Lactobacillus paracasei ZK-35 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus paracasei ZK-35 and application thereof. The lactobacillus paracasei ZK-35 is separated from a human oral cavity flushing fluid, has good compatibility with the human intestinal environment, high safety and excellent acid and cholate resistance, has an effect of relieving side effects of various antibiotics such as penicillin, cephalosporin and quinolones, can regulate various intestinal florae at the same time, improves constipation and diarrhea, and has a good application prospect in the field of oral cavity flushing fluid, oral cavity flushing fluid, oral cavity flushing fluid, oral cavity flushing fluid, oral cavity flushing fluid, oral cavity flushing fluid, oral cavity flushing fluid, oral cavity flushing fluid and oral cavity flushing fluid. The intestinal barrier function is enhanced, and nutrient absorption is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a lactobacillus paracasei ZK-35 and application thereof. BACKGROUND

[0002] As the largest digestive and immune organ of the human body, the health status of the intestinal tract is closely related to the overall health. Different degrees of intestinal health problems exist in adults, among which constipation (incidence rate 18%-25%), chronic diarrhea (incidence rate 8%-12%), and intestinal flora imbalance (incidence rate 30%-40%) are the most common; the incidence rate of inflammatory bowel disease (IBD, including ulcerative colitis UC and Crohn's disease CD) is gradually increasing and tends to be younger.

[0003] A high-sugar, high-fat, low-dietary fiber diet leads to a decrease in intestinal flora diversity and 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, non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and other drugs can also damage the intestinal mucosa; environmental pollutants (such as heavy metals and pesticide residues) can disrupt the balance of intestinal flora. SUMMARY

[0004] In view of the above problems of the prior art, the present application aims to provide a lactobacillus paracasei ZK-35 and application thereof, which has a broad-spectrum antibiotic side effect alleviating ability and a comprehensive intestinal flora regulating function.

[0005] To solve the above problems, the present application adopts the following technical scheme:

[0006] In a first aspect, the present application provides a lactobacillus paracasei ZK-35, which has been preserved in the China General Microbiological Culture Collection Center on July 9, 2025, with a preservation number of CGMCC NO.35142.

[0007] In a second aspect, the present application provides application of the lactobacillus paracasei ZK-35 and / or its metabolites in alleviating intestinal problems caused by antibiotics.

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

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

[0010] In a third aspect, the present application provides application of the Lactobacillus paracasei ZK-35 and / or metabolites thereof in intestinal improvement.

[0011] In a fourth aspect, the present application provides a composition for relieving ulcerative colitis, comprising the Lactobacillus paracasei ZK-35, Lactobacillus acidophilus and Bifidobacterium longum.

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

[0013] In a fifth aspect, the present application provides application of the Lactobacillus paracasei ZK-35 and / or metabolites thereof or the composition in preparation of an intestinal improvement product.

[0014] Further, the form of the intestinal improvement product comprises a freeze-dried powder, an oral liquid, a tablet, a capsule, a granule or a drop.

[0015] The present application has the following beneficial effects: the Lactobacillus paracasei ZK-35 of the present application is isolated from human oral rinse, has good compatibility with the intestinal environment of the human body, is highly safe, has excellent acid and bile salt resistance, has relieving effects on side effects of various antibiotics such as penicillins, cephalosporins and quinolones, can simultaneously regulate various intestinal flora (increase Lactobacillus, Bifidobacterium, Akk bacteria and Clostridium coccoides, and reduce Escherichia coli and Clostridium difficile), improve constipation and diarrhea, enhance intestinal barrier function, and promote nutrient absorption. DETAILED DESCRIPTION

[0016] The present application will be further described in detail below in combination with specific examples.

[0017] It should be noted that these examples are only used to illustrate the present application, but not to limit the present application, and simple improvements of the present method under the concept of the present application all fall within the scope of the present application.

[0018] The Lactobacillus paracasei ZK-35 of the present application has been preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, on July 9, 2025, and the preservation number is CGMCC NO. 35142.

[0019] Example 1

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

[0021] 1.1 Test materials

[0022] Sample source: oral rinse of healthy adults (2 males, aged 28 and 32 years; 2 females, aged 25 and 30 years), all volunteers had no use of antibiotics, probiotics and immunomodulators in the past 3 months, and had no history of intestinal diseases and oral diseases.

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

[0024] Reagents: Gram staining solution (crystal violet staining solution, iodine solution, decolorizing solution, counterstaining solution), API 50 CH carbohydrate fermentation kit (Biomerieux, France), DNA extraction kit (Beijing Tian Gen Biochemical Technology Co., Ltd.), 16S rRNA gene amplification primers (upper primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', lower primer 1492R: 5'-GGTTACCTTGTTACGACTT-3', Shanghai Sangon Biological Engineering Co., Ltd.), PCR Master Mix (Bao Biological 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 method

[0027] Sample collection and processing:

[0028] The volunteers rinsed their mouths with 100 mL of sterile normal saline (37°C) for 30 seconds, and then collected the rinse liquid in a sterile centrifuge tube to obtain the oral rinse sample.

[0029] 10 mL of the sample was taken and diluted 10 times with 90 mL of sterile normal saline, and then diluted by 10 times gradient (10 -1 ~10-6 ).

[0030] Isolation and purification:

[0031] Take 10 -4 , 10 -5 , 10 -6 0.1 mL of each dilution sample was coated on MRS solid medium plate, and 3 repeats were made for each dilution.

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

[0033] Observe the colony morphology, select the colonies that meet the characteristics of lactobacillus (circular, milky white, neat edge, smooth surface), inoculate into MRS liquid medium, 37℃ anaerobic culture for 24h, and obtain pure culture.

[0034] Repeat the streaking purification for 3 times to ensure the purity of the strain.

[0035] Morphological identification:

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

[0037] Physiological and biochemical identification:

[0038] According to the API 50 CH carbohydrate fermentation kit instructions, inoculate the pure culture into the kit reaction tube, incubate at 37℃ for 48h, and observe the acid production (the reaction tube changes from purple to yellow is positive).

[0039] Acid tolerance experiment: inoculate the activated bacterial suspension (1.0×10 9 CFU / mL) into MRS liquid medium with pH 2.0, 3.0, 4.0, respectively, incubate at 37℃ for 2h, detect the viable bacteria count by plate counting method, and calculate the survival rate (survival rate = viable bacteria count after treatment / viable bacteria count before treatment x 100%).

[0040] Bile salt tolerance experiment: inoculate the activated bacterial suspension (1.0×10 9 CFU / mL) into MRS liquid medium containing 0.1%, 0.3%, 0.5% bile salt, respectively, incubate at 37℃ for 2h, detect the viable bacteria count by plate counting method, and calculate the survival rate.

[0041] Growth temperature experiment: inoculate the activated bacterial suspension into MRS liquid medium, incubate at 15℃, 25℃, 37℃, 42℃, 45℃, respectively, for 24h, measure the OD 600OD values were measured to determine the growth condition. 600 ≥0.5 is positive for growth).

[0042] Molecular biology identification:

[0043] The genomic DNA of the strain was extracted using a DNA extraction kit.

[0044] PCR amplification was performed using 27F / 1492R primers with genomic DNA as the template: the reaction system (50 μL) included PCR Master Mix 25 μL, upstream primer (10 μmol / L) 2 μL, downstream primer (10 μmol / L) 2 μL, DNA template 5 μL, and sterile water 16 μL; 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; 72 ℃ final extension for 10 min.

[0045] After 1% agarose gel electrophoresis verification of the PCR product, sequencing was performed by Shanghai Shengong Bioengineering Co., Ltd.

[0046] The sequencing results were submitted to the GenBank database, and homology comparison was performed with the 16S rRNA gene sequences of known strains using the BLAST tool 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 rinse samples, numbered ZK-1~ZK-42.

[0049] Morphological identification results:

[0050] The colonies of 12 strains on MRS solid medium were round, milky white, with a neat edge, smooth surface, and a diameter of 1.0~1.5 mm.

[0051] Gram staining was positive, the bacterial cells were short rods, without spores, flagella, and motility.

[0052] Physiological and biochemical identification results:

[0053] Carbohydrate fermentation results: ZK-35 strain could ferment glucose, lactose, sucrose, maltose, fructose, galactose, and mannose to produce acid and gas; it could not ferment xylose, arabinose, raffinose, rhamnose, and sorbose; the remaining strains could not ferment lactose or maltose, which was different from ZK-35.

[0054] The acid resistance experiment results: the survival rate of ZK-35 was 52.3%±3.5% after 2h treatment at pH 2.0; the survival rate was 85.6%±4.2% after 2h treatment at pH 3.0; the survival rate was 96.8%±2.1% after 2h treatment at pH 4.0; significantly higher than other strains.

[0055] The bile salt resistance experiment results: the survival rate of ZK-35 was 92.5%±3.1% after 2h treatment at 0.1% bile salt; the survival rate was 41.8%±3.7% after 2h treatment at 0.3% bile salt; the survival rate was 15.6%±2.4% after 2h treatment at 0.5% bile salt; significantly higher than other strains.

[0056] The growth temperature experiment results: ZK-35 can grow (OD 600 =0.62±0.05, 1.25±0.08, 2.18±0.12, 0.85±0.06) at 15℃, 25℃, 37℃, 42℃, respectively, and cannot grow (OD 600 =0.21±0.03) at 45℃; the optimum growth temperature is 37℃.

[0057] Molecular biology identification results:

[0058] The 16S rRNA gene of ZK-35 strain is sequenced, and the sequence is submitted to GenBank; the BLAST comparison results show that the homology of ZK-35 and the model strain of Paracasei (GenBank accession number NR_074515.1) is 99.8%, and the homology with other Lactobacillus strains (such as Lactobacillus acidophilus, Lactobacillus plantarum) is ≤95%.

[0059] The phylogenetic tree analysis shows that ZK-35 and the model strain of Paracasei are clustered into one branch, and the bootstrap value is 99%, which further confirms that it is Paracasei.

[0060] In summary, a Paracasei with excellent acid and bile salt resistance is isolated from human oral rinse, named ZK-35, and preserved.

[0061] Example 2

[0062] Paracasei ZK-35 alleviates the side effects of amoxicillin

[0063] 2.1 Test materials

[0064] Strain: Paracasei ZK-35 (CGMCC NO. 35142, prepared by the laboratory, live bacteria number 1.0×10 11CFU / g); Lactobacillus rhamnosus was commercially available, and the viable count was 1.0×10 11 CFU / g, as a positive control).

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

[0066] Reagents: Amoxicillin capsules (North China Pharmaceutical Co., Ltd., specification 0.25 g / particle, prepared into a 20 mg / mL solution with sterile normal saline); Clostridioides difficile (ATCC 9689, preserved in the 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 (American Abeam Company); Western blot detection kit (Bi Yun Tian Biotechnology Institute).

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

[0068] 2.2 Test method

[0069] Strain activation and bacterial suspension preparation:

[0070] Subcutaneous cheese Lactobacillus ZK-35 and Lactobacillus rhamnosus were inoculated into MRS liquid medium, 37°C anaerobic culture for 16h, and activated for three generations.

[0071] Centrifugation (4°C, 6000r / min, 10min) to collect bacterial cells, washed twice with sterile normal saline, and the concentration of bacterial suspension was adjusted to 1.0×10 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, 20 mice in each group, half male and half female.

[0074] Control group: normal saline 0.2 mL per mouse per day, for 21 days;

[0075] AMX group: amoxicillin 0.2 mL per mouse per day (dose 200 mg / kg) for the first 7 days, and normal saline 0.2 mL per mouse per day for the next 14 days.

[0076] ZK-35-L group: amoxicillin 0.2 mL per mouse per day for the first 7 days, and ZK-35 0.2 mL per mouse per day (dose 1.0 x 10 8 CFU per mouse, equivalent to 5.0 x 10 9 CFU / kg) for the next 14 days.

[0077] ZK-35-M group: amoxicillin 0.2 mL per mouse per day for the first 7 days, and ZK-35 0.2 mL per mouse per day (dose 5.0 x 10 8 CFU per mouse, equivalent to 2.5 x 10 10 CFU / kg) for the next 14 days.

[0078] ZK-35-H group: amoxicillin 0.2 mL per mouse per day for the first 7 days, and ZK-35 0.2 mL per mouse per day (dose 1.0 x 10 9 CFU per mouse, equivalent to 5.0 x 10 10 CFU / kg) for the next 14 days.

[0079] GG group: amoxicillin 0.2 mL per mouse per day for the first 7 days, and GG 0.2 mL per mouse per day (dose 1.0 x 10 9 CFU per mouse, equivalent to 5.0 x 10 10 CFU / kg) for the next 14 days.

[0080] All mice were housed in SPF level animal rooms with temperature 22-25℃, humidity 40%-60%, 12h light / 12h dark cycle, free access to food and water.

[0081] Detection index and method:

[0082] General clinical observation: observe the mental state (active / depressed), appetite (food intake), defecation (stool shape, diarrhea occurrence) of mice daily, and record the incidence of diarrhea and diarrhea score.

[0083] Diarrhea scoring criteria: 0 points: stool is formed and granular; 1 point: stool is loose and not formed; 2 points: stool is loose and sticky to the anus; 3 points: stool is watery and jet-like.

[0084] Body weight change: weigh once a week (1st day, 7th day, 14th day, 21st day), calculate the body weight gain rate (body weight gain rate = (body weight on the day - initial body weight) / initial body weight x 100%);

[0085] Intestinal flora analysis: on the 21st day, 6 mice were randomly selected from each group and sacrificed by cervical dislocation, 0.5g of cecal contents was taken aseptically, total DNA was extracted using the CTAB method, the 16S rRNA gene V4 region was amplified (primers 515F: 5'-GTGCCAGCMGCCGCGGTAA-3', 907R: 5'-CCGTCAATTCMTTTRAGTTT-3'), Illumina MiSeq high-throughput sequencing was performed, and the alpha diversity (Chao1 index, Shannon index) and beta diversity (PCoA analysis) of intestinal flora were analyzed, as well as the relative abundance of key flora.

[0086] Intestinal barrier function detection:

[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 as internal reference, relative expression amount was calculated);

[0088] Serum endotoxin (LPS) content: the mouse eyeball was taken for blood, centrifuged (37°C, 3000r / min, 10min) to separate serum, and the LPS content was detected by limulus reagent method.

[0089] Mucosal sIgA content: 0.5g of colon mucosa was taken, homogenized with PBS buffer (containing protease inhibitors), centrifuged (4°C, 12000r / min, 15min) to take the supernatant, and the sIgA content was detected by ELISA method.

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

[0091] Short-chain fatty acids (SCFAs) detection: Take 0.5 g of cecal contents, add 1 mL of ultrapure water homogenate, centrifuge (4℃, 12000r / min, 15min) to take the supernatant, filter through 0.22μm filter membrane, and then detect the content of acetic acid, propionic acid and butyric acid by gas chromatograph (model GC-2014, Japan Shimadzu company).

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

[0093] C. difficile colonization detection: On the 14th day, 6 mice were randomly taken from each group, and C. difficile bacterial suspension (1.0 x 10 6 CFU / only) was administered by gavage, and on the 21st day, the cecal contents were taken, C. difficile was isolated and cultured using CCFA medium (cycloserine-cefoxitin-fructose-agar), and the viable bacterial count was detected by plate counting method.

[0094] 2.3 Test results

[0095] General clinical observation results:

[0096] Blank control group: The mice were active, had normal appetite, formed stools, and no diarrhea occurred;

[0097] Amoxicillin model group: Diarrhea occurred on the 3rd day, the incidence of diarrhea reached 85% (17 / 20) on the 7th day, and the diarrhea score reached 2.8±0.3; the mice were listless, and the appetite decreased (food intake decreased by 40%-50% compared with the blank group).

[0098] ZK-35 dose groups: The incidence of diarrhea and diarrhea score decreased significantly with increasing dose; the incidence of diarrhea in ZK-35-H group was 20% (4 / 20) on the 7th day, and the diarrhea score was 0.6±0.2, which was significantly lower than that of 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 was 45% (9 / 20) on the 7th day, 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 the 21st day, the incidence of diarrhea in the ZK-35-H group decreased 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] Body weight change results:

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

[0103] Amoxicillin model group: the body weight gain rate was -8.5% ± 2.3% (body weight loss) on day 7, and 12.3% ± 3.2% on day 21, which was significantly lower than that of the blank group (P < 0.01).

[0104] ZK-35 dose groups: the body weight gain rate increased significantly with the increase of the dose; the body 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) and significantly higher than the model group and the control group (P < 0.01).

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

[0106] Intestinal flora analysis results:

[0107] Alpha diversity analysis: the Chao1 index (285.6 ± 25.3) and Shannon index (2.1 ± 0.3) of the model group were significantly lower than those of the blank 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) of the ZK-35-H group were not significantly different from those of the blank group (P > 0.05) and significantly higher than those of the model group and the control group (Chao1 index 352.7 ± 28.6, Shannon index 2.9 ± 0.3) (P < 0.01).

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

[0109] Relative abundance of key flora:

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

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

[0112] Intestinal barrier function detection results:

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

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

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

[0116] Inflammatory factor detection results:

[0117] Proinflammatory factors: the serum TNF-α content (65.8 ± 8.5 pg / mL) and IL-6 content (45.2 ± 6.3 pg / mL) of the model group, and the colon tissue TNF-α content (85.6 ± 10.2 pg / g) and IL-6 content (65.3 ± 8.1 pg / g) were significantly higher than those of 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 above-mentioned proinflammatory factor contents of the ZK-35-H group were not significantly different from those of the blank group (P > 0.05), and were significantly lower than those of the model group and the control group (P < 0.01).

[0118] Anti-inflammatory factors: the serum IL-10 content (8.5 ± 1.5 pg / mL) and colon tissue IL-10 content (12.3 ± 2.1 pg / g) of the model group were significantly lower than those of 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 above-mentioned anti-inflammatory factor contents of the ZK-35-H group were not significantly different from those of the blank group (P > 0.05), and were significantly higher than those of the model group and the control group (P < 0.01).

[0119] Short-chain fatty acid detection results:

[0120] The acetic acid content (15.2 ± 2.3 mmol / L), propionic acid content (5.8 ± 1.1 mmol / L), and butyric acid content (2.1 ± 0.5 mmol / L) of the cecal contents of the model group were significantly lower than those of 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 SCFA 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 had no significant difference (P>0.05) with the blank group, and were significantly higher than those in the model group and the control group (acetic acid 25.3±3.5 mmol / L, propionic acid 8.5±1.5 mmol / L, and butyric acid 4.2±0.8 mmol / L) (P<0.01).

[0122] C. difficile colonization detection results:

[0123] The number of viable C. difficile in the cecal contents of the model group (8.5×10 5 ±1.2×10 5 CFU / g) was significantly higher than that in the blank group (not detected) (P<0.01).

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

[0125] In summary, L. paracasei ZK-35 can significantly alleviate the side effects of amoxicillin, including reducing the incidence of AAD, restoring body weight gain, regulating intestinal flora balance, repairing intestinal barrier function, reducing inflammatory response, increasing SCFA synthesis, and inhibiting C. difficile colonization, and the effect is better than that of commercially available L. rhamnosus GG, and the high dose (5.0×10 10 CFU / kg) has the best effect.

[0126] Example 3

[0127] L. paracasei ZK-35 alleviates the side effects of cefixime

[0128] 3.1 Test materials

[0129] Strains: L. paracasei ZK-35 (CGMCC NO. 35142, viable bacteria 1.0×10 11 CFU / g); L. acidophilus (as a positive control).

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

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

[0132] Instruments: Full-automatic biochemical analyzer (model Cobas 6000, Roche, Switzerland); flow cytometer (model BD FACSCanto II, BD, USA).

[0133] 3.2 Test method

[0134] Strain activation and bacterial suspension preparation: same as Example 2, adjust the ZK-35 and control bacterial suspension concentration to 5.0×10 9 CFU / mL (medium dose), 1.0×10 10 CFU / mL (high dose).

[0135] Animal grouping and treatment:

[0136] 96 rats were randomly divided into 6 groups, 16 rats in each group, half male and half female.

[0137] Blank control group: intragastrically administered with sterile normal saline 2 mL / rat per day, for 28 consecutive days.

[0138] Cefixime model group (CTX): intragastrically administered with cefixime solution 2 mL / rat (dose 100 mg / kg) for the first 10 days, and intragastrically administered with sterile normal saline 2 mL / rat for the 11th to 28th day.

[0139] ZK-35 medium dose group (ZK-35-M): intragastrically administered with cefixime solution 2 mL / rat for the first 10 days, and intragastrically administered with ZK-35 bacterial suspension 2 mL / rat (dose 1.0×10 10 CFU / rat, equivalent to 5.0×10 10 CFU / kg) for the first 28 days.

[0140] ZK-35 high dose group (ZK-35-H): intragastrically administered with cefixime solution 2 mL / rat for the first 10 days, and intragastrically administered with ZK-35 bacterial suspension 2 mL / rat (dose 2.0×10 10 CFU / rat, equivalent to 1.0×10 11 CFU / kg) for the first 28 days.

[0141] Control medium dose group (-M): 2 mL of cefixime solution was given by gavage from day 1 to day 10, and 2 mL of control bacteria suspension was given by gavage from day 1 to day 28 (dose 1.0 x 10 10 CFU / rat, equivalent to 5.0 x 10 10 CFU / kg).

[0142] Control high dose group (-H): 2 mL of cefixime solution was given by gavage from day 1 to day 10, and 2 mL of control bacteria suspension was given by gavage from day 1 to day 28 (dose 2.0 x 10 10 CFU / rat, equivalent to 1.0 x 10 11 CFU / kg).

[0143] All rats were raised in SPF level animal room, temperature 22-25℃, humidity 40%-60%, 12h light / 12h dark cycle, free access to standard rat feed and drinking water.

[0144] The gavage time was fixed at 9:00 am every day, and the interval between cefixime solution and probiotic bacteria suspension was 2h (if both liquids need to be given on the same day), to avoid direct killing of probiotics by cefixime.

[0145] Detection index and method:

[0146] Diarrhea symptom monitoring:

[0147] From the first day of the test to the 28th day, the rats were observed and recorded daily for defecation, including stool shape (formed / loose / liquid / watery), defecation frequency (times / day), and diarrhea occurrence.

[0148] Diarrhea determination criteria: 3 or more consecutive liquid or watery stools were considered diarrhea, and the number of rats with diarrhea in each group was recorded daily, and the diarrhea incidence rate was calculated (diarrhea incidence rate = number of rats with diarrhea on the day / total number of rats in the group x 100%).

[0149] The average duration of diarrhea in each group was calculated once a week (from the occurrence of diarrhea to the recovery of stool shape).

[0150] Body weight and food intake monitoring:

[0151] The rats were weighed on an empty stomach every Monday morning (1st, 7th, 14th, 21st, 28th day), and the body weight was recorded, and the body weight change rate was calculated (body weight change rate = (current body weight - initial body weight / initial body weight x 100%).

[0152] The remaining feed weight was weighed before feeding every Monday morning, and the average food intake of the previous week was calculated (average food intake = (the weight of the feed put in the previous week - the remaining feed weight this week) / the total number of rats in the group / 7 days).

[0153] Intestinal permeability detection:

[0154] On day 28, 8 rats were randomly selected from each group, and after 12h of fasting and water deprivation, 4kDa fluorescein isothiocyanate-dextran (FITC-dextran, Sigma, USA) solution was administered by gavage (dose 600mg / kg, concentration 200mg / mL).

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

[0156] 50μL of plasma was diluted with 950μL of sterile saline, and the fluorescence intensity was detected using a fluorescence spectrophotometer (model F-4600, Hitachi, Japan) (excitation wavelength 485nm, emission wavelength 520nm). The plasma FITC-dextran concentration was calculated according to the standard curve, reflecting the intestinal permeability (the higher the concentration, the stronger the intestinal permeability, and the worse the barrier function).

[0157] At the same time, the contents of D-lactate (a marker of intestinal mucosal damage) and diamine oxidase (DAO, a marker of intestinal mucosal integrity) in the serum were detected using ELISA kits, and the operation was strictly according to the instructions of the kits.

[0158] Intestinal flora functional metabolism analysis:

[0159] On day 28, 6 rats were randomly selected from each group, and after carbon dioxide euthanasia, 1g of cecal contents was taken aseptically and placed in a sterile centrifuge tube.

[0160] The intestinal flora metabolites in the cecal contents were detected using high performance liquid chromatography-mass spectrometry (HPLC-MS, Agilent 1290-6460, Agilent, USA), including:

[0161] Short chain fatty acids (SCFAs): Acetic acid, Propionic acid, Butyric acid, Isobutyric acid, Valeric acid, Isovaleric acid, Chromatographic conditions: Column ZORBAX SB-Aq (150 mm x 4.6 mm x 5 pm), mobile phase 0.1% formic acid in water - acetonitrile (95:5, v / v), flow rate 1 mL / min, column temperature 30 °C, injection volume 10 pL; 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: Column ZORBAX Eclipse Plus C18 (150 mm x 4.6 mm x 5 pm), mobile phase 0.1% formic acid in water - acetonitrile (gradient elution), flow rate 1 mL / min, column temperature 35 °C, injection volume 10 pL; Mass spectrometry conditions: ESI positive ion mode, MRM.

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

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

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

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

[0168] Escherichia coli: primer 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°C pre-denaturation 30 s; 95°C denaturation 5 s, 60°C annealing 30 s, a total of 40 cycles; melting curve analysis: 95°C 15 s, 60°C 1 min, 95°C 15 s; according to the standard curve, the copy number (copies / g content) of each bacterium was calculated.

[0170] Nutrient absorption function detection:

[0171] On days 26-28, 6 rats were randomly selected from each group and individually fed in metabolic cages. The 24h feces were collected and the feces weight was recorded.

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

[0173] The atomic absorption spectrophotometer (model AA-6300, Shimadzu, Japan) was used to detect the calcium and iron content in feed and feces, and the apparent calcium and iron absorption rates were calculated (calculation formula same as apparent protein digestibility).

[0174] The rat serum was collected, and the total protein, albumin, calcium, iron, and vitamin B 12 content in the serum were detected by automatic biochemical analyzer to reflect the absorption and utilization of nutrients in the body.

[0175] Liver and kidney function detection:

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

[0177] Part of the liver and kidney tissues were fixed with 4% neutral formaldehyde, paraffin-embedded, sectioned (thickness 5 μm), HE stained, and observed under optical microscope for histopathological changes. The injury degree was evaluated by semi-quantitative scoring method (0 points: no injury; 1 point: mild injury, local cell degeneration; 2 points: moderate injury, local cell necrosis; 3 points: severe injury, extensive cell necrosis).

[0178] The rat serum was collected, and the liver function indexes (glutamic-pyruvic transaminase ALT, glutamic-oxalacetic transaminase AST, total bilirubin TBIL) and kidney function indexes (serum creatinine Scr, urea nitrogen BUN) were detected by using a fully automatic biochemical analyzer to evaluate the potential damage of cefixime to liver and kidney and the protective effect of ZK-35.

[0179] 3.3 Test results

[0180] Diarrhea symptom monitoring results:

[0181] Blank control group: During the whole test period, the feces of rats were formed, no diarrhea occurred, and the daily defecation frequency was stable at 6-8 times / day.

[0182] Cefixime model group: Diarrhea occurred from the 3rd day, and the incidence of diarrhea reached a peak of 75.0% (12 / 16) on the 7th-10th day (the end of cefixime administration), with mainly loose or watery feces, and the daily defecation frequency increased to 12-15 times / day; after drug withdrawal (from the 11th day), the incidence of diarrhea gradually decreased, but 25.0% (4 / 16) of the rats still had diarrhea by the 28th day, with an average diarrhea duration of 8.5±1.2 days.

[0183] ZK-35 dose groups: The incidence of diarrhea, severity of diarrhea, and duration of diarrhea were significantly reduced with the increase of ZK-35 dose; the highest incidence of diarrhea in the ZK-35-H group during cefixime administration (1st-10th day) was only 18.8% (3 / 16), which was significantly lower than that of the model group (P<0.01), and the feces were mostly loose, with a daily defecation frequency of 8-10 times / day; by the 5th day after drug withdrawal (15th day), the feces of all rats returned to normal, with an average diarrhea duration of 2.1±0.5 days, which was significantly shorter than that of the model group (P<0.01).

[0184] Control dose groups: The highest incidence of diarrhea in the -H group was 43.8% (7 / 16), and the average diarrhea duration was 5.3±0.8 days, which was lower than that of the model group (P<0.05), but significantly higher than that of the ZK-35-H group (P<0.05), indicating that the effect of ZK-35 in relieving cefixime-related diarrhea was better than that of the control.

[0185] Body weight and food intake monitoring results:

[0186] Body weight change: The body weight of the blank control group rats increased steadily, and the body weight change rate was 38.5±3.2% on day 28. The body weight of the model group rats decreased significantly during the cefixime medication period (days 1-10), and the body weight change rate was -6.2±1.8% on day 10. The body weight change rate was only 15.3±2.5% on day 28, which was significantly lower than that of the blank group (P<0.01). The body weight change rate of the ZK-35-H group was -1.3±0.9% on day 10, and the body weight change rate was 36.8±2.8% on day 28, which had no significant difference with the blank group (P>0.05) and was significantly higher than that of the model group and the -H group (the body weight change rate was 22.6±2.1% on day 28) (P<0.01).

[0187] Food intake: The average food intake of the blank control group was stable at 22-25 g / (rat·day). The average food intake of the model group decreased to 15-18 g / (rat·day) on days 1-10, which was significantly lower than that of the blank group (P<0.01). The average food intake of the ZK-35-H group was 20-22 g / (rat·day) on days 1-10, 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 food intake of the -H group was 17-19 g / (rat·day) on days 1-10, 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, which was significantly higher than that of the blank group (0.32±0.05 μg / mL) (P<0.01). The ZK-35-H group was 0.41±0.08 μg / mL, which had no significant difference with the blank group (P>0.05) and was significantly lower than that of the model group and the -H group (1.02±0.15 μg / mL) (P<0.01).

[0190] Serum D-lactic acid content: The model group was 85.6±10.2 μg / mL, which was significantly higher than that of the blank group (15.3±2.1 μg / mL) (P<0.01). The ZK-35-H group was 18.5±3.2 μg / mL, which had no significant difference with the blank group (P>0.05) and was significantly lower than that of 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, which was 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, which had no significant difference with the blank group (P>0.05), and was significantly lower than the model group and the -H group (7.8±1.2 U / L) (P<0.01). The above results show that cefixime can significantly increase the intestinal permeability of rats, damage the intestinal mucosa, while ZK-35 can effectively repair the intestinal mucosal damage, reduce the intestinal permeability, and restore the intestinal barrier function, and the effect is better than that of the control.

[0192] Intestinal flora functional metabolism analysis results:

[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, which was significantly lower than that of the blank group (65.3±5.8 mmol / kg) (P<0.01), and the butyric acid content was only 1.8±0.3 mmol / kg, which was 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 had no significant difference with the blank group (P>0.05), and was significantly higher than that of the model group and the -H group (total SCFA 42.3±3.8 mmol / kg, butyric acid 4.5±0.8 mmol / kg) (P<0.01).

[0194] Bile acid content: the secondary bile acid (DCA, LCA) content in the serum of the model group was significantly increased (DCA 15.8±2.3 μmol / L, LCA 8.5±1.2 μmol / L), which was significantly higher than that of 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 of the ZK-35-H group (DCA 4.1±0.7 μmol / L, LCA 2.3±0.5 μmol / L) had no significant difference with the blank group (P>0.05), and was significantly lower than that of 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] Intestinal flora quantity (qPCR results): the number of Bifidobacterium (2.5×108±0.5×108 copies / g) and Lactobacillus (3.2×108±0.6×108 copies / g) in the model group was significantly lower than that in the blank group (Bifidobacterium 8.5×108±0.5×108 copies / g, Lactobacillus 4.2×108±0.8×108 copies / g) (P<0.01); the number of Bifidobacterium (1.2×109±0.6×109 copies / g) and Lactobacillus (2.5×109±0.5×109 copies / g) in the ZK-35-H group was significantly higher than that in the model group and the -H group (Bifidobacterium 2.5×108±0.5×108 copies / g, Lactobacillus 3.2×108±0.6×108 copies / g) (P<0.01). 8 8 9 ​​±1.2 x 10 9 copies / g, Lactobacillus 6.8 x 10 9 ±1.0 x 10 9 copies / g) (P < 0.01); the number of E. coli (1.8 x 10 9 ±0.3 x 10 9 copies / g) was significantly higher than the blank group (3.5 x 10 7 ±0.8 x 10 7 copies / g) (P < 0.01); the number of Bifidobacterium (7.8 x 10 9 ±1.0 x 10 9 copies / g), Lactobacillus (6.5 x 10 9 ±0.8 x 10 9 copies / g) in the ZK-35-H group had no significant difference with the blank group (P > 0.05), and the number of E. coli (4.2 x 10 7 ±0.6 x 10 7 copies / g) was significantly lower than the model group and the -H group (Bifidobacterium 4.2 x 10 9 ±0.8 x 10 9 copies / g, Lactobacillus 3.5 x 10 9 ±0.7 x 10 9 copies / g, E. coli 8.5 x 10 8 ±1.2 x 10 8 copies / g) (P < 0.01).

[0196] The results of the nutritional absorption function test are as follows:

[0197] The apparent digestibility: the apparent digestibility of protein (70.2 ± 3.5%), the apparent absorption rate of calcium (45.3 ± 4.2%), and the apparent absorption rate of iron (38.5 ± 3.8%) in the model group were significantly lower than those in the blank group (the apparent digestibility of protein 85.6 ± 2.8%, the apparent absorption rate of calcium 68.5 ± 4.5%, and the apparent absorption rate of iron 58.2 ± 4.1%) (P < 0.01); the above-mentioned digestion and absorption rates in the ZK-35-H group (protein 84.2 ± 2.5%, calcium 66.8 ± 4.1%, and iron 56.5 ± 3.8%) had no significant difference with the blank group (P > 0.05) and were significantly higher than those in the model group and the -H group (protein 76.5 ± 3.2%, calcium 55.2 ± 3.8%, and iron 45.8 ± 3.5%) (P < 0.01).

[0198] Serum nutrition indicators: the 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), vitamin B 12 (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 B 12 325.8 ± 35.6 pg / mL) (P < 0.01); the above indicators of 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 12 308.5 ± 32.1 pg / mL) had no significant difference with the blank group (P > 0.05), and were significantly higher than the model group and -H group (P < 0.01).

[0199] Liver and kidney function test results:

[0200] Organ index: the liver index (3.8 ± 0.3%) and kidney index (0.85 ± 0.08%) of the model group were significantly higher than those of 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%) of the ZK-35-H group had no significant difference with the blank group (P > 0.05), and were significantly lower than those of the model group (P < 0.05); the liver index (3.6 ± 0.2%) and kidney index (0.80 ± 0.07%) of the -H group were lower than those of the model group, but were still higher than those of the ZK-35-H group (P < 0.05).

[0201] Histopathology score: the liver tissue of the model group showed mild fatty degeneration 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 of the blank group (0) (P < 0.01); the liver pathological score of the ZK-35-H group was 0.3 ± 0.1, and the kidney pathological score was 0.2 ± 0.1, which were significantly lower than those of the model group (P < 0.01); the liver pathological score of the -H group was 1.0 ± 0.2, and the kidney pathological score was 0.8 ± 0.1, which were higher than those of the ZK-35-H group (P < 0.05).

[0202] Biochemical indicators: The 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) of the model group were significantly higher than those of 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, and BUN 6.2 ± 0.8 mmol / L) (P < 0.01); the above indicators of 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, and BUN 6.5 ± 0.7 mmol / L) showed no significant difference from the blank group (P > 0.05) and were significantly lower than those of the model group and the -H group (P < 0.01).

[0203] In summary, L. 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 intestinal flora structure and metabolism, improving nutrient absorption, and protecting liver and kidney tissues, and the high-dose (1.0 × 10¹¹ CFU / kg) effect is the best, and the overall effect is better than that of the commercially available Lactobacillus acidophilus control.

[0204] Example 4

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

[0206] 4.1 Test materials

[0207] Strain: L. paracasei ZK-35 (CGMCC NO. 35142, freeze-dried powder prepared in the laboratory, viable bacterial count 1.0 × 10 11 CFU / g, prepared into 1.0 × 10 9 CFU / mL, 5.0 × 10 9 CFU / mL, and 1.0 × 10 10 CFU / mL bacterial suspension).

[0208] Experimental animals: SPF Kunming mice, 6-8 weeks old, half male and half female, weighing 20-22 g, a total of 100, purchased from Shanghai Slek Experimental Animal Co., Ltd., Animal Production License No. SCXK (Shanghai) 2023-0001, Animal Qualification Certificate No. 20230605001.

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

[0210] Instruments: Small animal live imaging instrument (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 method

[0212] Constipation model establishment and animal grouping:

[0213] After 3 days of adaptive feeding, the mice in the remaining groups were given compound loperamide suspension 0.2 mL / mouse (dose 10 mg / kg) by gavage daily for 7 consecutive days to establish a mouse constipation model; the success criteria for the model were that the number of defecation was reduced by more than 50% compared to the blank group, the water content of the feces was reduced by more than 30%, and the time for the first black feces to be discharged was extended by more than 50%.

[0214] One hundred mice were randomly divided into 5 groups, 20 mice in each group, half male and half female:

[0215] Blank control group (Control): 0.2 mL / mouse of 0.5% CMC-Na solution was given by gavage daily for 14 consecutive days.

[0216] Constipation model group (Model): Compound loperamide suspension 0.2 mL / mouse was given by gavage for the first 7 days, and 0.2 mL / mouse of 0.5% CMC-Na solution was given by gavage for the next 7 days.

[0217] ZK-35 low-dose group (ZK-35-L): Compound loperamide suspension 0.2 mL / mouse was given by gavage for the first 7 days, and ZK-35 suspension 0.2 mL / mouse (dose 1.0 x 10 8 CFU / mouse, equivalent to 5.0 x 10 9 CFU / kg) was given by gavage for the next 7 days.

[0218] ZK-35 medium dose group (ZK-35-M): Compound Loperamide suspension 0.2 mL per mouse was given by gavage from day 1 to day 7, and ZK-35 bacterial suspension 0.2 mL per mouse was given by gavage from day 1 to day 14 (dose 5.0x10 8 CFU per mouse, equivalent to 2.5x10 10 CFU / kg).

[0219] ZK-35 high dose group (ZK-35-H): Compound Loperamide suspension 0.2 mL per mouse was given by gavage from day 1 to day 7, and ZK-35 bacterial suspension 0.2 mL per mouse was given by gavage from day 1 to day 14 (dose 1.0x10 9 CFU per mouse, equivalent to 5.0x10 10 CFU / kg).

[0220] All mice were raised under the same conditions as in Example 2, and were given gavage at a fixed time every day (9:00 am);

[0221] Detection index and method:

[0222] Constipation symptom evaluation:

[0223] Defecation frequency and fecal characteristics: On day 7 (after model establishment) and day 14 (end of the test), 10 mice were randomly selected from each group and individually raised in metabolic cages, and were fasted but not water deprived. The 24h defecation frequency and fecal weight (accurate to 0.01g) were recorded. Five fresh feces were taken and the fecal water content was detected by drying method (Fecal water content = (fecal wet weight - fecal dry weight) / fecal wet weight x 100%).

[0224] Time of the first black feces: On day 14, after 12h fasting and water deprivation, activated carbon suspension (containing 5% activated carbon and 10% acacia) 0.2 mL per mouse was given by gavage, and the time from gavage to the first black feces was recorded, reflecting the intestinal transport function.

[0225] Small intestine propulsion rate: After the first black feces discharge time test was completed, the mice were executed 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 carbon in the small intestine were measured, and the small intestine propulsion rate was calculated (Small intestine propulsion rate = activated carbon propulsion distance / total length of small intestine x 100%).

[0226] Detection of intestinal motility related indicators:

[0227] Gastrointestinal hormone content: On day 14, 8 mice were randomly selected from each group, and blood was collected from the eyeball. The serum was separated by centrifugation (4°C, 3000 r / min, 10 min), and the contents of MTL (promoting gastrointestinal peristalsis), VIP (inhibiting gastrointestinal peristalsis), and SS (inhibiting gastrointestinal peristalsis) in the serum were detected by an ELISA kit.

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

[0229] Intestinal flora and metabolite analysis:

[0230] Intestinal flora alpha diversity: On day 14, 6 mice were randomly selected from each group, and 0.5 g of cecal content was taken for total DNA extraction. The 16S rRNA gene V4-V5 region (primer 515F: 5'-GTGCCAGCMGCCGCGGTAA-3', 907R: 5'-CCGTCAATTCMTTTRAGTTT-3') was amplified, and Illumina NovaSeq 6000 high-throughput sequencing platform was used for sequencing. Chao1 index (flora richness) and Shannon index (flora diversity) were analyzed.

[0231] Relative abundance of key flora: The relative abundance of Lactobacillus, Bifidobacterium, Akk, Faecalibacterium (butyrate-producing bacteria), and Desulfovibrio (harmful bacteria associated with constipation) was analyzed by sequencing results.

[0232] Short-chain fatty acid (SCFA) content: 0.5 g of cecal content was homogenized with 1 mL of ultrapure water, and the supernatant was obtained by centrifugation (4°C, 12000 r / min, 15 min). After filtration through a 0.22 μm filter membrane, the contents of acetic acid, propionic acid, and butyric acid were detected by a gas chromatograph (model GC-2030, Shimadzu, Japan).

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

[0234] Intestinal barrier function detection:

[0235] Colon histopathology observation: The colon tissue (2 cm from the anus) of mice was taken, fixed with 4% neutral formaldehyde, paraffin-embedded, sectioned, HE stained, and observed under an optical microscope for the colon mucosa thickness, villus integrity, and inflammatory cell infiltration. The pathological scoring standard (0 points: normal; 1 point: mild mucosal edema, a small amount of inflammatory cell infiltration; 2 points: moderate mucosal edema, partial villus rupture, moderate inflammatory cell infiltration; 3 points: severe mucosal edema, massive villus rupture, severe inflammatory cell infiltration) was used for scoring.

[0236] Tight junction protein expression: 0.5 g of colon tissue was taken to extract total protein, 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 amount, and the operation was the same as in Example 2.

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

[0238] 4.3 Test results

[0239] Constipation symptom evaluation results:

[0240] Defecation frequency and stool characteristics: On day 7, the 24h defecation frequency (3.2±0.8 times), stool weight (0.35±0.08 g), and stool water content (35.2±4.5%) of the model group were significantly lower than those of the blank group (defecation frequency 8.5±1.2 times, stool weight 1.25±0.15 g, and stool water content 65.3±5.8%) (P<0.01). On day 14, the above-mentioned indexes of the ZK-35 dose groups were significantly increased in a dose-dependent manner. The 24h defecation frequency (7.8±1.0 times), stool weight (1.18±0.12 g), and stool water content (62.5±4.2%) of the ZK-35-H group had no significant difference from those of the blank group (P>0.05), and were significantly higher than those of the model group (defecation frequency 4.5±0.9 times, stool weight 0.52±0.10 g, and stool water content 42.8±5.1%) (P<0.01).

[0241] Time for the first black stool to be discharged: The model group was 158.5±15.2 min, which was significantly longer than that of the blank group (65.3±8.5 min) (P<0.01). The ZK-35-H group was 72.5±9.8 min, which had no significant difference from the blank group (P>0.05) and was significantly shorter than the model group (P<0.01).

[0242] Small intestine propulsion 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), and was significantly higher than the model group (P<0.01). The above results show that the compound diphenoxylate can successfully establish a mouse constipation model, and L. paracasei ZK-35 can significantly improve the constipation symptoms and promote the intestinal transport function.

[0243] Intestinal motility-related index detection results:

[0244] Gastrointestinal hormone content: The serum MTL content of the model group (85.6±10.2 pg / mL) was significantly lower than that of the blank group (158.5±15.3 pg / mL) (P<0.01), and the VIP (125.3±15.8 pg / mL) and SS (65.8±8.5 pg / mL) contents were significantly higher than those of the blank group (VIP 68.5±8.2 pg / mL, SS 35.2±5.8 pg / mL) (P<0.01); the serum MTL content (148.2±12.5 pg / mL), VIP content (72.5±7.8 pg / mL), and SS content (38.5±6.2 pg / mL) of the ZK-35-H group had no significant difference from the blank group (P>0.05), and were significantly better than the model group (P<0.01).

[0245] Intestinal smooth muscle contraction function: The duodenal smooth muscle spontaneous contraction amplitude (0.85±0.15 g) and frequency (2.5±0.5 times / min) of the model group were significantly lower than those of the blank group (contraction amplitude 2.15±0.25 g, frequency 5.8±0.8 times / min) (P<0.01); after adding ACh, the model group had a significantly lower contraction amplitude increase (50.2±8.5%) than the blank group (125.3±15.8%) (P<0.01); the spontaneous contraction amplitude (1.98±0.22 g), frequency (5.5±0.7 times / min), and ACh-induced contraction amplitude increase (118.5±12.5%) of the ZK-35-H group had no significant difference from the blank group (P>0.05), and were significantly higher than the model group (P<0.01).

[0246] Intestinal flora and metabolite analysis results:

[0247] Intestinal flora a diversity: the Chao1 index of the model group (295.6±28.5), Shannon index (2.3±0.3) was significantly lower than that of the blank 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) of ZK-35-H group had no significant difference with the blank group (P>0.05), and were significantly higher than those of the model group (P<0.01).

[0248] The relative abundance of key flora: the relative abundance of Lactobacillus (2.1±0.5%), Bifidobacterium (1.8±0.4%), Akk (0.4±0.1%) and Faecalibacterium (0.6±0.2%) in the model group was significantly lower than that in the blank group (Lactobacillus 14.8±1.6%, Bifidobacterium 12.3±1.5%, Akk 3.5±0.5%, Faecalibacterium 5.2±0.8%) (P<0.01), and Desulfovibrio (8.5±1.2%) was significantly higher than that in the blank group (1.2±0.3%) (P<0.01); the relative abundance of the above beneficial bacteria in ZK-35-H group (Lactobacillus 13.5±1.5%, Bifidobacterium 11.8±1.4%, Akk 3.2±0.4%, Faecalibacterium 4.8±0.7%) had no significant difference with the blank group (P>0.05), and Desulfovibrio (1.8±0.4%) was significantly lower than that in the model group (P<0.01).

[0249] SCFAs content: the content of acetic acid (18.5±2.3mmol / L), propionic acid (6.2±1.1mmol / L) and butyric acid (2.5±0.5mmol / L) in the cecum content of the model group was significantly lower than that in the blank group (acetic acid 38.5±4.2mmol / L, propionic acid 13.5±1.8mmol / L, butyric acid 9.2±1.2mmol / L) (P<0.01); the content of the above SCFAs in ZK-35-H group (acetic acid 36.8±3.8mmol / L, propionic acid 12.8±1.7mmol / L, butyric acid 8.8±1.1mmol / L) had no significant difference with the blank group (P>0.05), and was significantly higher than that in the model group (P<0.01).

[0250] Intestinal barrier function detection results:

[0251] Colon histopathological score: the model group was 2.3±0.3, which was significantly higher than the blank group (0) (P<0.01), showing moderate edema of the colon mucosa, partial rupture of the villi, and moderate inflammatory cell infiltration; the ZK-35-H group had a pathological score of 0.3±0.1, which was significantly lower than the model group (P<0.01), and the structure of the colon mucosa was basically restored to normal;

[0252] Tight junction protein expression: the relative expression of occludin (0.35±0.08), ZO-1 (0.28±0.07), and claudin-1 (0.32±0.06) in the model group was significantly lower than that in the blank group (all 1.0±0.1) (P<0.01); the relative expression 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) had no significant difference with the blank group (P>0.05), and was significantly higher than 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 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 had no significant difference with the blank group (P>0.05), and was significantly higher than the model group (P<0.01).

[0254] In summary, Lactobacillus paracasei ZK-35 can effectively promote the intestinal health of constipation model mice by improving the intestinal transport function, regulating the balance of gastrointestinal hormones, enhancing the function of intestinal smooth muscle contraction, restoring the diversity and metabolism of intestinal flora, and repairing the intestinal barrier function, and the high dose (5.0×10 10 CFU / kg) has the best effect.

[0255] Example 5

[0256] Lactobacillus paracasei ZK-35 and Lactobacillus acidophilus, Bifidobacterium longum are compounded to synergistically relieve ulcerative colitis

[0257] 5.1 Test materials

[0258] Strains:

[0259] Lactobacillus paracasei ZK-35 (CGMCC NO. 35142, freeze-dried powder, viable bacteria number 1.0×10 11 CFU / g);

[0260] Lactobacillus acidophilus (Lactobacillus acidophilus, commercially available freeze-dried powder, viable bacteria number 1.0×10 11 CFU / g);

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

[0262] Preparation of compound bacterial powder: ZK-35, Lactobacillus acidophilus and Bifidobacterium longum freeze-dried powder were weighed according to the weight ratio of 2-10:1-2:1-2, preferably 4:1:1, mixed uniformly, which was the compound bacterial powder, and the total viable bacterial count was 1.0 x 10 11 - 8.0 x 10 11 CFU / g.

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

[0264] Reagents: 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma Company, USA, purity ≥98%); ethanol (analytical pure, National Pharmaceutical Group 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 Solaybao Technology Co., Ltd.).

[0265] Instruments: pathological section machine (model RM2235, Leica Company, Germany); image analysis system (model Image-Pro Plus 6.0, Media Cybernetics Company, USA); flow cytometer (model BD FACSAria III, BD Company, USA).

[0266] 5.2 Test method

[0267] Ulcerative colitis (UC) model establishment:

[0268] Except for the blank control group, the rest of the rats were used to establish the UC model by the TNBS / ethanol method: after fasting for 24h, the rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (1.5mL / kg), a soft tube with a diameter of 2mm was inserted into the colon 8cm through the anus, and TNBS / ethanol solution (TNBS concentration 70mg / mL, ethanol concentration 50%) was slowly injected at a dose of 0.25mL / 100g body weight; after injection, the tail of the rat was lifted and inverted for 30s to make the modeling agent fully contact the colon mucosa; modeling was performed 3 times at 15 days, 22 days and 29 days of the test to enhance the stability of the model.

[0269] Animal grouping and treatment:

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

[0271] Control group (Control): No modeling, daily intragastric administration of sterile normal saline 2mL / rat, for 34 consecutive days.

[0272] UC model group (Model): Modeling, daily intragastric administration of sterile normal saline 2mL / rat, for 34 consecutive days.

[0273] ZK-35 alone group (ZK-35): Modeling, daily intragastric administration of ZK-35 bacterial suspension 2mL / rat (dose 2.0×10 10 CFU / rat, equivalent to 1.0×10 11 CFU / kg), for 34 consecutive days.

[0274] Lactobacillus acidophilus alone group: Modeling, daily intragastric administration of Lactobacillus acidophilus bacterial suspension 2mL / rat (dose 2.0×10 10 CFU / rat), for 34 consecutive days.

[0275] Bifidobacterium longum alone group: Modeling, daily intragastric administration of Bifidobacterium longum bacterial suspension 2mL / rat (dose 2.0×10 10 CFU / rat), for 34 consecutive days.

[0276] ZK-35+Lactobacillus acidophilus two-compound group: Modeling, daily intragastric administration of ZK-35 and Lactobacillus acidophilus mixed bacterial suspension 2mL / rat (total dose 2.0×10 10 CFU / rat, weight ratio 4:1), for 34 consecutive days.

[0277] ZK-35+Bifidobacterium longum two-compound group: Modeling, daily intragastric administration of ZK-35 and Bifidobacterium longum mixed bacterial suspension 2mL / rat (total dose 2.0×10 10 CFU / rat, weight ratio 4:1), for 34 consecutive days.

[0278] Three-compound group (ZK-35+Lactobacillus acidophilus+Bifidobacterium longum): Modeling, daily intragastric administration of compound bacterial suspension 2mL / rat (total dose 2.0×10 10 CFU / rat, weight ratio 4:1:1), for 34 consecutive days.

[0279] All rats were raised under the same conditions as in Example 3, and the intragastric administration time was 3:00 pm daily. The intragastric administration time on the modeling day was adjusted to 2h after modeling.

[0280] Detection index and method:

[0281] Disease activity index (DAI) score:

[0282] DAI score was performed on the 1st day, 3rd day, 5th day after modeling (i.e. 16th day, 18th day, 20th day, 23rd day, 25th day, 27th day, 30th day, 32nd day, 34th day of the test), the scoring criteria combined with the percentage of body weight loss, stool character, degree of hematochezia, total score 0-4 points, the higher the score, the more severe the UC symptoms.

[0283] Percentage of body weight loss calculation: (pre-modeling body weight - current body weight) / pre-modeling body weight x 100%, body weight gain was recorded as 0 points.

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

[0285] Hematochezia score: 0 points (no blood), 1 point (positive occult blood), 2 points (small amount of visible blood stool), 3 points (large amount of visible blood stool).

[0286] DAI = (body weight loss score + stool character score + hematochezia score) / 3.

[0287] Colonic tissue morphology detection:

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

[0289] The middle section of the colon tissue (1 cm) was taken, fixed with 4% neutral formaldehyde, paraffin-embedded, sectioned (thickness 5 μm), HE stained, and the colon mucosa damage was observed under a light microscope. The histopathological scoring criteria were used to score from four dimensions of inflammation, ulcer depth, mucosal hyperplasia, and edema, with a total score of 0-12 points, the higher the score, the more severe the tissue damage.

[0290] Intestinal inflammatory factor detection:

[0291] Serum inflammatory factors: The rat eyeball was bled, the serum was separated by centrifugation, and the contents of pro-inflammatory factors (TNF-α, IL-6, IL-17) and anti-inflammatory factors (IL-10) in the serum were detected by ELISA kit.

[0292] Colonic tissue inflammatory factors: Take 0.5 g of colonic tissue, add PBS buffer (containing protease inhibitors) and homogenize, centrifuge (4°C, 12000 r / min, 15 min) to take the supernatant, and use an ELISA kit to detect the content of the above inflammatory factors.

[0293] Intestinal flora structure analysis:

[0294] Take 0.5 g of cecal contents, extract total DNA, and use 16S rRNA gene high-throughput sequencing (Illumina MiSeq platform) to analyze intestinal flora β diversity (PCoA analysis) and evaluate the differences in flora structure of each group.

[0295] Analysis of the relative abundance of key flora (Lactobacillus, Bifidobacterium, Akk, Faecalibacterium, Escherichia coli, Enterococcus).

[0296] Synergistic mechanism verification:

[0297] Intestinal colonization ability: qPCR method was used to detect the viable count of ZK-35, Lactobacillus acidophilus and Bifidobacterium longum on the surface of colonic mucosa (copies / g mucosal tissue), and 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] Metabolite complementation: HPLC-MS was used to detect the content of bacteriocin (paracasei lactobacillin), lactic acid, acetic acid and butyric acid in the colonic contents, and the synergistic effect of the metabolites of the three strains was analyzed.

[0302] Signal pathway protein expression: The colon tissue was taken, 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 of inflammation pathway) were detected by Western blot, with β-actin as the internal reference.

[0303] 5.3 Test results

[0304] DAI score results:

[0305] Blank control group: The DAI score was 0 throughout the entire test period, and there was no UC symptom.

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

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

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

[0309] Three-compound group: The DAI score on day 34 was 0.5±0.1, which was significantly lower than that of all other groups (P<0.01), and only showed mild weight loss (average decrease of 3.2±0.8%), loose stool, and no gross blood stool. The results showed that the three-strain compound had a significantly better effect on UC symptoms than the single-strain and two-compound groups, and there was a synergistic effect.

[0310] Colon tissue morphology test results:

[0311] Colon length: The colon length of the model group was 12.5±1.2 cm, which was significantly shorter than that of the blank group (18.5±0.8 cm) (P<0.01); the colon length of the three-compound group was 17.8±0.7 cm, which had no significant difference with the blank group (P>0.05), and was significantly longer than that of the single-strain groups (ZK-35 group 14.8±1.0 cm) and the two-compound groups (ZK-35+Bifidobacterium longum group 16.2±0.9 cm) (P<0.01).

[0312] Pathohistological score: The total score of model group was 9.8±1.2, showing severe transmural inflammation, a large number of ulcers (>4), significant mucosal hyperplasia and edema; the total score of three-compound group was 2.1±0.3, showing only mild mucosal inflammation and slight edema, which was significantly lower than that of single-strain group (ZK-35 group 5.8±0.8) and two-compound groups (ZK-35+ L. longus group 3.5±0.5) (P<0.01).

[0313] Intestinal inflammatory factor detection results:

[0314] Serum inflammatory factors: The contents 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 model group were significantly higher than those in 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), and the content of IL-10 (10.2±1.8 pg / mL) was significantly lower than that in blank group (35.6±4.8 pg / mL) (P<0.01); the contents of 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 32.8±4.2 pg / mL) in three-compound group had no significant difference with blank group (P>0.05), and were significantly better than those in other groups (P<0.01).

[0315] Colonic tissue inflammatory factors: The results were consistent with serum. The contents of pro-inflammatory factors in colonic tissue of three-compound group were significantly reduced, and the contents of anti-inflammatory factors were significantly increased, which were better than those in single-strain and two-compound groups.

[0316] Intestinal flora structure analysis results:

[0317] Beta diversity analysis: The flora structure of blank group and three-compound group was clustered into one branch, and the model group was clustered into one branch alone, and the single-strain group and two-compound groups were between the two, indicating that three-compound could significantly restore the intestinal flora imbalance caused by UC.

[0318] Key flora relative abundance: The relative abundance of Lactobacillus (1.8 ± 0.4%), Bifidobacterium (1.5 ± 0.3%), Akk (0.2 ± 0.1%), and Faecalibacterium (0.3 ± 0.1%) in the model group was significantly lower than that in the blank group (P < 0.01), and the relative abundance of Escherichia coli (15.8 ± 2.3%) and Enterococcus (12.5 ± 1.8%) was significantly higher than that in the blank group (P < 0.01); the relative abundance of the above-mentioned beneficial bacteria in the three-strain complex group (Lactobacillus 14.2 ± 1.6%, Bifidobacterium 11.5 ± 1.4%, Akk 3.1 ± 0.4%, and Faecalibacterium 4.5 ± 0.7%) had no significant difference from that in the blank group (P > 0.05), the relative abundance of harmful bacteria (Escherichia coli 3.8 ± 0.7% and Enterococcus 2.5 ± 0.6%) was significantly lower than that in the model group (P < 0.01), and was better than that in the single-strain group and the two-strain complex group.

[0319] Synergistic mechanism verification results:

[0320] Intestinal colonization ability: The viable cell count of ZK-35 (8.5 × 10 8 ± 1.2 × 10 8 copies / g), Lactobacillus acidophilus (2.2 × 10 8 ± 0.5 × 10 8 copies / g), and Bifidobacterium longum (2.1 × 10 8 ± 0.4 × 10 8 copies / g) on the colon mucosa surface in the three-strain complex group was significantly higher than that in the single-strain group (ZK-35 single-strain group 4.2 × 10 8 ± 0.8 × 10 8 copies / g, Lactobacillus acidophilus single-strain group 0.8 × 10 8 ± 0.2 × 10 8 copies / g, and Bifidobacterium longum single-strain group 0.7 × 10 8 ± 0.2 × 10 8 copies / g) (P < 0.01), indicating that the three-strain complex could promote intestinal colonization.

[0321] Metabolite complementation: The contents of paracasein (15.8 ± 2.3 μg / mL), lactic acid (25.3 ± 3.2 mmol / L), acetic acid (38.5 ± 4.2 mmol / L), and butyric acid (12.5 ± 1.5 mmol / L) in the colon contents in the three-strain complex group were significantly higher than those in the single-strain group and the two-strain complex group (P < 0.01), indicating that the metabolites of the three strains could be complementary and enhance the overall function.

[0322] Signal 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), and the relative expression level of NF-κB (0.32±0.07) was significantly lower than that in the single-strain group (P<0.01), indicating that the three strains can activate the TLR2, Nrf2, and TGF-β signal pathways and inhibit the NF-κB inflammatory pathway, thereby reducing intestinal inflammation.

[0323] In summary, after the Lactobacillus paracasei ZK-35, Lactobacillus acidophilus, and Bifidobacterium longum are combined at a weight ratio of 4:1:1, the three strains can significantly alleviate the symptoms of rats with ulcerative colitis, repair colon tissue damage, and regulate intestinal flora balance by synergistically enhancing intestinal colonization ability, complementary metabolites, and signal pathways, and the effect is significantly better than that of single strains and two combined groups, and has a clear synergistic effect.

[0324] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described by referring to the preferred embodiments of the present application, it should be understood by those skilled in the art that various changes can be made in form and detail without departing from the spirit and scope of the present application as defined by the appended claims.

Claims

1. A type of Lactobacillus paracasei ZK-35, characterized in that, It was deposited at the China General Microbiological Culture Collection Center on July 9, 2025, with accession number CGMCC NO.35142.

2. The use of Lactobacillus paracasei ZK-35 and / or its metabolites as described in claim 1 in alleviating antibiotic-induced intestinal problems.

3. The application according to claim 2, characterized in that, The antibiotics mentioned include amoxicillin and cefixime.

4. The application according to claim 2, characterized in that, 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.

5. The use of Lactobacillus paracasei ZK-35 and / or its metabolites as described in claim 1 in intestinal improvement.

6. A composition for relieving ulcerative colitis, characterized in that, It includes Lactobacillus paracasei ZK-35, Lactobacillus acidophilus, and Bifidobacterium longum as described in claim 1.

7. The composition according to claim 6, characterized in that, 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.

8. The use of Lactobacillus paracasei ZK-35 of claim 1 and / or its metabolites or the composition of claim 6 in the preparation of intestinal improvement products.

9. The application according to claim 8, characterized in that, The intestinal improvement products are available in various forms, including freeze-dried powder, oral liquid, tablets, capsules, granules, and drops.