is 1.00×10 6 -1.00×10 12 CFU / mL or 1.00×10 6 -1.00×10 12 CFU / g.

15. The use according to claim 14, characterized in that The viable count of Lactobacillus acidophilus LIHUO 1978 in the product is 1.00×10 8 CFU / mL or 1.00×10 8 CFU / g.

16. The use according to any one of claims 11 to 15, characterized in that: The product has at least one of the following functions: (1) Significantly increase the number of fecal particles and fecal water content; (2) Significantly improve gastrointestinal propulsion rate; (3) Significantly reduce serum endothelin-1, serum vasoactive intestinal peptide, serum somatostatin, and serum nitric oxide synthase concentrations; (4) Significantly increased serum motilin, serum substance P, serum acetylcholine, colon tissue 5-hydroxytryptamine, and colon tissue interleukin-10 concentrations; (5) Significantly increased the content of mucus-secreting goblet cells; (6) Significantly increase the concentration of aquaporin 9 in colon tissue; (7) Regulate the intestinal flora of cecal contents; (8) Significantly increase the concentrations of isobutyric acid, isovaleric acid, and valeric acid in the colon contents.

17. A product for improving constipation, characterized in that: The product comprises the Lactobacillus acidophilus LIHUO 1978 of claim 1 or the Lactobacillus acidophilus LIHUO of any one of claims 2-3. 1978 preparation or the microbial agent according to claim 4.

18. Use of the Lactobacillus acidophilus LIHUO 1978 according to claim 1, the Lactobacillus acidophilus LIHUO 1978 preparation according to any one of claims 2 to 3, or the microbial agent according to claim 4 in the preparation of a product for regulating intestinal flora.

19. The use according to claim 18, characterized in that The product can significantly increase the relative abundance of Lactobacillus and Lactobacillus mucis in the intestine.

20. A product for regulating intestinal flora, characterized in that: The product comprises the Lactobacillus acidophilus LIHUO 1978 of claim 1 or the Lactobacillus acidophilus LIHUO of any one of claims 2-3. 1978 preparation or the microbial agent according to claim 4.

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