Lactobacillus paracasei zj-35 and application thereof

Abstract

This invention relates to the field of microbial technology, specifically to Lactobacillus paracasei ZK-35 and its applications. The Lactobacillus paracasei ZK-35 of this invention is isolated from human oral rinsing fluid, exhibiting good compatibility with the human intestinal environment and high safety. It possesses excellent acid and bile salt resistance, and can alleviate the side effects of various antibiotics such as penicillins, cephalosporins, and quinolones. It can simultaneously regulate multiple intestinal flora, improve constipation and diarrhea, enhance intestinal barrier function, and promote nutrient absorption.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to Lactobacillus paracasei ZK-35 and its applications. Background Technology

[0002] As the largest digestive and immune organ in the human body, the health of the gut is closely related to overall health. Adults experience varying degrees of gut health problems, with constipation (18%–25%), chronic diarrhea (8%–12%), and gut microbiota imbalance (30%–40%) being the most common. Meanwhile, the incidence of inflammatory bowel disease (IBD, including ulcerative colitis (UC) and Crohn's disease (CD)) is gradually increasing and showing a trend towards affecting younger people.

[0003] A diet high in sugar, fat, and fiber leads to a decrease in gut microbiota diversity and the proliferation of harmful bacteria. Long-term sleep deprivation, high stress, and lack of exercise can inhibit intestinal peristalsis and reduce the colonization of beneficial bacteria through the hypothalamic-pituitary-adrenal axis (HPA axis). In addition to antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and other medications can also damage the intestinal mucosa. Environmental pollutants (such as heavy metals and pesticide residues) can disrupt the balance of gut microbiota. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention aims to provide Lactobacillus paracasei ZK-35 and its applications, which possesses the ability to alleviate the side effects of broad-spectrum antibiotics and has a comprehensive function of regulating intestinal flora.

[0005] To solve the above problems, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a Lactobacillus paracasei ZK-35, which was deposited on July 9, 2025 at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.35142.

[0007] Secondly, the present invention provides the use of the aforementioned Lactobacillus paracasei ZK-35 and / or its metabolites in alleviating antibiotic-induced intestinal problems.

[0008] Furthermore, the antibiotics include amoxicillin and cefixime.

[0009] Furthermore, the intestinal problems caused by the antibiotics include the occurrence of AAD, intestinal flora imbalance, impaired intestinal barrier function, intestinal inflammatory response, Clostridium difficile colonization, reduced secretion of short-chain fatty acids, decreased nutrient absorption, or diarrhea.

[0010] Thirdly, the present invention provides the application of the aforementioned Lactobacillus paracasei ZK-35 and / or its metabolites in intestinal improvement.

[0011] Fourthly, the present invention provides a composition for relieving ulcerative colitis, comprising the aforementioned Lactobacillus paracasei ZK-35, Lactobacillus acidophilus, and Bifidobacterium longum.

[0012] Furthermore, the weight ratio of *Lactobacillus paracasei* ZK-35, *Lactobacillus acidophilus*, and *Bifidobacterium longum* is 2-10:1-2:1-2; the total viable count of the composition is 1.0 × 10⁻⁶. 11 - 8.0×10 11 CFU / g.

[0013] Fifthly, the present invention provides the use of the aforementioned Lactobacillus paracasei ZK-35 and / or its metabolites or the aforementioned composition in the preparation of intestinal improvement products.

[0014] Furthermore, the intestinal improvement products may be in the form of lyophilized powder, oral liquid, tablets, capsules, granules, or drops.

[0015] The beneficial effects of this invention are as follows: Lactobacillus paracasei ZK-35 of this invention is isolated from human oral rinsing fluid, has good compatibility with the human intestinal environment, high safety, excellent acid and bile salt resistance, and can alleviate the side effects of various antibiotics such as penicillins, cephalosporins, and quinolones. It can simultaneously regulate multiple intestinal flora (increasing Lactobacillus, Bifidobacterium, Akk bacteria, and Faecalibacterium, and decreasing Escherichia coli and Clostridium difficile), improve constipation and diarrhea, enhance intestinal barrier function, and promote nutrient absorption. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to specific embodiments.

[0017] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0018] This invention relates to Lactobacillus paracasei ZK-35, which was deposited on July 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.35142.

[0019] Example 1

[0020] Isolation, purification and identification of Lactobacillus paracasei ZK-35

[0021] 1.1 Test Materials

[0022] Sample source: Oral rinsing solution from healthy adults (2 males, aged 28 and 32; 2 females, aged 25 and 30). All volunteers had not used antibiotics, probiotics, or immunomodulators in the past 3 months and had no history of intestinal or oral diseases.

[0023] Culture media: MRS solid medium (10g tryptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 2g diammonium citrate, 1mL Tween-80, 0.58g MgSO4·7H2O, 0.25g MnSO4·4H2O, 15g agar, 1000mL distilled water, pH 6.2~6.4, sterilized at 121℃ for 20min); MRS liquid medium (without agar, other components are the same as MRS solid medium).

[0024] Reagents: Gram staining solution (crystal violet staining solution, iodine solution, destaining solution, counterstaining solution), API 50 CH carbohydrate fermentation kit (bioMérieux, France), DNA extraction kit (Tiangen Biotech Beijing Co., Ltd.), 16S rRNA gene amplification primers (upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3', Shanghai Sangon Biotech Co., Ltd.), PCR Master Mix (Takara Bio Engineering Dalian Co., Ltd.).

[0025] Instruments: Anaerobic incubator (model YQX-II, Shanghai Xinmiao Medical Instrument Co., Ltd.), biological microscope (model BX53, Olympus China Co., Ltd.), PCR instrument (model C1000, Bio-Rad, USA), gene sequencer (model ABI 3730xl, Applied Biosystems, USA).

[0026] 1.2 Test Methods

[0027] Sample collection and processing:

[0028] Volunteers rinsed their mouths with 100 mL of sterile saline (37°C) for 30 seconds, and then collected the rinsing solution in a sterile centrifuge tube, which became the oral rinsing solution sample.

[0029] Take 10 mL of sample, add 90 mL of sterile physiological saline, and perform a 10-fold serial dilution (10... -1 ~10-6 ).

[0030] Separation and purification:

[0031] Take 10 -4 10 -5 10 -6 0.1 mL of each dilution was spread onto MRS solid culture medium plates, and each dilution was performed in triplicate.

[0032] The plates were placed in an anaerobic incubator (37℃, 85% N2, 10% H2, 5% CO2) and incubated for 48 hours.

[0033] Observe the colony morphology, select colonies that conform to the characteristics of Lactobacillus (round, milky white, with neat edges and smooth surface), inoculate them into MRS liquid medium, and anaerobic culture at 37℃ for 24 h to obtain pure culture.

[0034] Repeat the streak purification process three times to ensure the purity of the strain.

[0035] Morphological identification:

[0036] Take a smear of pure culture, perform Gram staining, and observe the morphology of the bacteria under an optical microscope (1000×).

[0037] Physiological and biochemical identification:

[0038] Following the instructions of the API 50 CH Carbohydrate Fermentation Kit, the pure culture was inoculated into the reaction tubes of the kit and incubated at 37°C for 48 hours. The acid production was then observed (the reaction tube turning from purple to yellow indicates a positive result).

[0039] Acid resistance test: The activated bacterial suspension (1.0 × 10⁻⁶) was subjected to acid resistance test. 9 CFU / mL were inoculated into MRS liquid medium at pH 2.0, 3.0, and 4.0, respectively, and incubated at 37°C for 2 h. The number of viable bacteria was detected by plate counting method, and the survival rate was calculated (survival rate = number of viable bacteria after treatment / number of viable bacteria before treatment × 100%).

[0040] Bile salt tolerance test: The activated bacterial suspension (1.0 × 10⁻⁶) was subjected to a bile salt tolerance test. 9 CFU / mL were inoculated into MRS liquid medium containing 0.1%, 0.3%, and 0.5% bile salts, respectively, and incubated at 37°C for 2 hours. The viable count was determined by plate counting, and the survival rate was calculated.

[0041] Growth temperature experiment: The activated bacterial suspension was inoculated into MRS liquid medium and cultured at 15℃, 25℃, 37℃, 42℃, and 45℃ for 24 h, respectively. OD was then measured. 600Value, used to determine growth status (OD) 600 ≥0.5 indicates positive growth.

[0042] Molecular biological identification:

[0043] Genomic DNA was extracted from the strain using a DNA extraction kit.

[0044] Using genomic DNA as a template, PCR amplification was performed using 27F / 1492R primers: The reaction system (50 μL) included 25 μL PCR Master Mix, 2 μL upstream primer (10 μmol / L), 2 μL downstream primer (10 μmol / L), 5 μL DNA template, and 16 μL sterile water; the reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; and a final extension at 72℃ for 10 min.

[0045] After verification by 1% agarose gel electrophoresis, the PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0046] The sequencing results were submitted to the GenBank database, and the BLAST tool was used to compare the homology with the 16S rRNA gene sequences of known strains to construct a phylogenetic tree.

[0047] 1.3 Test Results

[0048] Isolation results: A total of 42 lactobacillus-like strains were isolated from 4 oral rinsing fluid samples, numbered ZK-1 to ZK-42.

[0049] Morphological identification results:

[0050] All 12 strains formed round, milky-white colonies with neat edges and smooth surfaces on MRS solid medium, with a diameter of 1.0–1.5 mm.

[0051] All bacteria were Gram-positive, and the cells were short rod-shaped, non-spore-forming, non-flagellated, and non-motile.

[0052] Physiological and biochemical identification results:

[0053] Carbohydrate fermentation results: Strain ZK-35 can ferment glucose, lactose, sucrose, maltose, fructose, galactose, and mannose to produce acid and gas; it cannot ferment xylose, arabinose, raffinose, rhamnose, or sorbitol; other strains cannot ferment lactose or maltose, which differs from ZK-35.

[0054] Acid resistance test results: After treatment at pH 2.0 for 2 hours, the survival rate of ZK-35 was 52.3%±3.5%; after treatment at pH 3.0 for 2 hours, the survival rate was 85.6%±4.2%; after treatment at pH 4.0 for 2 hours, the survival rate was 96.8%±2.1%; significantly higher than other strains.

[0055] Results of bile salt tolerance test: After treatment with 0.1% bile salt for 2 hours, the survival rate of ZK-35 was 92.5%±3.1%; after treatment with 0.3% bile salt for 2 hours, the survival rate was 41.8%±3.7%; after treatment with 0.5% bile salt for 2 hours, the survival rate was 15.6%±2.4%; which was significantly higher than that of other strains.

[0056] Growth temperature experiment results: ZK-35 can grow at 15℃, 25℃, 37℃, and 42℃ (OD). 600 The values ​​were 0.62±0.05, 1.25±0.08, 2.18±0.12, and 0.85±0.06, respectively, and no growth was observed at 45℃ (OD). 600 =0.21±0.03); the optimal growth temperature is 37℃.

[0057] Molecular biological identification results:

[0058] The 16S rRNA gene of strain ZK-35 was sequenced and its sequence was submitted to GenBank. BLAST alignment results showed that ZK-35 had 99.8% homology with the Lactobacillus paracasei model strain (GenBank accession number NR_074515.1) and ≤95% homology with other Lactobacillus strains (such as Lactobacillus acidophilus and Lactobacillus plantarum).

[0059] Phylogenetic analysis showed that ZK-35 clustered with the Lactobacillus paracasei type strain, with a bootstrap value of 99%, further confirming that it is Lactobacillus paracasei.

[0060] In summary, a strain of Lactobacillus paracasei with excellent acid and bile salt resistance was isolated from human oral rinsing fluid, named ZK-35, and preserved.

[0061] Example 2

[0062] Lactobacillus paracasei ZK-35 relieves amoxicillin side effects

[0063] 2.1 Test Materials

[0064] Strain: Lactobacillus paracasei ZK-35 (CGMCC NO.35142, prepared in our laboratory, viable count 1.0 × 10⁻⁶) 11CFU / g); Lactobacillus rhamnosus, commercially available, viable count 1.0 × 10⁻⁶. 11 CFU / g, as a positive control).

[0065] Experimental animals: SPF grade ICR mice, 6-8 weeks old, half male and half female, weighing 20-22g, totaling 120 mice.

[0066] Reagents: Amoxicillin capsules (North China Pharmaceutical Group Co., Ltd., 0.25g / capsule, prepared into a 20mg / mL solution with sterile physiological saline); Clostridium difficile (ATCC 9689, preserved in our laboratory); Mouse TNF-α ELISA kit, Mouse IL-6 ELISA kit, Mouse IL-10 ELISA kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd.); Mouse occludin antibody, Mouse ZO-1 antibody (Abcam, USA); Western blot detection kit (Beyotime Biotechnology Research Institute).

[0067] Instruments: Electronic balance (model FA2004, Shanghai Precision Scientific Instruments Co., Ltd.); Microplate reader (model Multiskan FC, Thermo Fisher Scientific, USA); High-throughput sequencer (model Illumina MiSeq, Illumina, USA); Cryostat (model CM1950, Leica, Germany).

[0068] 2.2 Test Methods

[0069] Strain activation and bacterial suspension preparation:

[0070] Lactobacillus paracasei ZK-35 and Lactobacillus rhamnosus were inoculated into MRS liquid medium and cultured anaerobically at 37°C for 16 h to activate three generations.

[0071] Collect bacterial cells by centrifugation (4℃, 6000 r / min, 10 min), wash twice with sterile physiological saline, and adjust the bacterial suspension concentration to 1.0 × 10⁻⁶. 9 CFU / mL (low dose), 5.0×10 9 CFU / mL (medium dose), 1.0×10 10 CFU / mL (high dose).

[0072] Animal grouping and treatment:

[0073] 120 mice were randomly divided into 6 groups of 20 each, with half males and half females.

[0074] Control group: 0.2 mL of sterile saline per animal was administered by gavage daily for 21 consecutive days;

[0075] Amoxicillin model group (AMX): 0.2 mL amoxicillin solution per animal (dose 200 mg / kg) was administered by gavage from day 1 to day 7, and 0.2 mL sterile saline per animal was administered by gavage from day 8 to day 21.

[0076] ZK-35 low-dose group (ZK-35-L): Amoxicillin solution 0.2 mL / animal was administered by gavage from days 1 to 7, and ZK-35 bacterial suspension 0.2 mL / animal was administered by gavage from days 1 to 21 (dose 1.0 × 10⁻⁶). 8 CFU / each, equivalent to 5.0 × 10 9 CFU / kg).

[0077] ZK-35 medium-dose group (ZK-35-M): Days 1-7, administer 0.2 mL amoxicillin solution per animal via gavage; Days 1-21, administer 0.2 mL ZK-35 bacterial suspension per animal via gavage (dose 5.0 × 10⁻⁶). 8 CFU / each, equivalent to 2.5 × 10 10 CFU / kg).

[0078] ZK-35 high-dose group (ZK-35-H): Amoxicillin solution 0.2 mL / animal was administered by gavage on days 1-7, and ZK-35 bacterial suspension 0.2 mL / animal was administered by gavage on days 1-21 (dose 1.0 × 10⁻⁶). 9 CFU / each, equivalent to 5.0 × 10 10 CFU / kg).

[0079] GG control group (GG): 0.2 mL / bird of amoxicillin solution was administered by gavage from day 1 to 7, and 0.2 mL / bird of bacterial suspension was administered by gavage from day 1 to 21 (dose 1.0 × 10⁻⁶). 9 CFU / each, equivalent to 5.0 × 10 10 CFU / kg).

[0080] All mice were housed in an SPF-grade animal facility at a temperature of 22-25°C and a humidity of 40-60%, with a 12-hour light / 12-hour dark cycle and free access to food and water.

[0081] Detection indicators and methods:

[0082] General clinical observation: Observe the mice's mental state (active / listless), appetite (food intake), and defecation (stool characteristics, occurrence of diarrhea) daily, and record the incidence of diarrhea and diarrhea score.

[0083] Diarrhea scoring criteria: 0 points: formed stool, granular; 1 point: loose stool, unformed; 2 points: loose stool, sticky to the anus; 3 points: watery stool, projectile stool.

[0084] Weight change: Weigh yourself once a week (day 1, day 7, day 14, day 21) and calculate the weight growth rate (weight growth rate = (daily weight - initial weight) / initial weight × 100%).

[0085] Gut microbiota analysis: On day 21, 6 mice from each group were randomly selected and euthanized by cervical dislocation. 0.5g of cecal contents were aseptically collected, and total DNA was extracted using the CTAB method. The V4 region of the 16S rRNA gene was amplified (primers 515F: 5'-GTGCCAGCMGCCGCGGTAA-3', 907R: 5'-CCGTCAATTCMTTTRAGTTT-3'). Illumina MiSeq high-throughput sequencing was performed to analyze the α-diversity (Chao1 index, Shannon index) and β-diversity (PCoA analysis) of the gut microbiota, as well as the relative abundance of key microbiota.

[0086] Intestinal barrier function testing:

[0087] Tight junction protein expression: 0.5g of colon tissue was taken, total protein was extracted, and the protein expression levels of occludin and ZO-1 were detected by Western blot (β-actin was used as an internal reference, and the relative expression level was calculated).

[0088] Serum endotoxin (LPS) content: Blood was collected from mouse eyeballs, and serum was separated by centrifugation (37℃, 3000r / min, 10min). LPS content was detected by the Limulus Amebocyte Lysate (LAL) method.

[0089] Mucosal sIgA content: Take 0.5g of colonic mucosal tissue, add PBS buffer (containing protease inhibitor) to homogenize, centrifuge (4℃, 12000r / min, 15min), take the supernatant, and detect the sIgA content by ELISA.

[0090] Inflammatory factor detection: Serum and colon tissue homogenate supernatant were collected, and the levels of TNF-α, IL-6 (pro-inflammatory factor) and IL-10 (anti-inflammatory factor) were detected by ELISA.

[0091] Detection of short-chain fatty acids (SCFAs): Take 0.5g of cecal contents, add 1mL of ultrapure water to homogenize, centrifuge (4℃, 12000r / min, 15min), collect the supernatant, filter through a 0.22μm filter membrane, and use a gas chromatograph (model GC-2014, Shimadzu Corporation, Japan) to detect the contents of acetic acid, propionic acid and butyric acid.

[0092] Chromatographic conditions: DB-FFAP capillary column (30m×0.25mm×0.25μm), column temperature 40℃ for 5 min, then increased to 150℃ at 5℃ / min and held for 2 min; injection port temperature 200℃, detector temperature 250℃; carrier gas nitrogen, flow rate 1 mL / min.

[0093] Clostridium difficile colonization detection: On day 14, 6 mice from each group were randomly selected and administered Clostridium difficile bacterial suspension (1.0 × 10⁻⁶) by gavage. 6 CFU / animal), cecal contents were collected on day 21, and Clostridium difficile was isolated and cultured on CCFA medium (cycloserine-cefoxitin-fructose-agar), and viable count was determined by plate count method.

[0094] 2.3 Test Results

[0095] General clinical observation results:

[0096] Blank control group: Mice were energetic, had normal appetite, formed stools, and no diarrhea occurred;

[0097] Amoxicillin model group: Diarrhea began to appear on day 3, and the incidence of diarrhea reached 85% (17 / 20) on day 7, with a diarrhea score of 2.8±0.3; mice were lethargic and had a decreased appetite (food intake was reduced by 40%~50% compared with the control group).

[0098] In each dose group of ZK-35, the incidence of diarrhea and the diarrhea score decreased significantly with increasing dose; the incidence of diarrhea in the ZK-35-H group on day 7 was 20% (4 / 20), and the diarrhea score was 0.6±0.2, which was significantly lower than that in the model group (P<0.01); the mental state and appetite of the mice were close to those of the blank group.

[0099] Control group: The incidence of diarrhea on day 7 was 45% (9 / 20), and the diarrhea score was 1.5±0.3, which was significantly higher than that of the ZK-35-H group (P<0.05).

[0100] On day 21, the incidence of diarrhea in the ZK-35-H group dropped to 0, while 30% (6 / 20) of the mice in the model group still had diarrhea, and the incidence of diarrhea in the GG control group was 15% (3 / 20).

[0101] Weight change results:

[0102] Blank control group: Weight continued to increase, with a weight increase rate of 35.2% ± 4.1% on day 21.

[0103] The amoxicillin model group showed a weight gain of -8.5% ± 2.3% on day 7 (weight loss) and a weight gain of 12.3% ± 3.2% on day 21, which were significantly lower than the control group (P < 0.01).

[0104] In each dose group of ZK-35, the weight gain rate increased significantly with increasing dose; the weight gain rate of the ZK-35-H group was -2.1%±1.5% on day 7 and 32.5%±3.8% on day 21, which was not significantly different from the blank group (P>0.05) but significantly higher than the model group and the control group (P<0.01).

[0105] The control group had a weight gain of -5.8% ± 2.1% on day 7 and a weight gain of 22.6% ± 3.5% on day 21, which was significantly lower than that of the ZK-35-H group (P<0.05).

[0106] Gut microbiota analysis results:

[0107] Alpha diversity analysis: The Chao1 index (285.6±25.3) and Shannon index (2.1±0.3) in the model group were significantly lower than those in the control group (Chao1 index 452.8±32.5, Shannon index 3.8±0.4) (P<0.01); The Chao1 index (421.5±30.2) and Shannon index (3.6±0.3) in the ZK-35-H group were not significantly different from those in the control group (P>0.05), but were significantly higher than those in the model group and the control group (Chao1 index 352.7±28.6, Shannon index 2.9±0.3) (P<0.01).

[0108] β-diversity analysis: PCoA analysis showed that the gut microbiota structure of the blank group and the ZK-35-H group clustered into one branch, the model group clustered into a separate branch, and the control group was in between, indicating that ZK-35 can significantly restore the gut microbiota structure imbalance caused by amoxicillin.

[0109] Relative abundance of key bacterial communities:

[0110] Beneficial bacteria: The abundance of Lactobacillus (2.1%±0.5%), Bifidobacterium (1.8%±0.4%), Akk bacteria (0.3%±0.1%), and Faecalibacterium (0.5%±0.2%) in the model group was significantly lower than that in the control group (Lactobacillus 15.2%±1.8%, Bifidobacterium 12.5%±1.5%, Akk bacteria 3.2%±0.5%, Faecalibacterium 4.8%±0.8%) (P<0.01); The abundance of the above beneficial bacteria in the ZK-35-H group was 14.8%±1.6%, 11.9%±1.4%, 2.9%±0.4%, and 4.5%±0.7%, respectively, which was not significantly different from the control group (P>0.05), but significantly higher than that in the model group and the control group (P<0.01).

[0111] Harmful bacteria: The abundance of Escherichia coli (18.5%±2.3%), Enterococcus spp. (12.3%±1.8%), and Clostridium difficile (5.2%±0.8%) in the model group was significantly higher than that in the control group (Escherichia coli 3.2%±0.6%, Enterococcus spp. 2.1%±0.5%, Clostridium difficile 0.1%±0.05%) (P<0.01); The abundance of the above harmful bacteria in the ZK-35-H group was 3.5%±0.7%, 2.3%±0.6%, and 0.2%±0.08%, respectively, which were not significantly different from those in the control group (P>0.05), but significantly lower than those in the model group and the control group (P<0.01).

[0112] Results of intestinal barrier function test:

[0113] Tight junction protein expression: The relative expression levels of occludin (0.3±0.1) and ZO-1 (0.2±0.1) in the model group were significantly lower than those in the blank group (occludin 1.0±0.1, ZO-1 1.0±0.1) (P<0.01); The relative expression levels of occludin (0.9±0.1) and ZO-1 (0.8±0.1) in the ZK-35-H group were not significantly different from those in the blank group (P>0.05), but were significantly higher than those in the model group and the control group (occludin 0.6±0.1, ZO-1 0.5±0.1) (P<0.01).

[0114] Serum LPS levels: The serum LPS level in the model group (85.6±10.2 EU / mL) was significantly higher than that in the blank group (15.2±2.5 EU / mL) (P<0.01); the serum LPS level in the ZK-35-H group (18.5±3.2 EU / mL) was not significantly different from that in the blank group (P>0.05), but was significantly lower than that in the model group and the control group (45.3±8.5 EU / mL) (P<0.01).

[0115] Mucosal sIgA content: The mucosal sIgA content in the model group (5.2±1.1 μg / mL) was significantly lower than that in the blank group (15.8±2.3 μg / mL) (P<0.01); the mucosal sIgA content in the ZK-35-H group (14.9±2.1 μg / mL) was not significantly different from that in the blank group (P>0.05), but was significantly higher than that in the model group and the control group (9.8±1.8 μg / mL) (P<0.01).

[0116] Results of inflammatory factor detection:

[0117] Pro-inflammatory factors: The serum TNF-α level (65.8±8.5 pg / mL) and IL-6 level (45.2±6.3 pg / mL) in the model group, and the colon tissue TNF-α level (85.6±10.2 pg / g) and IL-6 level (65.3±8.1 pg / g) were significantly higher than those in the blank group (serum TNF-α 15.2±2.5 pg / mL, IL-6 12.3±2.1 pg / mL; colon TNF-α 25.3±3.5 pg / g, IL-6 18.5±2.8 pg / g) (P<0.01). The levels of the above pro-inflammatory factors in the ZK-35-H group were not significantly different from those in the blank group (P>0.05), but were significantly lower than those in the model group and the control group (P<0.01).

[0118] Anti-inflammatory factors: The serum IL-10 level (8.5±1.5 pg / mL) and colon tissue IL-10 level (12.3±2.1 pg / g) in the model group were significantly lower than those in the blank group (serum IL-10 25.3±3.5 pg / mL, colon IL-10 35.6±4.8 pg / g) (P<0.01); the levels of the above anti-inflammatory factors in the ZK-35-H group were not significantly different from those in the blank group (P>0.05), but were significantly higher than those in the model group and the control group (P<0.01).

[0119] Short-chain fatty acid test results:

[0120] The acetic acid (15.2±2.3 mmol / L), propionic acid (5.8±1.1 mmol / L), and butyric acid (2.1±0.5 mmol / L) contents in the cecal contents of the model group were significantly lower than those in the blank group (acetic acid 35.6±4.2 mmol / L, propionic acid 12.5±1.8 mmol / L, butyric acid 8.5±1.2 mmol / L) (P<0.01).

[0121] The SCFAs contents in the ZK-35-H group were 34.8±4.1 mmol / L, 11.9±1.7 mmol / L, and 8.2±1.1 mmol / L, respectively, which were not significantly different from the blank group (P>0.05), but significantly higher than those in the model group and control group (acetic acid 25.3±3.5 mmol / L, propionic acid 8.5±1.5 mmol / L, butyric acid 4.2±0.8 mmol / L) (P<0.01).

[0122] Clostridium difficile colonization test results:

[0123] The viable count of Clostridium difficile in the cecal contents of the model group was 8.5 × 10⁻⁶. 5 ±1.2×10 5 The CFU / g ratio was significantly higher than that in the control group (not detected) (P<0.01).

[0124] The viable count of Clostridium difficile in group ZK-35-H was 1.2 × 10⁻⁶. 3 ±0.3×10 3 The CFU / g level was significantly lower than that of the model group and the control group (3.5×10⁻⁶). 5 ±0.8×10 5 CFU / g)(P<0.01).

[0125] In summary, *Lactobacillus paracasei* ZK-35 can significantly alleviate the side effects caused by amoxicillin, including reducing the incidence of acute exacerbations of amoxicillin-induced diseases (AAD), restoring weight gain, regulating gut microbiota balance, repairing intestinal barrier function, reducing inflammatory responses, increasing SCFA synthesis, and inhibiting *Clostridium difficile* colonization. Its efficacy is superior to that of commercially available *Lactobacillus rhamnosus* GG, especially at high doses (5.0 × 10⁻⁶). 10 The best results are achieved with CFU / kg.

[0126] Example 3

[0127] Lactobacillus paracasei ZK-35 alleviates cefixime side effects

[0128] 3.1 Test Materials

[0129] Strain: Lactobacillus paracasei ZK-35 (CGMCC NO.35142, viable count 1.0 × 10⁻⁶) 11 CFU / g); Lactobacillus acidophilus (as a positive control).

[0130] Experimental animals: SPF grade SD rats, 6-8 weeks old, half male and half female, weighing 180-200g, a total of 96 rats.

[0131] Reagents: Cefixime dispersible tablets (Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd., specification 0.1g / tablet, prepared into a 10mg / mL solution with sterile physiological saline); Rat D-lactic acid ELISA kit, Rat diamine oxidase (DAO) ELISA kit (Shanghai Xinfan Biotechnology Co., Ltd.).

[0132] Instruments: Fully automated biochemical analyzer (model Cobas 6000, Roche, Switzerland); flow cytometer (model BD FACSCanto II, BD Laboratories, USA).

[0133] 3.2 Test Methods

[0134] Strain activation and bacterial suspension preparation: Same as in Example 2, except the concentrations of ZK-35 and control bacterial suspensions were adjusted to 5.0 × 10⁻⁶. 9 CFU / mL (medium dose), 1.0×10 10 CFU / mL (high dose).

[0135] Animal grouping and treatment:

[0136] Ninety-six rats were randomly divided into six groups of 16 each, with half being male and half female.

[0137] Blank control group: 2 mL of sterile saline per animal was administered by gavage daily for 28 consecutive days.

[0138] Cefixime model group (CTX): 2 mL / animal of cefixime solution (100 mg / kg) was administered by gavage from day 1 to day 10, and 2 mL / animal of sterile saline was administered by gavage from day 11 to day 28.

[0139] ZK-35 medium-dose group (ZK-35-M): 2 mL / animal gavage with cefixime solution on days 1-10, and 2 mL / animal gavage with ZK-35 bacterial suspension on days 1-28 (dose 1.0 × 10⁻⁶). 10 CFU / each, equivalent to 5.0 × 10 10 CFU / kg).

[0140] ZK-35 high-dose group (ZK-35-H): 2 mL / animal gavage with cefixime solution on days 1-10, and 2 mL / animal gavage with ZK-35 bacterial suspension on days 1-28 (dose 2.0 × 10⁻⁶). 10 CFU / each, equivalent to 1.0 × 10 11 CFU / kg).

[0141] Control group (-M): 2 mL / animal gavage with cefixime solution on days 1-10, and 2 mL / animal gavage with control bacterial suspension on days 1-28 (dose 1.0 × 10⁻⁶). 10 CFU / each, equivalent to 5.0 × 10 10 CFU / kg).

[0142] High-dose control group (-H): 2 mL / animal gavage with cefixime solution on days 1-10, and 2 mL / animal gavage with control bacterial suspension on days 1-28 (dose 2.0 × 10⁻⁶). 10 CFU / each, equivalent to 1.0 × 10 11 CFU / kg).

[0143] All rats were housed in an SPF-grade animal facility at a temperature of 22–25°C and a humidity of 40%–60%, with a 12-hour light / 12-hour dark cycle, and were given free access to standard rat feed and water.

[0144] The gavage time is fixed at 9:00 AM every day. The cefixime solution and the probiotic suspension are administered 2 hours apart (if both liquids need to be administered at the same time on the same day), to avoid cefixime directly killing the probiotics.

[0145] Detection indicators and methods:

[0146] Monitoring diarrhea symptoms:

[0147] From day 1 to day 28 of the experiment, the rats' defecation was observed and recorded daily, including stool characteristics (formed / loose / loose / watery), number of defecations (times / day), and the occurrence of diarrhea.

[0148] Diarrhea criteria: Three or more consecutive loose or watery stools are considered diarrhea. The number of rats with diarrhea in each group per day is recorded, and the diarrhea incidence rate is calculated (diarrhea incidence rate = number of rats with diarrhea on the day / total number of rats in the group × 100%).

[0149] The average duration of diarrhea in each group was recorded weekly (the number of days from the onset of diarrhea to the return of formed stool).

[0150] Weight and food intake monitoring:

[0151] Weigh the rats on an empty stomach every Monday morning (days 1, 7, 14, 21, and 28), record their weight, and calculate the weight change rate (weight change rate = (daily weight - initial weight / initial weight × 100%).

[0152] Every Monday morning before changing the feed, weigh the remaining feed and calculate the average feed intake of the previous week (average feed intake = (weight of feed given last week - weight of remaining feed this week / total number of rats in the group / 7 days).

[0153] Intestinal permeability testing:

[0154] On day 28, eight rats were randomly selected from each group. After fasting for 12 hours but not water, they were given a 4 kDa fluorescein isothiocyanate-dextran (FITC-dextran, Sigma, USA) solution by gavage (dosage 600 mg / kg, concentration 200 mg / mL).

[0155] Four hours after gavage, 1 mL of blood was collected through the orbital venous plexus and placed in a centrifuge tube containing heparin sodium. The plasma was then separated by centrifugation (4℃, 3000 r / min, 10 min).

[0156] Take 50 μL of plasma and dilute it with 950 μL of sterile saline. Use a fluorescence spectrophotometer (model F-4600, Hitachi, Japan) to detect the fluorescence intensity (excitation wavelength 485 nm, emission wavelength 520 nm). Calculate the plasma FITC-dextran concentration according to the standard curve to reflect intestinal permeability (the higher the concentration, the stronger the intestinal permeability and the worse the barrier function).

[0157] Meanwhile, the levels of D-lactic acid (a marker of intestinal mucosal damage) and diamine oxidase (DAO, a marker of intestinal mucosal integrity) in serum were detected using an ELISA kit, and the procedure was strictly performed in accordance with the kit instructions.

[0158] Gut microbiota functional metabolic analysis:

[0159] On day 28, six rats were randomly selected from each group, euthanized with carbon dioxide, and 1g of cecal contents were aseptically dissected and placed in a sterile centrifuge tube.

[0160] High-performance liquid chromatography-mass spectrometry (HPLC-MS, Agilent 1290-6460, Agilent Technologies, USA) was used to detect the microbial metabolites in the cecal contents, including:

[0161] Short-chain fatty acids (SCFAs): acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid; chromatographic conditions: ZORBAX SB-Aq column (150 mm × 4.6 mm × 5 μm), mobile phase 0.1% formic acid aqueous solution - acetonitrile (95:5, v / v), flow rate 1 mL / min, column temperature 30 ℃, injection volume 10 μL; mass spectrometry conditions: electrospray ionization source (ESI), negative ion mode, multiple reaction monitoring (MRM).

[0162] Bile acids: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA).

[0163] Chromatographic conditions: ZORBAX Eclipse Plus C18 column (150 mm × 4.6 mm × 5 μm), mobile phase 0.1% formic acid aqueous solution - acetonitrile (gradient elution), flow rate 1 mL / min, column temperature 35 ℃, injection volume 10 μL; Mass spectrometry conditions: ESI positive ion mode, MRM.

[0164] The number of key functional bacteria in cecal contents was detected using real-time quantitative PCR (qPCR), including:

[0165] Total bacteria: primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3'), 518R (5'-ATTACCGCGGCTGCTGG-3');

[0166] Bifidobacterium: primers Bif-164F (5'-GGTGTTCTTCCCGATATCTACA-3'), Bif-662R (5'-CCACATCCAGCRTCCACCA-3');

[0167] Lactobacillus: Primers Lab-0159F (5'-GGAAACAGRTGCTAATACCG-3'), Lab-0677R (5'-CACCGCTACACATGGAG-3');

[0168] Escherichia coli: Primers Eco-1447F (5'-CAGGATTAGATACCCTGGTAGTCC-3'), Eco-1522R (5'-TATTAACTTTACTCCCTTCCTCC-3').

[0169] Reaction system (20 μL): SYBR Green qPCR Master Mix 10 μL, upstream primer (10 μmol / L) 0.8 μL, downstream primer (10 μmol / L) 0.8 μL, DNA template 2 μL, sterile water 6.4 μL; Reaction program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, for a total of 40 cycles; Melting curve analysis: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s; Calculate the copy number (copies / g contents) of each bacterium according to the standard curve.

[0170] Nutritional absorption function test:

[0171] From day 26 to day 28, six rats were randomly selected from each group and housed individually in metabolic cages. Feces were collected over 24 hours, and the weight of the feces was recorded.

[0172] The crude protein content in feed and feces was determined by the Kjeldahl method, and the apparent protein digestibility was calculated (apparent protein digestibility = (total protein intake - total fecal protein) / total protein intake × 100%).

[0173] The calcium and iron content in feed and feces was detected using an atomic absorption spectrophotometer (model AA-6300, Shimadzu Corporation, Japan), and the apparent absorption rates of calcium and iron were calculated (the calculation formula is the same as that of apparent protein digestibility).

[0174] Rat serum was collected, and total protein, albumin, calcium, iron, and vitamin B were detected using a fully automated biochemical analyzer. 12 The content of nutrients reflects the absorption and utilization of nutrients in the body.

[0175] Liver and kidney function tests:

[0176] On day 28, after the rats were euthanized, liver and kidney tissues were collected, weighed, and the organ index was calculated (organ index = organ weight / rat body weight × 100%).

[0177] Partial liver and kidney tissues were collected, fixed with 4% neutral formaldehyde, embedded in paraffin, sectioned (5 μm thick), stained with hematoxylin and eosin (HE), and histopathological changes were observed under an optical microscope. The degree of damage was assessed using a semi-quantitative scoring method (0 points: no damage; 1 point: mild damage, local cell degeneration; 2 points: moderate damage, local cell necrosis; 3 points: severe damage, extensive cell necrosis).

[0178] Rat serum was collected, and liver function indicators (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL)) and kidney function indicators (serum creatinine (Scr) and blood urea nitrogen (BUN)) were detected using a fully automated biochemical analyzer to assess the potential liver and kidney damage caused by cefixime and the protective effect of ZK-35.

[0179] 3.3 Test Results

[0180] Diarrhea symptom monitoring results:

[0181] Blank control group: Throughout the experiment, the rats' stools were formed, no diarrhea occurred, and the number of defecations per day remained stable at 6-8 times / day.

[0182] Cefixime model group: Diarrhea began to appear on day 3, and the incidence of diarrhea peaked on days 7-10 (end of cefixime administration), at 75.0% (12 / 16). Stools were mainly loose or watery, and the number of defecations per day increased to 12-15 times / day. After drug withdrawal (from day 11), the incidence of diarrhea gradually decreased, but 25.0% (4 / 16) of rats still had diarrhea on day 28, with an average duration of diarrhea of ​​8.5±1.2 days.

[0183] In all ZK-35 dosage groups, the incidence, severity, and duration of diarrhea all decreased significantly with increasing ZK-35 dosage. In the ZK-35-H group, the highest incidence of diarrhea during cefixime treatment (days 1-10) was only 18.8% (3 / 16), significantly lower than that in the model group (P<0.01). The stools were mostly loose, with 8-10 defecations per day. On day 5 (day 15) after drug withdrawal, the stools of all rats returned to formed, and the average duration of diarrhea was 2.1±0.5 days, significantly shorter than that in the model group (P<0.01).

[0184] Compared with the control group, the highest incidence of diarrhea in the -H group was 43.8% (7 / 16), and the average duration of diarrhea was 5.3±0.8 days. Although it was lower than the model group (P<0.05), it was significantly higher than the ZK-35-H group (P<0.05), indicating that ZK-35 was more effective than the control in relieving cefixime-related diarrhea.

[0185] Results of weight and food intake monitoring:

[0186] Body weight change: In the blank control group, the body weight of rats increased steadily, with a change rate of 38.5±3.2% on day 28. In the model group, the body weight of rats decreased significantly during cefixime treatment (days 1-10), with a change rate of -6.2±1.8% on day 10 and only 15.3±2.5% on day 28, significantly lower than that of the blank group (P<0.01). In the ZK-35-H group, the body weight change rate was -1.3±0.9% on day 10 and 36.8±2.8% on day 28, which was not significantly different from that of the blank group (P>0.05), but significantly higher than that of the model group and the -H group (body weight change rate of 22.6±2.1% on day 28) (P<0.01).

[0187] Feed intake: The average feed intake of the blank control group remained stable at 22-25 g / (animal·day); the average feed intake of the model group decreased to 15-18 g / (animal·day) from day 1 to day 10, which was significantly lower than that of the blank group (P<0.01); the average feed intake of the ZK-35-H group from day 1 to day 10 was 20-22 g / (animal·day), which was close to that of the blank group (P>0.05) and significantly higher than that of the model group (P<0.01); the average feed intake of the -H group from day 1 to day 10 was 17-19 g / (animal·day), which was lower than that of the ZK-35-H group (P<0.05).

[0188] Intestinal permeability test results:

[0189] Plasma FITC-dextran concentration: The model group was 1.85±0.23 μg / mL, significantly higher than the blank group (0.32±0.05 μg / mL) (P<0.01); the ZK-35-H group was 0.41±0.08 μg / mL, with no significant difference from the blank group (P>0.05), but significantly lower than the model group and the -H group (1.02±0.15 μg / mL) (P<0.01).

[0190] Serum D-lactate levels: The model group was 85.6±10.2 μg / mL, significantly higher than the blank group (15.3±2.1 μg / mL) (P<0.01); the ZK-35-H group was 18.5±3.2 μg / mL, with no significant difference from the blank group (P>0.05), but significantly lower than the model group and the -H group (45.8±6.5 μg / mL) (P<0.01).

[0191] Serum DAO activity: The model group was 12.5±1.8 U / L, significantly higher than the blank group (3.2±0.5 U / L) (P<0.01); the ZK-35-H group was 3.8±0.7 U / L, with no significant difference from the blank group (P>0.05), but significantly lower than the model group and the -H group (7.8±1.2 U / L) (P<0.01). These results indicate that cefixime significantly increases intestinal permeability and damages the intestinal mucosa in rats, while ZK-35 effectively repairs intestinal mucosal damage, reduces intestinal permeability, and restores intestinal barrier function, with better effects than the control.

[0192] Results of gut microbiota functional metabolism analysis:

[0193] Short-chain fatty acid (SCFA) content: The total SCFA content in the cecal contents of the model group was 28.5±3.2 mmol / kg, significantly lower than that of the blank group (65.3±5.8 mmol / kg) (P<0.01), of which the butyric acid content was only 1.8±0.3 mmol / kg, significantly lower than that of the blank group (12.5±1.5 mmol / kg) (P<0.01); The total SCFA content of the ZK-35-H group was 62.1±4.5 mmol / kg, and the butyric acid content was 11.8±1.2 mmol / kg, which was not significantly different from that of the blank group (P>0.05), but significantly higher than that of the model group and the -H group (total SCFAs 42.3±3.8 mmol / kg, butyric acid 4.5±0.8 mmol / kg) (P<0.01).

[0194] Bile acid content: The serum secondary bile acid (DCA, LCA) levels in the model group were significantly increased (DCA 15.8±2.3 μmol / L, LCA 8.5±1.2 μmol / L), significantly higher than those in the blank group (DCA 3.2±0.5 μmol / L, LCA 1.8±0.3 μmol / L) (P<0.01); the secondary bile acid content in the ZK-35-H group (DCA 4.1±0.7 μmol / L, LCA 2.3±0.5 μmol / L) was not significantly different from that in the blank group (P>0.05), but significantly lower than that in the model group and the -H group (DCA 9.5±1.5 μmol / L, LCA 5.2±0.8 μmol / L) (P<0.01).

[0195] Gut microbiota count (qPCR results): Model group Bifidobacterium spp. (2.5 × 10⁻⁶) 8 ±0.5×10 8 The number of *Lactobacillus* (3.2 × 10⁸ ± 0.6 × 10⁸ copies / g) was significantly lower than that of the control group (*Bifidobacterium* 8.5 × 10⁸ copies / g). 9±1.2×10 9 copies / g, Lactobacillus 6.8 × 10 9 ±1.0×10 9 copies / g) (P<0.01), Escherichia coli count (1.8×10) 9 ±0.3×10 9 The number of copies / g was significantly higher than that of the control group (3.5×10). 7 ±0.8×10 7 copies / g)(P<0.01); Bifidobacterium spp. of group ZK-35-H (7.8×10 copies / g) 9 ±1.0×10 9 copies / g), Lactobacillus (6.5×10) 9 ±0.8×10 9 The number of copies / g was not significantly different from the control group (P>0.05), while the number of Escherichia coli (4.2×10⁻⁶) was significantly higher. 7 ±0.6×10 7 The number of copies / g was significantly lower than that of the model group and the -H group (Bifidobacterium 4.2 × 10⁻⁶). 9 ±0.8×10 9 copies / g, Lactobacillus spp. 3.5 × 10 9 ±0.7×10 9 copies / g, Escherichia coli 8.5×10 8 ±1.2×10 8 (copies / g) (P<0.01).

[0196] Results of nutrient absorption function test:

[0197] Apparent digestibility: The apparent digestibility of protein (70.2±3.5%), apparent absorption of calcium (45.3±4.2%), and apparent absorption of iron (38.5±3.8%) in the model group were significantly lower than those in the control group (apparent digestibility of protein 85.6±2.8%, apparent absorption of calcium 68.5±4.5%, apparent absorption of iron 58.2±4.1%) (P<0.01); The above digestibility and absorption rates of the ZK-35-H group (protein 84.2±2.5%, calcium 66.8±4.1%, iron 56.5±3.8%) were not significantly different from those of the control group (P>0.05), but were significantly higher than those of the model group and the -H group (protein 76.5±3.2%, calcium 55.2±3.8%, iron 45.8±3.5%) (P<0.01).

[0198] Serum nutritional indicators: In the model group, serum total protein (55.2±4.2 g / L), albumin (32.5±3.1 g / L), calcium (2.0±0.2 mmol / L), iron (15.8±2.3 μmol / L), and vitamin B12 were present. 12 The levels of (185.6±25.3 pg / mL) were significantly lower than those in the control group (total protein 70.5±3.8 g / L, albumin 42.8±2.5 g / L, calcium 2.5±0.1 mmol / L, iron 28.5±3.2 μmol / L, vitamin B12) and (185.6±25.3 pg / mL). 12 325.8±35.6 pg / mL (P<0.01); The above indicators in the ZK-35-H group (total protein 68.8±3.5 g / L, albumin 41.2±2.3 g / L, calcium 2.4±0.1 mmol / L, iron 26.8±2.8 μmol / L, vitamin B...) 12 The concentration of 308.5±32.1 pg / mL was not significantly different from the blank group (P>0.05), but was significantly higher than the model group and the -H group (P<0.01).

[0199] Liver and kidney function test results:

[0200] Organ indices: The liver index (3.8±0.3%) and kidney index (0.85±0.08%) in the model group were significantly higher than those in the blank group (liver index 3.2±0.2%, kidney index 0.72±0.05%) (P<0.05); The liver index (3.3±0.2%) and kidney index (0.73±0.06%) in the ZK-35-H group were not significantly different from those in the blank group (P>0.05), but were significantly lower than those in the model group (P<0.05); Although the liver index (3.6±0.2%) and kidney index (0.80±0.07%) in the -H group were lower than those in the model group, they were still higher than those in the ZK-35-H group (P<0.05).

[0201] Histopathological scores: In the model group, the liver tissue showed mild steatosis and inflammatory cell infiltration, with a pathological score of 1.8±0.3; the kidney tissue showed renal tubular epithelial cell edema, with a pathological score of 1.5±0.2; both were significantly higher than those in the blank group (0 points) (P<0.01); In the ZK-35-H group, the liver pathological score was 0.3±0.1 and the kidney pathological score was 0.2±0.1, significantly lower than those in the model group (P<0.01); In the -H group, the liver pathological score was 1.0±0.2 and the kidney pathological score was 0.8±0.1, higher than those in the ZK-35-H group (P<0.05).

[0202] Biochemical indexes: In the model group, the levels of serum ALT (85.6±10.2 U / L), AST (125.3±15.8 U / L), TBIL (15.8±2.3 μmol / L), Scr (85.3±8.5 μmol / L), and BUN (10.5±1.2 mmol / L) were significantly higher than those in the blank group (ALT 35.2±5.8 U / L, AST 65.3±8.2 U / L, TBIL 8.5±1.2 μmol / L, Scr 55.2±6.3 μmol / L, BUN 6.2±0.8 mmol / L) (P<0.01); for the above indexes in the ZK-35-H group (ALT 38.5±6.2 U / L, AST 68.5±7.5 U / L, TBIL 9.2±1.1 μmol / L, Scr 58.3±5.8 μmol / L, BUN 6.5±0.7 mmol / L), there was no significant difference compared with the blank group (P>0.05), and they were significantly lower than those in the model group and the -H group (P<0.01).

[0203] In summary, Lactobacillus paracasei ZK-35 can significantly alleviate the side effects of cefixime by reducing the incidence of cefixime-related diarrhea, restoring body weight and food intake, repairing intestinal barrier function, regulating the structure and metabolism of intestinal flora, improving the absorption of nutrients, and protecting liver and kidney tissues. The high-dose group (1.0×10¹¹ CFU / kg) has the best effect, and the overall effect is better than that of the commercially available Lactobacillus acidophilus control.

[0204] Example 4

[0205] Lactobacillus paracasei ZK-35 promotes intestinal health in constipation model mice

[0206] 4.1 Test materials

[0207] Strains: Lactobacillus paracasei ZK-35 (CGMCC NO.35142, freeze-dried powder prepared in our laboratory, viable count 1.0×10 11 CFU / g, formulated into bacterial suspensions of 1.0×10 9 CFU / mL, 5.0×10 9 CFU / mL, and 1.0×10 10 CFU / mL with sterile saline).

[0208] Experimental animals: SPF-grade Kunming mice, 6-8 weeks old, half male and half female, weighing 20-22 g, a total of 100 mice, purchased from Shanghai Slack Experimental Animal Co., Ltd., animal production license number SCXK (Shanghai) 2023-0001, animal certificate number 20230605001.

[0209] Reagents: Compound diphenoxylate tablets (Shanghai Xinyi Pharmaceutical Co., Ltd., specification 2.5mg / tablet, prepared into a 0.1mg / mL suspension with 0.5% sodium carboxymethyl cellulose (CMC-Na) solution for establishing a constipation model); activated charcoal (Sinopharm Chemical Reagent Co., Ltd., particle size 200 mesh); gum arabic (Sinopharm Chemical Reagent Co., Ltd.); mouse gastrointestinal hormone (motilin MTL, vasoactive intestinal peptide VIP, somatostatin SS) ELISA kit (Wuhan Yilairuit Biotechnology Co., Ltd.).

[0210] Instruments: Small animal in vivo imaging system (model IVIS Lumina III, PerkinElmer, USA); intestinal motility monitoring system (model RM6240BD, Chengdu Instrument Factory); refrigerated centrifuge (model Centrifuge 5810R, Eppendorf, Germany).

[0211] 4.2 Test Methods

[0212] Establishment of a constipation model and animal grouping:

[0213] After 3 days of acclimatization, except for the blank control group, the other mice were administered 0.2 mL / mouse of compound diphenoxylate suspension (dose 10 mg / kg) by gavage for 7 consecutive days to establish a mouse constipation model. The criteria for successful model establishment were: the number of defecations of mice decreased by more than 50% compared with the blank control group, the water content of feces decreased by more than 30%, and the time to excretion of the first black feces was prolonged by more than 50%.

[0214] One hundred mice were randomly divided into 5 groups of 20 each, with half males and half females:

[0215] Control group: 0.2 mL / animal was administered 0.5% CMC-Na solution by gavage daily for 14 consecutive days.

[0216] Constipation model group (Model): 0.2 mL / animal of compound diphenoxylate suspension was administered by gavage from day 1 to day 7, and 0.2 mL / animal of 0.5% CMC-Na solution was administered by gavage from day 8 to day 14.

[0217] ZK-35 low-dose group (ZK-35-L): 0.2 mL / animal of compound diphenoxylate suspension was administered by gavage on days 1-7, and 0.2 mL / animal of ZK-35 bacterial suspension was administered by gavage on days 1-14 (dose 1.0 × 10⁻⁶). 8 CFU / each, equivalent to 5.0 × 10 9 CFU / kg).

[0218] ZK-35 medium-dose group (ZK-35-M): 0.2 mL / animal of compound diphenoxylate suspension was administered by gavage on days 1-7, and 0.2 mL / animal of ZK-35 bacterial suspension was administered by gavage on days 1-14 (dose 5.0 × 10⁻⁶). 8 CFU / each, equivalent to 2.5 × 10 10 CFU / kg).

[0219] ZK-35 high-dose group (ZK-35-H): 0.2 mL / animal of compound diphenoxylate suspension was administered by gavage on days 1-7, and 0.2 mL / animal of ZK-35 bacterial suspension was administered by gavage on days 1-14 (dose 1.0 × 10⁻⁶). 9 CFU / each, equivalent to 5.0 × 10 10 CFU / kg).

[0220] All mice were housed under the same conditions as in Example 2, and were administered gavage at the same time every day (9:00 AM).

[0221] Detection indicators and methods:

[0222] Constipation symptom assessment:

[0223] Defecation frequency and fecal characteristics: On day 7 (after model establishment) and day 14 (end of experiment), 10 mice were randomly selected from each group and housed in metabolic cages. They were fasted but allowed free access to water. The number of defecations and fecal weight (accurate to 0.01g) were recorded over 24 hours. Five fresh fecal pellets were taken and the fecal moisture content was determined by drying method (fecal moisture content = (fecal wet weight - fecal dry weight / fecal wet weight × 100%).

[0224] Time of first black stool excretion: On day 14, after the mice were fasted for 12 hours but allowed free access to water, they were given 0.2 mL of activated charcoal suspension (containing 5% activated charcoal and 10% gum arabic) by gavage. The time from gavage to the excretion of the first black stool was recorded to reflect intestinal transit function.

[0225] Small intestinal propulsion rate: After the time of first black stool excretion was measured, the mice were euthanized by cervical dislocation, and the small intestine (from the pylorus to the ileocecal junction) was dissected. The total length of the small intestine and the propulsion distance of activated charcoal in the small intestine were measured, and the small intestinal propulsion rate was calculated (small intestinal propulsion rate = activated charcoal propulsion distance / total length of small intestine × 100%).

[0226] Intestinal motility-related markers detection:

[0227] Gastrointestinal hormone levels: On day 14, 8 mice were randomly selected from each group, blood was collected from their eyeballs, and serum was separated by centrifugation (4℃, 3000r / min, 10min). The levels of MTL (promotes gastrointestinal motility), VIP (inhibits gastrointestinal motility), and SS (inhibits gastrointestinal motility) in the serum were detected by ELISA kit.

[0228] Intestinal smooth muscle contraction function: A 1cm segment of isolated mouse duodenum was placed in Krebs-Henseleit nutrient solution at 37℃ (containing NaCl 118mmol / L, KCl 4.7mmol / L, CaCl2 2.5mmol / L, MgSO4 1.2mmol / L, NaHCO3 25mmol / L, Glucose 11mmol / L, pH 7.4, and 95% O2 + 5% CO2). A tension transducer and an intestinal motility monitoring system were connected to record the amplitude and frequency of spontaneous contractions of duodenal smooth muscle. Acetylcholine (ACh, final concentration 10) was then added. -6 (mol / L), recording changes in contraction amplitude and frequency to assess the responsiveness of intestinal smooth muscle to agonists.

[0229] Analysis of gut microbiota and metabolites:

[0230] Gut microbiota α diversity: On day 14, 6 mice were randomly selected from each group, sacrificed, and 0.5g of cecal contents were collected. Total DNA was extracted, and the V4-V5 region of the 16S rRNA gene was amplified (primers 515F: 5'-GTGCCAGCMGCCGCGGTAA-3', 907R: 5'-CCGTCAATTCMTTTRAGTTT-3'). Sequencing was performed using the Illumina NovaSeq 6000 high-throughput sequencing platform, and the Chao1 index (microbiota richness) and Shannon index (microbiota diversity) were analyzed.

[0231] Relative abundance of key bacterial groups: The relative abundance of Lactobacillus, Bifidobacterium, Akk bacteria, Faecalibacterium (butyric acid-producing bacteria), and Desulfovibrio (harmful bacteria, associated with constipation) was analyzed based on sequencing results.

[0232] Short-chain fatty acid (SCFA) content: Take 0.5g of cecal contents, add 1mL of ultrapure water to homogenize, centrifuge (4℃, 12000r / min, 15min), take the supernatant, filter through a 0.22μm filter membrane, and use a gas chromatograph (model GC-2030, Shimadzu Corporation, Japan) to detect the content of acetic acid, propionic acid and butyric acid.

[0233] The chromatographic conditions were the same as in Example 2.

[0234] Intestinal barrier function testing:

[0235] Histopathological observation of colon tissue: Mouse colon tissue (2 cm from the anus) was taken, fixed in 4% neutral formaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope for colonic mucosal thickness, villus integrity, and inflammatory cell infiltration. The pathological scoring criteria (0 points: normal; 1 point: mild mucosal edema, a small amount of inflammatory cell infiltration; 2 points: moderate mucosal edema, partial villus breakage, moderate inflammatory cell infiltration; 3 points: severe mucosal edema, extensive villus breakage, severe inflammatory cell infiltration) were used for scoring.

[0236] Tight junction protein expression: 0.5g of colon tissue was taken, total protein was extracted, and the protein expression levels of occludin, ZO-1, and claudin-1 were detected by Western blot. β-actin was used as an internal reference to calculate the relative expression levels. The operation was the same as in Example 2.

[0237] Mucosal immune function: Colonic mucosal tissue was taken, homogenized, centrifuged, and the supernatant was collected. The sIgA content of the mucosa was detected using an ELISA kit to reflect the intestinal mucosal immune barrier function.

[0238] 4.3 Test Results

[0239] Constipation symptom assessment results:

[0240] Defecation frequency and stool characteristics: On day 7, the model group had significantly lower 24-hour defecation frequency (3.2±0.8 times), stool weight (0.35±0.08g), and stool water content (35.2±4.5%) than the control group (defecation frequency 8.5±1.2 times, stool weight 1.25±0.15g, stool water content 65.3±5.8%) (P<0.01). On day 14, all the above indicators in each ZK-35 dosage group increased significantly in a dose-dependent manner. The ZK-35-H group had no significant difference in 24-hour defecation frequency (7.8±1.0 times), stool weight (1.18±0.12g), and stool water content (62.5±4.2%) compared to the control group (P>0.05), but significantly higher than the model group (defecation frequency 4.5±0.9 times). The fecal weight was 0.52±0.10g and the fecal water content was 42.8±5.1% (P<0.01).

[0241] Time to excretion of the first black stool: The model group was 158.5±15.2 min, which was significantly longer than the blank group (65.3±8.5 min) (P<0.01); The ZK-35-H group was 72.5±9.8 min, which was not significantly different from the blank group (P>0.05) and was significantly shorter than the model group (P<0.01).

[0242] Small intestinal transit rate: The model group was 35.2±4.5%, significantly lower than the blank group (68.5±5.8%) (P<0.01); the ZK-35-H group was 65.8±5.2%, with no significant difference from the blank group (P>0.05), but significantly higher than the model group (P<0.01). These results indicate that compound diphenoxylate can successfully establish a mouse constipation model, while Lactobacillus paracasei ZK-35 can significantly improve constipation symptoms and promote intestinal transit function.

[0243] Results of intestinal motility-related indicators:

[0244] Gastrointestinal hormone levels: The serum MTL level in the model group (85.6±10.2 pg / mL) was significantly lower than that in the blank group (158.5±15.3 pg / mL) (P<0.01), while the VIP (125.3±15.8 pg / mL) and SS (65.8±8.5 pg / mL) levels were significantly higher than those in the blank group (VIP 68.5±8.2 pg / mL, SS 35.2±5.8 pg / mL) (P<0.01). The serum MTL level (148.2±12.5 pg / mL), VIP level (72.5±7.8 pg / mL), and SS level (38.5±6.2 pg / mL) in the ZK-35-H group were not significantly different from those in the blank group (P>0.05), but were significantly better than those in the model group (P<0.01).

[0245] Intestinal smooth muscle contraction function: The spontaneous contraction amplitude (0.85±0.15g) and frequency (2.5±0.5 times / min) of duodenal smooth muscle in the model group were significantly lower than those in the blank group (contraction amplitude 2.15±0.25g, frequency 5.8±0.8 times / min) (P<0.01); after the addition of ACh, the increase in contraction amplitude in the model group (50.2±8.5%) was significantly lower than that in the blank group (125.3±15.8%) (P<0.01); the spontaneous contraction amplitude (1.98±0.22g), frequency (5.5±0.7 times / min), and ACh-induced increase in contraction amplitude (118.5±12.5%) in the ZK-35-H group were not significantly different from those in the blank group (P>0.05), but were significantly higher than those in the model group (P<0.01).

[0246] Results of gut microbiota and metabolite analysis:

[0247] Gut microbiota α diversity: The Chao1 index (295.6±28.5) and Shannon index (2.3±0.3) in the model group were significantly lower than those in the control group (Chao1 index 468.5±35.2, Shannon index 3.9±0.4) (P<0.01); The Chao1 index (442.8±32.1) and Shannon index (3.7±0.3) in the ZK-35-H group were not significantly different from those in the control group (P>0.05), but were significantly higher than those in the model group (P<0.01).

[0248] Relative abundance of key bacterial groups: The relative abundance of *Lactobacillus* (2.1±0.5%), *Bifidobacterium* (1.8±0.4%), *Akk* (0.4±0.1%), and *Femtobacter* (0.6±0.2%) in the model group was significantly lower than that in the control group (*Lactobacillus* 14.8±1.6%, *Bifidobacterium* 12.3±1.5%, *Akk* 3.5±0.5%, *Femtobacter* 5.2±0.8%) (P<0.01), while *Desulfovibrio* (8.5±1.2%) was significantly higher than that in the control group (1.2±0.3%) (P<0.01); The relative abundance of the above beneficial bacteria in the ZK-35-H group (*Lactobacillus* 13.5±1.5%, *Bifidobacterium* 11.8±1.4%, *Akk* 0.4±0.1%), and *Femtobacter* 0.6±0.2%) was significantly higher than that in the control group (P<0.01). The percentages of bacteria (3.2±0.4%) and fecal bacteria (4.8±0.7%) were not significantly different from the control group (P>0.05), while the percentages of desulfurized Vibrio (1.8±0.4%) were significantly lower than those in the model group (P<0.01).

[0249] SCFAs content: The cecal contents of the model group contained significantly lower levels of acetic acid (18.5±2.3 mmol / L), propionic acid (6.2±1.1 mmol / L), and butyric acid (2.5±0.5 mmol / L) than those of the blank group (acetic acid 38.5±4.2 mmol / L, propionic acid 13.5±1.8 mmol / L, butyric acid 9.2±1.2 mmol / L) (P<0.01); The SCFAs content of the ZK-35-H group (acetic acid 36.8±3.8 mmol / L, propionic acid 12.8±1.7 mmol / L, butyric acid 8.8±1.1 mmol / L) was not significantly different from that of the blank group (P>0.05), but was significantly higher than that of the model group (P<0.01).

[0250] Results of intestinal barrier function test:

[0251] Colonic histopathological score: The model group scored 2.3±0.3 points, significantly higher than the blank group (0 points) (P<0.01), showing moderate colonic mucosal edema, partial villus breakage, and moderate inflammatory cell infiltration; The ZK-35-H group scored 0.3±0.1 points, significantly lower than the model group (P<0.01), and the colonic mucosal structure basically returned to normal.

[0252] Tight junction protein expression: The relative expression levels of occludin (0.35±0.08), ZO-1 (0.28±0.07), and claudin-1 (0.32±0.06) in the model group were significantly lower than those in the blank group (all 1.0±0.1) (P<0.01); The relative expression levels of the above proteins in the ZK-35-H group (occludin 0.92±0.09, ZO-1 0.85±0.08, claudin-1 0.88±0.07) were not significantly different from those in the blank group (P>0.05), but were significantly higher than those in the model group (P<0.01).

[0253] Mucosal sIgA content: The model group was 6.5±1.2 μg / mL, which was significantly lower than that of the blank group (16.8±2.3 μg / mL) (P<0.01); the ZK-35-H group was 15.5±2.1 μg / mL, which was not significantly different from that of the blank group (P>0.05) but significantly higher than that of the model group (P<0.01).

[0254] In summary, *Lactobacillus paracasei* ZK-35 can effectively promote intestinal health in constipation model mice by improving intestinal transit function, regulating gastrointestinal hormone balance, enhancing intestinal smooth muscle contraction function, restoring intestinal flora diversity and metabolism, and repairing intestinal barrier function. Furthermore, high doses (5.0 × 10⁻⁶) are particularly effective. 10 The best results are achieved with CFU / kg.

[0255] Example 5

[0256] Lactobacillus paracasei ZK-35, in combination with Lactobacillus acidophilus and Bifidobacterium longum, synergistically alleviates ulcerative colitis.

[0257] 5.1 Test Materials

[0258] Strains:

[0259] Lactobacillus paracasei ZK-35 (CGMCC NO.35142, lyophilized powder, viable count 1.0 × 10⁻⁶) 11 CFU / g);

[0260] Lactobacillus acidophilus (commercially available lyophilized powder, viable count 1.0 × 10⁻⁶) 11 CFU / g);

[0261] Bifidobacterium longum subsp. infantis, commercially available freeze-dried powder, viable count 1.0 × 10⁻⁶ 11 CFU / g).

[0262] Preparation of compound bacterial powder: Weigh out ZK-35, Lactobacillus acidophilus, and Bifidobacterium longum freeze-dried powders separately at a weight ratio of 2-10:1-2:1-2, preferably 4:1:1, mix them evenly to obtain the compound bacterial powder, with a total viable count of 1.0 × 10⁻⁶. 11 - 8.0×10 11 CFU / g.

[0263] Experimental animals: SPF grade SD rats, 6-8 weeks old, half male and half female, weighing 180-200g, a total of 128 rats.

[0264] Reagents: 2,4,6-Trinitrobenzenesulfonic acid (TNBS, Sigma-Aldrich, USA, purity ≥98%); ethanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); rat TNF-α, IL-6, IL-10, IL-17 ELISA kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd.); rat colon tissue HE staining kit (Beijing Solarbio Science & Technology Co., Ltd.).

[0265] Instruments: Pathology microtome (model RM2235, Leica GmbH, Germany); Image analysis system (model Image-Pro Plus 6.0, Media Cybernetics, USA); Flow cytometer (model BD FACSAria III, BD Systems, USA).

[0266] 5.2 Test Methods

[0267] Establishment of an ulcerative colitis (UC) model:

[0268] Except for the blank control group, the remaining rats were used to establish the UC model using the TNBS / ethanol method: After fasting for 24 hours but not water, the rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (1.5 mL / kg). A 2 mm diameter tube was inserted into the colon 8 cm deep through the anus, and TNBS / ethanol solution (TNBS concentration 70 mg / mL, ethanol concentration 50%) was slowly injected at a dose of 0.25 mL / 100 g body weight. After injection, the rats were lifted by the tail and inverted for 30 seconds to allow the modeling agent to fully contact the colonic mucosa. The model was established three times on days 15, 22, and 29 of the experiment to enhance the stability of the model.

[0269] Animal grouping and treatment:

[0270] 128 rats were randomly divided into 8 groups, with 16 rats in each group, half male and half female:

[0271] Control group: No model was established. Each animal was given 2 mL of sterile saline by gavage daily for 34 consecutive days.

[0272] UC Model Group (Model): Modeling was performed by gavage of 2 mL of sterile saline per animal daily for 34 consecutive days.

[0273] ZK-35 monotherapy group (ZK-35): Modeling, daily gavage administration of 2 mL / animal of ZK-35 bacterial suspension (dose 2.0 × 10⁻⁶). 10 CFU / each, equivalent to 1.0 × 10 11 CFU / kg), for 34 consecutive days.

[0274] Lactobacillus acidophilus alone group: Model established, Lactobacillus acidophilus suspension was administered orally to each animal daily at a dose of 2.0 × 10⁻⁶ mL / day (dose 2.0 × 10⁻⁶). 10 CFU / each), for 34 consecutive days.

[0275] Bifidobacterium longum alone group: Model established, 2 mL of Bifidobacterium longum bacterial suspension per animal was administered by gavage daily (dose 2.0 × 10⁻⁶). 10 CFU / each), for 34 consecutive days.

[0276] Two groups of ZK-35 + Lactobacillus acidophilus combination groups: Modeling was performed, and each animal was administered 2 mL of a mixed bacterial suspension of ZK-35 and Lactobacillus acidophilus daily by gavage (total dose 2.0 × 10⁻⁶). 10 CFU / animal (weight ratio 4:1), for 34 consecutive days.

[0277] Two groups of ZK-35 + Bifidobacterium longum combination groups: Modeling was performed, and each animal was administered 2 mL of a mixed bacterial suspension of ZK-35 and Bifidobacterium longum via gavage daily (total dose 2.0 × 10⁻⁶). 10 CFU / animal (weight ratio 4:1), for 34 consecutive days.

[0278] The three-component combination group (ZK-35 + Lactobacillus acidophilus + Bifidobacterium longum): Modeling was performed, with 2 mL of the combined bacterial suspension administered orally per animal daily (total dose 2.0 × 10⁻⁶). 10 CFU / animal, weight ratio 4:1:1), for 34 consecutive days.

[0279] All rats were fed under the same conditions as in Example 3. The gavage time was 3:00 PM daily, and the gavage time on the day of modeling was adjusted to 2 hours after modeling.

[0280] Detection indicators and methods:

[0281] Disease Activity Index (DAI) score:

[0282] DAI scores were assessed on days 1, 3, and 5 after each modeling procedure (i.e., days 16, 18, 20, 23, 25, 27, 30, 32, and 34 of the trial). The scoring criteria combined percentage weight loss, stool characteristics, and degree of rectal bleeding, with a total score of 0-4. The higher the score, the more severe the UC symptoms.

[0283] Weight loss percentage calculation: (Pre-model weight - Today's weight) / Pre-model weight × 100%, weight gain is scored as 0 points.

[0284] Stool characteristics score: 0 points (formed), 1 point (loose), 2 points (loose), 3 points (watery).

[0285] Score for degree of rectal bleeding: 0 points (no blood), 1 point (positive for occult blood), 2 points (small amount of visible blood in stool), 3 points (large amount of visible blood in stool).

[0286] DAI = (Weight loss score + Stool characteristics score + Rectal bleeding score) / 3.

[0287] Colonic histological examination:

[0288] On day 34 of the experiment, rats were euthanized with carbon dioxide, and their colons (from the ileocecal junction to the anus) were dissected and their length was measured (accurate to 0.1 cm).

[0289] A 1cm section of mid-colon tissue was taken, fixed in 4% neutral formaldehyde, embedded in paraffin, sectioned (5μm thick), stained with hematoxylin and eosin (HE), and the colonic mucosal damage was observed under an optical microscope. The pathological histological scoring criteria were used to score the tissue damage from four dimensions: degree of inflammation, ulcer depth, mucosal hyperplasia, and edema. The total score ranged from 0 to 12 points, with higher scores indicating more severe tissue damage.

[0290] Intestinal inflammatory factor detection:

[0291] Serum inflammatory factors: Blood was collected from rat eyeballs, and serum was separated by centrifugation. The levels of pro-inflammatory factors (TNF-α, IL-6, IL-17) and anti-inflammatory factors (IL-10) in the serum were detected using an ELISA kit.

[0292] Inflammatory factors in colon tissue: Take 0.5g of colon tissue, add PBS buffer (containing protease inhibitor) to homogenize, centrifuge (4℃, 12000r / min, 15min), collect the supernatant, and use an ELISA kit to detect the content of the above inflammatory factors.

[0293] Gut microbiota structure analysis:

[0294] 0.5g of cecal contents were collected, and total DNA was extracted. 16S rRNA gene high-throughput sequencing (Illumina MiSeq platform) was used to analyze the β diversity of gut microbiota (PCoA analysis) to assess the differences in microbiota structure among the groups.

[0295] The relative abundance of key bacterial groups (Lactobacillus, Bifidobacterium, Akk bacteria, Faecalibacterium, Escherichia coli, Enterococcus) was analyzed.

[0296] Validation of the synergistic mechanism:

[0297] Intestinal colonization capacity: The viable counts (copies / g mucosal tissue) of ZK-35, Lactobacillus acidophilus, and Bifidobacterium longum on the colonic mucosa were detected by qPCR. The primers were as follows:

[0298] ZK-35: Upstream primer ZK-F (5'-GGTGAAGCTGCGGTTGATAC-3'), downstream primer ZK-R (5'-CCACGCTCACCGGCTTATAC-3');

[0299] Lactobacillus acidophilus: upstream primer LA-F (5'-AGAGTTTGATCCTGGCTCAG-3'), downstream primer LA-R (5'-GCTGATCCGCGATTACTAGC-3');

[0300] Bifidobacterium longum: upstream primer BB-F (5'-GCGTGCTTAACACATGCAAG-3'), downstream primer BB-R (5'-TGGATCCGCGATTACTAGC-3').

[0301] Metabolic product complementarity: The contents of bacteriocin (Lactobacillus paracasei), lactic acid, acetic acid and butyric acid in colon contents were detected by HPLC-MS to analyze the synergistic effect of the metabolites of the three strains.

[0302] Signaling pathway protein expression: Colon tissue was collected, total protein was extracted, and the expression levels of TLR2 (target of ZK-35), Nrf2 (target of Lactobacillus acidophilus), TGF-β (target of Bifidobacterium longum), and NF-κB (key protein in the inflammatory pathway) were detected by Western blot, with β-actin as an internal control.

[0303] 5.3 Test Results

[0304] DAI Score Results:

[0305] Blank control group: The DAI score was 0 throughout the trial, and there were no UC symptoms.

[0306] UC model group: The DAI score increased significantly after each modeling, and the DAI score on day 34 was 3.2±0.3, which manifested as severe weight loss (average weight loss of 15.8±2.3%), watery stool, and a large amount of visible bloody stool.

[0307] Individual strain groups: The DAI score of the ZK-35 group on day 34 was 1.8±0.2, the Lactobacillus acidophilus group was 2.3±0.3, and the Bifidobacterium longum group was 2.5±0.3, all of which were significantly lower than the model group (P<0.01), but still higher than the compound group.

[0308] The DAI scores of the two compound groups were 1.2±0.2 for the ZK-35+Lactobacillus acidophilus group and 1.0±0.2 for the ZK-35+Bifidobacterium longum group, which were significantly lower than those of the single strain groups (P<0.05).

[0309] The three-strain combination group had a DAI score of 0.5±0.1 on day 34, significantly lower than all other groups (P<0.01), with only slight weight loss (mean decrease of 3.2±0.8%), loose stools, and no visible bloody stools. The results indicate that the combination of the three strains significantly improved the relief of UC symptoms compared to single strains or the combination of two strains, demonstrating a synergistic effect.

[0310] Results of colon histological examination:

[0311] Colon length: The colon length in the model group was 12.5±1.2cm, significantly shorter than that in the control group (18.5±0.8cm) (P<0.01); the colon length in the three-component combination group was 17.8±0.7cm, which was not significantly different from that in the control group (P>0.05), but significantly longer than that in the single-strain group (ZK-35 group 14.8±1.0cm) and the two-component combination group (ZK-35+Bifidobacterium longum group 16.2±0.9cm) (P<0.01).

[0312] Pathological histological scores: The total score of the model group was 9.8±1.2 points, which showed severe transmural inflammation, numerous ulcers (>4), significant mucosal hyperplasia and edema; the total score of the three-in-one combination group was 2.1±0.3 points, which showed only mild mucosal inflammation and slight edema, significantly lower than the single strain group (ZK-35 group 5.8±0.8 points) and the two-in-one combination group (ZK-35+Bifidobacterium longum group 3.5±0.5 points) (P<0.01).

[0313] Results of intestinal inflammatory factor test:

[0314] Serum inflammatory factors: The levels of TNF-α (85.6±10.2 pg / mL), IL-6 (65.3±8.5 pg / mL), and IL-17 (45.8±6.2 pg / mL) in the model group were significantly higher than those in the blank group (TNF-α 15.2±2.5 pg / mL, IL-6 12.3±2.1 pg / mL, IL-17 8.5±1.5 pg / mL) (P<0.01), while the level of IL-10 (10.2±1.8 pg / mL) was significantly lower than that in the blank group (35.6±4.8 pg / mL) (P<0.01). In the combination group, the levels of the above pro-inflammatory factors (TNF-α 20.5±3.2 pg / mL, IL-6 15.8±2.3 pg / mL, IL-17 12.5±2.1 pg / mL) and anti-inflammatory factors (IL-10) were significantly higher. The concentration of 32.8±4.2 pg / mL was not significantly different from the blank group (P>0.05), but was significantly better than other groups (P<0.01).

[0315] Inflammatory factors in colon tissue: The results were consistent with those in serum. The content of pro-inflammatory factors in colon tissue was significantly reduced and the content of anti-inflammatory factors was significantly increased in the combination of the three, which was better than that of single strains and the combination of the two strains.

[0316] Results of gut microbiota structure analysis:

[0317] β-diversity analysis: The gut microbiota structure of the blank group and the three-component combination group clustered into one branch, the model group clustered into one branch alone, and the single strain group and the two-component combination group were in between, indicating that the three-component combination can significantly restore the gut microbiota structure imbalance caused by UC.

[0318] Relative abundance of key bacterial groups: In the model group, the relative abundance of Lactobacillus (1.8±0.4%), Bifidobacterium (1.5±0.3%), AkK bacteria (0.2±0.1%), and Faecalibacterium (0.3±0.1%) was significantly lower than that in the control group (P<0.01), while the relative abundance of Escherichia coli (15.8±2.3%) and Enterococcus (12.5±1.8%) was significantly higher than that in the control group (P<0.01). In the combination group, the relative abundance of the above beneficial bacteria (Lactobacillus 14.2±1.6%, Bifidobacterium 11.5±1.4%, AkK bacteria 0.2±0.1%), and Faecalibacterium (0.3±0.1%) was significantly higher than that in the control group (P<0.01). The abundance of bacteria (3.1±0.4% of bacteria and 4.5±0.7% of bacteria) was not significantly different from that of the control group (P>0.05). The relative abundance of harmful bacteria (Escherichia coli 3.8±0.7% and Enterococcus spp. 2.5±0.6%) was significantly lower than that of the model group (P<0.01) and better than that of the single strain group and the two-strain combination group.

[0319] Results of verification of synergistic mechanism:

[0320] Intestinal colonization capacity: ZK-35 (8.5×10) on the colonic mucosa surface of the three-component combination group 8 ±1.2×10 8 copies / g), Lactobacillus acidophilus (2.2×10) 8 ±0.5×10 8 copies / g), Bifidobacterium longum (2.1×10) 8 ±0.4×10 8 The viable count (copies / g) was significantly higher than that of each strain's individual groups (ZK-35 individual group: 4.2 × 10⁻⁶). 8 ±0.8×10 8 copies / g, Lactobacillus acidophilus alone group 0.8×10 8 ±0.2×10 8 copies / g, Bifidobacterium longum alone group 0.7×10 8 ±0.2×10 8 The results showed that the three strains combined could mutually promote intestinal colonization (copies / g) (P<0.01), indicating that the combination of the three strains could mutually promote intestinal colonization.

[0321] Metabolic product complementarity: The contents of paracasein (15.8±2.3μg / mL), lactic acid (25.3±3.2mmol / L), acetic acid (38.5±4.2mmol / L), and butyric acid (12.5±1.5mmol / L) in the colon contents of the three-strain combination group were significantly higher than those of the single-strain group and the two-strain combination group (P<0.01), indicating that the metabolites of the three strains can complement each other and enhance the overall function.

[0322] Signaling pathway protein expression: The relative expression levels of TLR2 (0.92±0.09), Nrf2 (0.88±0.08), and TGF-β (0.95±0.10) in the three-strain combination group were significantly higher than those in the single-strain group (P<0.01), while the relative expression level of NF-κB (0.32±0.07) was significantly lower than that in the single-strain group (P<0.01). This indicates that the three strains can alleviate intestinal inflammation by synergistically activating the TLR2, Nrf2, and TGF-β signaling pathways and inhibiting the NF-κB inflammatory pathway.

[0323] In summary, the combination of Lactobacillus paracasei ZK-35 with Lactobacillus acidophilus and Bifidobacterium longum in a weight ratio of 4:1:1 can significantly alleviate the symptoms of ulcerative colitis in rats, repair colonic tissue damage, and regulate intestinal flora balance by synergistically enhancing intestinal colonization capacity, complementing metabolites, and regulating signaling pathways. Its effect is significantly better than that of single strains and the combination of two groups, and it has a clear synergistic effect.

[0324] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Application of Lactobacillus paracasei ZK-35 in alleviating antibiotic-induced intestinal problems, wherein the application is for non-disease treatment purposes. Lactobacillus paracasei ZK-35 was deposited at the China General Microbiological Culture Collection Center on July 9, 2025, with accession number CGMCC NO.35142. The antibiotics include amoxicillin and cefixime; The intestinal problems caused by antibiotics include the occurrence of AAD, intestinal flora imbalance, impaired intestinal barrier function, intestinal inflammatory response, Clostridium difficile colonization, reduced secretion of short-chain fatty acids, decreased nutrient absorption, or diarrhea.