Lactobacillus muci gen HC1183, a bacterial agent and application thereof

Abstract

This invention relates to a strain of *Lactobacillus fermentans* HC1183, its inoculum, and its applications, belonging to the technical field of microbiology. *Lactobacillus fermentans* ( Limosilactobacillus fermentum HC1183 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26325, on December 26, 2022. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This invention also provides the application of fermented *Lactobacillus mucinus* HC1183 in the preparation of products for regulating blood pressure, lowering blood cholesterol, and preparing antioxidant products. The fermented *Lactobacillus mucinus* HC1183 provided by this invention can effectively help reduce excessively high systolic blood pressure; the effect is more significant when used earlier.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of fermenting Lactobacillus mucinus HC1183, its inoculum, and its applications. Background Technology

[0002] Hypertension is a common disease worldwide, a clinical syndrome characterized by elevated systolic or diastolic blood pressure. It can lead to functional or organic changes in organs such as arteriosclerosis, myocardial infarction, coronary heart disease, and stroke, and is often referred to as a "silent killer." Treatment for secondary hypertension primarily targets the underlying cause, while primary hypertension can be treated with medication and adjuvant therapy using bioactive factors. Commonly used antihypertensive drugs include diuretics, angiotensin-converting enzyme (ACE) inhibitors, beta-blockers, calcium channel blockers, angiotensin II receptor blockers (ARBs), and α1-receptor blockers. Recent studies on gut metagenomics and metabolomics have revealed an imbalance in the gut microbiota of hypertensive patients, with an increase in Firmicutes and a decrease in Bacteroidetes. Transplanting fecal microbiota from hypertensive patients into germ-free mice resulted in elevated blood pressure in the mice, suggesting that gut microbiota directly affects the host's blood pressure. Therefore, using probiotics to intervene in the gut microbiota for hypertension prevention or adjuvant therapy is an ideal option.

[0003] *Lactobacillus fermentum*, a member of the *Lactobacillus* genus, possesses unique biological characteristics. It is a Gram-positive facultative or obligate anaerobic bacterium. Its cells are rod-shaped, usually arranged in pairs or short chains, non-spore-forming, flagellated, and capable of free movement in liquid culture media. On solid media, colonies are round, smooth, with regular edges, and are milky white or pale yellow in color. The optimal growth temperature for this bacterium is 30℃~40℃, and it can grow under low pH conditions. As an anaerobic or facultative anaerobic bacterium, it grows well under anaerobic or low-oxygen conditions. However, in existing published technical literature and patents, no reports have been found regarding the use of *Lactobacillus fermentum* as an adjunct therapy or preventative treatment of essential hypertension. Its application value in regulating essential hypertension remains to be further explored. Summary of the Invention

[0004] To address the issue that existing technologies lack applications of *Lactobacillus fermentatus* in regulating essential hypertension, this invention provides a strain of *Lactobacillus fermentatus* HC1183, its inoculum, and its applications to solve the aforementioned problem.

[0005] In a first aspect, the present invention provides a strain of *Lactobacillus fermentatus* HC1183, wherein the *Lactobacillus fermentatus* ( Limosilactobacillus fermentumHC1183 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26325, on December 26, 2022. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0006] Furthermore, the 16S rDNA sequence of the fermenting Lactobacillus mucinus HC1183 is shown in SEQ ID NO.3.

[0007] In a second aspect, the present invention provides a bacterial agent, wherein the bacterial agent is a bacterial suspension of Lactobacillus fermentum HC1183, a lysate of a strain containing Lactobacillus fermentum HC1183, or a fermentation supernatant containing Lactobacillus fermentum HC1183; the lysate is obtained by ultrasonically disrupting the fermentation broth using an ultrasonic disruptor; and the fermentation supernatant is obtained by centrifuging the fermentation broth.

[0008] Furthermore, the fermentation broth is prepared by culturing Lactobacillus mucinus HC1183 in MRS liquid medium at 37°C for 24-28 hours.

[0009] Furthermore, the MRS liquid culture medium comprises the following components: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 5 g / L sodium acetate, 20 g / L glucose, 2 g / L K₂HPO₄, 1.0 mL / L Tween 80, 2.0 g / L diamine citrate, 0.02 g / L MgSO₄·7H₂O, 0.04 g / L MnSO₄·7H₂O, with pure water added to make up to 1 L, and the pH adjusted to 5.7 ± 0.2.

[0010] Thirdly, the present invention provides the application of fermented Lactobacillus mucinus HC1183 in the preparation of products that regulate blood pressure levels.

[0011] Fourthly, the present invention provides the application of fermented Lactobacillus mucinus HC1183 in the preparation of products that lower blood cholesterol.

[0012] Fifthly, the present invention provides an application of fermented Lactobacillus mucinus HC1183 in the preparation of antioxidant products.

[0013] The beneficial effects of this invention are as follows: The *Lactobacillus fermentum* HC1183 provided by this invention can effectively help reduce excessively high systolic blood pressure, with more significant effects when used earlier. Regarding diastolic blood pressure reduction, the combination of perindopril and *Lactobacillus fermentum* HC1183 is more effective than perindopril alone, and can also restore diastolic blood pressure to a healthy level. Furthermore, *Lactobacillus fermentum* HC1183 can reduce serum FITC-D, DAO, and D-lactate levels, decrease intestinal mucosal barrier damage and intestinal wall permeability, and improve intestinal wall barrier integrity, helping to reduce the risk of hypertension caused by elevated levels of endotoxins such as LPS, thus achieving a blood pressure-lowering effect. In addition, *Lactobacillus fermentum* HC1183 can alleviate vascular remodeling and thus relieve symptoms of essential hypertension in rats by reducing the expression levels of Ang II and ET-1 proteins, as well as the expression levels of ET-1, CoI-1, and α-SMA genes. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a colony morphology diagram of *Lactobacillus mucinus* HC1183 fermented in Example 1 of this invention.

[0016] Figure 2 This is a microscopic image of Lactobacillus HC1183 fermenting in Example 1 of the present invention.

[0017] Figure 3 This is the RAPD fingerprint of Lactobacillus HC1183 fermented in Example 1 of the present invention.

[0018] Figure 4 This is the rep-PCR fingerprint of Lactobacillus HC1183 fermented in Example 1 of this invention.

[0019] Figure 5 This is a comparison chart of the results of improving blood pressure levels in rats by fermenting Lactobacillus mucinus HC1183 in Example 8 of the present invention; (a) in the figure is a comparison chart of systolic blood pressure results, and (b) is a comparison chart of diastolic blood pressure results.

[0020] Figure 6 This is a comparison chart of the results of improving the intestinal barrier function of rats by fermenting Lactobacillus mucin HC1183 in Example 8 of the present invention; (a) is a comparison chart of the results of FITC-D, and (b) is a comparison chart of the results of endotoxin LPS.

[0021] Figure 7This is a comparison chart of the results of improving serum DAO and D-lactic acid levels in rats by fermenting Lactobacillus mucinus HC1183 in Example 8 of the present invention; (a) in the figure is a comparison chart of DAO level results; (b) is a comparison chart of D-lactic acid level results.

[0022] Figure 8 This is a comparison chart of the results of reducing vascular remodeling-related proteins in rats by fermenting Lactobacillus mucinus HC1183 in Example 8 of the present invention; (a) in the figure is a comparison chart of the results of Ang II protein expression level; (b) is a comparison chart of the results of ET-1 protein expression level.

[0023] Figure 9 This is a comparison chart of the results of reducing rat vascular remodeling-related genes by fermenting Lactobacillus mucinus HC1183 in Example 8 of the present invention; (a) is a comparison chart of the relative expression level of ET-1 mRNA; (b) is a comparison chart of the relative expression level of Col-1 mRNA; (c) is a comparison chart of the relative expression level of α-SMA mRNA. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0025] Example 1 Isolation, screening and identification of Lactobacillus fermentans HC1183 1. Sampling: Fermented pickled cowpeas were purchased from a vegetable market in Weihai City, Shandong Province on September 18, 2022.

[0026] 2. Isolation: Weigh out MRS solid culture medium (Man Rogosa Sharpe, Haibo) ® 66.2 g of the sample was added to 1L of pure water and stirred evenly. The mixture was then autoclaved at 121 degrees Celsius for 15 minutes. After cooling, the sample was poured into sterile plates to prepare MRS solid culture medium.

[0027] Weigh 50g of the fermented green beans and place them in a sampling bag containing 1L of sterile physiological saline. Repeat the tapping motion for 5 minutes. Spread 50 μL of the sample solution onto MRS solid medium and incubate anaerobically at 37℃ for 48 hours. After single colonies grow on the plate, pick a single colony and continue streaking. After three purification cultures, pick single colonies for rapid MALDI-TOF-MS identification.

[0028] MALDI-TOF-MS rapid identification of bacterial strains was performed according to the kit instructions, following these steps: Single clones of the bacterial strain were uniformly coated onto a target plate in the form of a thin film. 1 μL of lysis buffer was added to cover the sample, which was then dried. Another 1 μL of matrix solution was added to cover the sample, and the plate was dried again before placing the sample target into the mass spectrometer for identification. The co-crystallized film formed by the sample and matrix was irradiated with a laser, causing the proteins in the sample to ionize. The ions were accelerated through the flight tube under an electric field of 10–20 kV, and the molecular weight of the proteins was determined based on their flight time to the detector. Ribosomal protein fingerprints were obtained using Autofms 1000 Analyzer v1.0 software. Based on the MALDI-TOF-MS identification results, a total of 38 strains of Lactobacillus were screened from the samples.

[0029] 3. Screening 3.1 Preparation of reagents Tris-HC1 buffer: Accurately weigh 0.9105g Tris and 1.7550g NaCl and dissolve them in 85mL of sterile triple-distilled water. Adjust the pH to 7.5 with 1M hydrochloric acid, and then bring the volume to 100mL with triple-distilled water.

[0030] ACE (Angiotensin-converting enzyme): Dissolve 1 U of ACE in 4 ml of sterile triple-distilled water. The activity is 0.25 U / m. After aliquoting, store in a -20°C refrigerator for later use.

[0031] 0.88mM FAPGG (N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine): Accurately weigh 0.0035g of FAPGG, dissolve it in a 300mM NaCl-75mM Tris-HCl buffer solution at pH 7.5, bring the volume to 10mL, and store at 4°C protected from light.

[0032] 3.1 Preparation of test strain samples After the test strain was purified for 3 generations, it was cultured in MRS liquid medium at 37°C for 24 hours. The fermentation broth and bacterial cells were then collected and ultrasonically disrupted for 20 minutes to obtain the fermentation lysate of the test strain for later use.

[0033] 3.2 ACE Inhibition Rate Measurement The lysate samples of the test strains were precisely added to 96-well plates according to the sample loading amounts shown in Table 1. The plates were shaken for 30 seconds using a microplate reader, and the initial absorbance of each sample well was immediately measured at 340 nm, designated as a1, b1, c1, and d1. After incubation at 37℃ for 15 min, the absorbance at 340 nm was measured again, designated as a2, b2, c2, and d2. The decrease in absorbance for each sample well was A = (a1 - a2), B = (b1 - b2), C = (c1 - c2), and D = (d1 - d2), respectively. The ACE inhibition rate of the samples was calculated using the following formula: .

[0034] Table 1 - Reagent dosages for ACE inhibitory activity assay

[0035] After testing, 38 strains of Lactobacillus were screened, with ACE inhibition rates ranging from 8.53% to 89.77%. Among them, 15 strains had ACE inhibition rates below 10.00%, 20 strains had ACE inhibition rates between 10.00% and 80.00%, and 3 strains had ACE inhibition rates exceeding 80.00%, with strain HC1183 showing the highest inhibition rate at 89.77%. Therefore, strain HC1183, with the highest ACE inhibition rate, was selected for further identification and research.

[0036] 4. Identification (1) Identification of colony morphology Strain strain HC1183 was inoculated onto MRS solid medium and incubated at 37°C for 24 h. Single colonies of HC1183 were observed to be yellowish-white, with a size of 2.0-3.0 mm. The colony edges were neat, and the surface was raised and glossy. (See colony photograph for example.) Figure 1 As shown in the image. Under an optical microscope, strain HC1183 exhibits short rod-shaped cells at both ends, with relatively short cells of roughly uniform size, arranged in clusters or chains. The image under the optical microscope is shown below. Figure 2 As shown.

[0037] (2) Identification of physiological and biochemical characteristics The inoculum for strain HC1183 was prepared as follows: Activated strain HC1183 was inoculated into MRS liquid medium at a 2% inoculum size and cultured at 37°C for 24 hours until the viable cell count reached 10⁻⁶. 9 CFU / mL was used to obtain the inoculum for strain HC1183.

[0038] (2.1) Temperature growth range experiment The HC1183 strain inoculum was inoculated into 10 mL of MRS liquid medium at a 10% inoculation rate. 10 mL of MRS liquid medium without inoculation was used as a control. The medium was then placed in constant temperature incubators at 0℃, 15℃, 30℃, 37℃, 45℃ and 60℃ for 48 h for anaerobic incubation. The turbidity of the culture medium was then observed.

[0039] The results showed that after culturing strain HC1183 in constant temperature incubators at 0℃, 15℃, 45℃, and 60℃ for 48 hours, the culture medium remained clear; it became slightly turbid in a constant temperature incubator at 30℃; and a large number of bacterial cells were produced after 48 hours of constant temperature incubation at 37℃, with the highest turbidity observed in the culture medium. Therefore, the optimal growth temperature for strain HC1183 is 37℃.

[0040] (2.2) Salinity tolerance test Under aseptic conditions, the activated bacterial culture of strain HC1183 was inoculated at a rate of 10% into 5 mL of MRS liquid medium with NaCl concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%, respectively. 5 mL of MRS liquid medium without bacterial inoculation was used as a control. The medium was incubated at 37°C with shaking for 48 h, and the medium was observed to see if it became turbid.

[0041] The results showed that strain HC1183 grew in culture medium with a salt concentration of 1% to 2%, but did not grow in culture medium with a salt concentration of more than 3%. The highest salt concentration that strain HC1183 could tolerate was 2%.

[0042] (2.3) Hydrogen peroxide concentration tolerance test Hydrogen peroxide was diluted with double-distilled water to prepare solutions of different concentrations. 9 mL of MRS liquid medium was taken, and 1 mL of the diluted hydrogen peroxide solution was added to the medium to achieve final concentrations of 0.4 M, 0.7 M, and 1.0 M. The HC1183 inoculum was inoculated at a rate of 2% into MRS liquid medium containing different concentrations of hydrogen peroxide solution as the experimental group, with each concentration replicated three times. The HC1183 strain was inoculated into MRS liquid medium without hydrogen peroxide solution as the control group. After incubation at 37°C overnight, the viable count of HC1183 was determined using the spread plating method. The hydrogen peroxide tolerance of HC1183 was calculated using the following formula: .

[0043] In the formula: N0 and N1 are the number of surviving colonies (CFU / mL) in the control group and experimental group, respectively. The hydrogen peroxide tolerance results of strain HC1183 are shown in Table 2.

[0044] Table 2 - Results of hydrogen peroxide tolerance test for strain HC1183

[0045] As shown in Table 2, the tolerance of strain HC1183 to hydrogen peroxide gradually decreased with increasing hydrogen peroxide concentration. Furthermore, strain HC1183 could still grow in MRS liquid medium containing a 1M hydrogen peroxide solution.

[0046] (2.4) Antibiotic resistance and hemolytic test (2.4.1) Antibiotic tolerance test The minimum inhibitory concentration (MIC) of antibiotics against strain HC1183 was determined using the microbroth dilution method. The antibiotics were prepared as follows: ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, and tetracycline were all prepared as stock solutions of 2048 μg / mL and stored at -20℃ for later use. Before use, the stock solutions were serially diluted 2-fold with MRS liquid medium to obtain the working solutions, with concentrations ranging from 1 to 1024 μg / mL.

[0047] Add 190 μL of antibiotic-free MRS liquid medium to each of the eight wells in the first column of a 96-well plate as a negative control. Add 190 μL of MRS liquid medium with serially diluted antibiotic concentrations ranging from 1 to 1024 μg / mL to each of the eleven wells in columns 2-12. Then, inoculate each well with 10 μL of HC1183 inoculum. Each experiment was repeated four times. Wells without HC1183 inoculum served as blank controls. Add 50 μL of autoclaved paraffin oil to each well to prevent moisture evaporation during culture. Incubate the 96-well plate at 37°C and measure OD every 5 minutes. 600 The MIC values ​​of different antibiotics for strain HC1183 were statistically analyzed. Specific results are shown in Table 3.

[0048] Table 3 - MIC values ​​of antibiotic resistance test for strain HC1183

[0049] Note: MIC unit is μg / mL; R: drug resistance; S: sensitive.

[0050] As can be seen from the results in Table 3, strain HC1183 is sensitive to common antibiotics such as erythromycin, streptomycin, ampicillin, tetracycline and clindamycin, and has good biosafety.

[0051] (2.4.2) Hemolytic test Prepare TBS basal medium comprising the following components: 1 L distilled water, 17.0 g tryptone, 5.0 g NaCl, 3.0 g soybean peptone, 2.5 g KH₂PO₄, and 2.5 g glucose. After dissolving, autoclave at 121°C for 15 min. When the TBS medium cools to 50°C, add 5% sterile defibrinated sheep blood, mix well, and pour into plates. Streak strain HC1183 onto the prepared blood cell plates and incubate at 37°C for 24–48 h. Observe whether hemolysis occurs in strain HC1183.

[0052] The results showed that strain HC1183 could not grow and there were no changes on the blood cell plate, indicating that strain HC1183 does not produce hemolysin and cannot lyse blood cells, thus exhibiting good biosafety.

[0053] (3) Carbon source metabolism experiment Prepare the phenol red basal medium, comprising the following components: 1.5 g peptone, 0.6 g yeast extract, 0.1 g Tween 80, 0.5 mL salt solution, 18 mg phenol red, 100 mL distilled water, pH = 7.4. The salt solution composition is as follows: 11.5 g MgSO4·7H2O, 8 g MnSO4·4H2O, 100 mL distilled water.

[0054] Prepare a 10 g / mL solution of sugars, alcohols, and glycosides, and filter it through a 0.22 μm sterile filter.

[0055] Under aseptic conditions, 20 μL of sterilized carbohydrate solution was added to each well of a 96-well plate. Four parallel experiments were performed for each carbohydrate. Then, 170 μL of sterilized phenol red basal medium was added, followed by 10 μL of *Lactobacillus fermentum* HC1183 inoculum. Wells without HC1183 inoculum served as controls. 50 μL of liquid paraffin was added to each well to prevent moisture evaporation during culture. After incubation at 37°C for 2 days, the color change of the medium was observed using phenol red as an indicator; a yellow color indicated a positive result. The experimental results were compared with those in the *Physiological Characteristics Table - *Lactobacillus** to determine the genus of the tested bacteria. Detailed results are shown in Table 4.

[0056] Table 4 - Results of carbon source metabolism experiments for strain HC1183

[0057] Note: "+" indicates a positive result; "-" indicates a negative result.

[0058] Carbon source metabolism experiments showed that strain HC1183 can utilize sucrose, galactose, lactose, maltose, mannose, sodium gluconate, and ribose, which is consistent with the carbon source metabolism pattern of fermenting Lactobacillus mucilaginosus.

[0059] (4) Glucose acid and gas production test The culture medium used in the glucose acid production and gas generation experiment consisted of the following components: 0.5 g peptone, 0.3 g yeast extract, 0.1 mL Tween 80, 0.5 mL salt solution A, 0.5 mL salt solution B, 0.5 g sodium acetate, 2.5 g glucose, 0.05 mL 2% bromocresol green (w / v), and 100 mL distilled water; pH = 6.8~7.0. The prepared culture medium was dispensed into large test tubes containing inverted small test tubes, 3 mL / tube, and autoclaved at 121°C for 15 min.

[0060] Salt solution A comprises the following components: 10g KH2PO4 and 1.0g K2HPO4, dissolved in distilled water and brought to a final volume of 100mL.

[0061] Salt solution B comprises the following components: 11.5g MgSO4·7H2O, 2.4g MnSO4·2H2O, and 0.68g FeSO4·7H2O, dissolved in distilled water and brought to a final volume of 100mL.

[0062] Under aseptic conditions, the inoculum of strain HC1183 was inoculated into the above culture medium at an inoculation rate of 2%. The culture medium without inoculation of the strain was used as a control. The top was then sealed with 2 mL of sterile liquid paraffin and incubated at 37°C for 48 h. The color of the culture medium was then observed.

[0063] The results showed that after 48 hours of culture, the culture medium changed from green to yellow, and no gas was produced in the small inverted tube, indicating that strain HC1183 produced acid but not gas during glucose fermentation.

[0064] (5) Molecular biological identification A single colony of strain HC1183 was picked from the plate and placed in MRS liquid medium. After incubation at 37°C for 24 hours, 500 μL of the fermentation broth was taken and processed according to TIANGEN. ® The bacterial genomic DNA extraction kit (DP302) was used to obtain the genome of the strain for subsequent molecular biological identification.

[0065] (5.1) Identification of 16S rDNA gene sequence Using TIANGEN ® The 2×Taq PCR premix kit was used to amplify the 16S rDNA gene of Lactobacillus fermentans HC1183. The reaction system and reaction cycle settings were performed according to the kit instructions.

[0066] The upstream primer is 27F: AGAGTTTGATCCTGGCTCA; The downstream primer is 1492R: GGTTACCTTGTTACGACTT.

[0067] Electrophoresis confirmed that the PCR amplification product size was approximately 1500 bp, which met the requirements. 16S rDNA sequencing results showed that the 16S rDNA sequence of strain HC1183 is as shown in SEQ ID NO.3: The sequence was compared with BALST on the EzBioCloud website and it matched that of *Lactobacillus fermentum* (…). Limosilactobacillus fermentum The strain showed the highest similarity to *Lactobacillus fermentatus*. Therefore, strain HC1183 was identified as *Lactobacillus fermentatus*. Limosilactobacillus fermentum Lactobacillus fermentum HC1183 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26325, on December 26, 2022. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0068] (5.2) RAPD fingerprint identification Using TIANGEN ® The 2×Taq PCR premix kit was used to amplify strain HC1183. The reaction system and reaction cycle settings were performed according to the kit instructions. The M13 primer sequence was: 5'-GAGGGTGGCGGTTCT-3'.

[0069] A 1.5% agarose gel plate was prepared, with a DL2000 DNA Marker used as a result control. Electrophoresis was performed at a constant voltage of 100V for 80 minutes. Finally, the electrophoresis pattern was detected using a gel imaging system. The RAPD fingerprint of strain HC1183 is shown below. Figure 3 As shown. A comparison revealed no similarity in existing publicly available reports. Figure 3 The matching RAPD fingerprint pattern indicates that Lactobacillus fermentans HC1183 is a novel Lactobacillus fermentans strain.

[0070] (5.3) Rep-PCR fingerprint identification Using TIANGEN ® The 2×Taq PCR premix kit was used to amplify strain HC1183. The reaction system and reaction cycle settings were performed according to the kit instructions. The rep-PCR primer was 5'-GTGGTGGTGGTGGTG-3'.

[0071] Prepare 1.5% agarose gel plates, using a DL2000 DNA Marker as a result control. Electrophoresis was performed at 100V for 80 minutes to detect the amplification results. The rep-PCR fingerprint of *Lactobacillus mucilaginosus* HC1183 is shown below. Figure 4 As shown. A comparison revealed no similarity in existing publicly available reports. Figure 4 The matching rep-PCR fingerprint pattern indicates that the selected *Lactobacillus fermentatus* HC1183 is a new strain of *Lactobacillus fermentatus*.

[0072] Based on the combined results of MALDI-TOF-MS identification, molecular biology experiments, and carbon source metabolism experiments, it can be concluded that Lactobacillus fermentatus HC1183 is different from all currently reported Lactobacillus fermentatus strains and is a new strain of Lactobacillus fermentatus.

[0073] Example 2 Preparation of a suspension of *Lactobacillus mucinus* HC1183 fermentation bacteria: Activated Lactobacillus fermentum HC1183 colonies were inoculated into freshly prepared MRS liquid medium and cultured at 37°C with shaking for 24 hours. Then, 1% of the inoculum was transferred to MRS liquid medium and cultured at 37°C with shaking for another 24 hours. Finally, the culture was cultured at 6000× g Centrifuge for 10 min, collect the bacterial cells, wash twice with PBS, and resuspend the bacterial cells in MRS liquid medium to obtain a suspension of fermenting Lactobacillus mucilaginosus HC1183.

[0074] Example 3 Fermentation of Lactobacillus mucinus HC1183 simulated digestive tract tolerance test Preparation of artificial gastric juice: Add 5g peptone, 2.5g yeast extract, 1g glucose, and 2g NaCl to 1L of distilled water in sequence. After thorough mixing, adjust the pH of the system to 3.0 and sterilize at 115℃ for 20min. After sterilization, wait for the solution to cool to a suitable temperature, and add 3.2g of porcine mucosal pepsin powder before the experiment. Gently shake the container to mix the powder thoroughly.

[0075] Preparation of artificial intestinal fluid: Add 5g peptone, 2.5g yeast extract, 1g glucose, 6.8g potassium dihydrogen phosphate, and 3.0g bile salts sequentially to 1L distilled water. After stirring and dissolving, add 77mL NaOH solution (0.2M) to adjust the pH to 6.8±0.1, then sterilize at 115℃ for 20min. Before the experiment, add 1.0g trypsin powder to the cooled sterilized solution and gently shake to mix.

[0076] The absorbance OD of the *Lactobacillus fermentans* HC1183 suspension prepared in Example 2 was measured. 600 Adjust nm to 1.5. Take 1 mL of the above bacterial suspension and incubate at 4℃ for 6000× g Centrifuge for 10 min, discard the supernatant, and resuspend the bacterial culture in 1 mL of simulated gastric fluid. Incubate in an anaerobic environment at 37°C for 3 h, and perform plate counts on the samples at the beginning (0 h) and end (3 h) of the incubation. Subsequently, the bacterial culture in the simulated gastric fluid after 3 h of incubation is again incubated at 4°C at 6000 × 10⁻⁶. g Centrifuge for 10 min, discard the supernatant, and resuspend the bacterial cells in an equal volume of simulated intestinal fluid. Continue incubation at 37°C for 2 h, followed by plate colony counting. Viability is calculated using the following formula: .

[0077] In the formula: N0 and N1 are the number of surviving colonies (CFU / mL) of the strain before and after treatment with simulated gastrointestinal fluid, respectively. The results are shown in Table 5: Table 5 - Results of tolerance test of Lactobacillus fermentum HC1183 to simulated gastrointestinal fluid

[0078] Note: The unit for viable cell count is (Log). 10 (CFU / mL).

[0079] As shown in Table 5, *Lactobacillus fermentum* HC1183 exhibits high tolerance to simulated gastric and intestinal fluids. After digestion with simulated gastric fluid, the viable bacterial survival rate was 99.03% ± 0.19%. This indicates that *Lactobacillus fermentum* HC1183 can withstand the harsh acidic environment of the stomach in the human body with minimal loss of viable bacteria, successfully entering the small intestine. After further digestion with simulated intestinal fluid, the viable bacterial survival rate of *Lactobacillus fermentum* HC1183 remained at 98.15% ± 1.42%. This demonstrates that *Lactobacillus fermentum* HC1183 can not only tolerate the harsh acidic environment of the stomach but also the bile salt environment of the intestines, facilitating its survival to reach the gastrointestinal tract and thus exert its probiotic function.

[0080] Example 4 Hydrophobic cell surface assay of Lactobacillus fermentum HC1183 Take the *Lactobacillus mucinus* HC1183 bacterial suspension prepared in Example 2, 6000× g Centrifuge for 10 min, collect the bacterial cells, rinse twice with sterile physiological saline, and then resuspend the bacterial cells in 1 mL of sterile KNO3 (0.1 M) solution as the test bacterial solution.

[0081] Add 50 μL of the above bacterial suspension to 2450 μL of KNO3 (0.1 M) and record the OD. 600 For A0, mix 1.5 mL of bacterial suspension with 500 μL of xylene and let stand at room temperature for 10 min. Vortex the two-phase system for 2 min and then let it stand for 20 min to reform the aqueous and organic phases. Measure the absorbance A1 of the aqueous phase at 600 nm. Calculate the cell hydrophobicity using the following formula, and take the average of three measurements.

[0082] .

[0083] The surface hydrophobicity of *Lactobacillus fermentatus* HC1183 cells provided by this invention is 72.17% ± 5.55%. Hydrophobicity and adhesiveness of lactic acid bacteria surfaces are positively correlated; higher hydrophobicity indicates stronger adhesiveness. *Lactobacillus fermentatus* HC1183 has significant adhesive potential, which facilitates its adhesion to intestinal epithelial cells, thereby colonizing the intestinal environment and exerting its probiotic properties.

[0084] Example 5 Adhesion of fermenting Lactobacillus mucinus HC1183 to intestinal epithelial cells Intestinal epithelial Caco-2 cells were revived, passaged, and cultured until the number of cells increased to the required amount. Subsequent experiments were conducted when the cells were observed to have reached approximately 80% confluence under an inverted microscope.

[0085] Discard the original culture medium in the cell culture flask and rinse twice with PBS solution. Add an appropriate amount of trypsin to digest the cells. After adding trypsin, return the cells to the CO2 incubator. Once the cells are observed to have completely detached visually, add 2-3 times the volume of culture medium containing trypsin to stop the digestion. Repeat the pipetting and resuspension process about 10 times. Under a microscope, observe the cells to ensure they are as single-celled as possible. Transfer the single-cell suspension to a 15mL centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, gently tap to disperse the cell pellet, and resuspend the cells with an appropriate amount of fresh culture medium. Count the cells using a hemocytometer. Dilute the cell suspension with PBS solution as needed. In a 6-well plate, the number of cells in each well should be 2 × 10-1. 6 Add 2 mL of culture medium to each well. After placing the 6-well plate in a CO2 incubator for 24 hours, subsequent cell adhesion experiments can be performed.

[0086] The *Lactobacillus fermentans* HC1183 bacterial suspension prepared in Example 2 was resuspended to 5 × 10⁻⁶ using MRS liquid medium. 7 CFU / mL was prepared for use. The Caco-2 monolayer that had adhered to the wells of a 6-well plate was washed twice with PBS, and 1 mL of antibiotic-free cell culture medium and 1 mL of the above 5 × 10⁻⁶ CFU / mL solution were added. 7 CFU / mL bacterial suspension was incubated in a CO2 incubator for 2 hours.

[0087] After culturing, Caco-2 cells were washed three times with PBS solution to remove unadhered bacteria. PBS solution was added slowly along the cell wall to avoid washing away the cell layer. 500 μL of trypsin was added for 3 min of digestion, followed by 1.5 mL of cell culture medium to terminate the digestion. The cells were repeatedly pipetted and the resulting solution was collected into sterile EP tubes. The collected solution was serially diluted 10-fold, 100-fold, 1000-fold, and 10000-fold, and plate counts were performed to calculate the number of adhered bacteria. The adhesion ability of the tested strain was calculated using the following formula.

[0088] .

[0089] The adhesion ability of fermenting Lactobacillus mucinus HC1183 to intestinal epithelial cells Caco-2 was 82.18±7.36 (CFU / cell), indicating that fermenting Lactobacillus mucinus HC1183 has excellent adhesion performance to intestinal epithelial cells Caco-2, which is conducive to its colonization in the intestinal environment and exerting its probiotic properties.

[0090] Example 6 In vitro cholesterol degradation assay of Lactobacillus mucilaginosus HC1183 Weigh 1g of cholesterol, dissolve it in anhydrous ethanol and bring the volume to 100mL. Filter the solution under sterile conditions using a 0.22µm microporous membrane to obtain a cholesterol solution.

[0091] Weigh out 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 1.0mL of Tween 80, 5.0g of CH3COONa, 0.1g of MgSO4, 0.05g of MnSO4, 2.0g of K2HPO4, and 1000mL of distilled water. After dissolving, adjust the pH to 7.3 and sterilize at 115℃ for 30min. After the culture medium cools, add cholesterol solution to make the final cholesterol concentration 0.1% to obtain a cholesterol-containing liquid culture medium.

[0092] The *Lactobacillus fermentans* HC1183 bacterial suspension prepared in Example 2 was used at 0.1% ( v / v The inoculum was inoculated into a cholesterol-containing liquid culture medium and cultured statically at 37°C for 48 hours. Then, 0.2 mL of bacterial culture was added to 1.8 mL of anhydrous ethanol, mixed well, and allowed to stand for 10 minutes. After centrifugation at 3000 rpm for 5 minutes, the supernatant was collected and the cholesterol content was determined according to the method specified in GB 5009.128-2016 "Determination of Cholesterol in Food". The cholesterol degradation rate was then calculated.

[0093] Cholesterol is an essential nutrient for the human body, but excessive cholesterol in the blood can easily induce cardiovascular and cerebrovascular diseases. The cholesterol degradation rate of *Lactobacillus fermentum* HC1183 was 82.85% ± 6.24%, indicating that this strain helps lower blood cholesterol levels and promote cardiovascular and cerebrovascular health.

[0094] Example 7 Antioxidant function assay of Lactobacillus mucinus HC1183 The culture of *Lactobacillus fermentans* HC1183 and the preparation of fermentation supernatant and lysis buffer are as follows: *Lactobacillus fermentans* HC1183, activated for 3 generations, was cultured in MRS liquid medium at 37℃ for 24 h, followed by centrifugation at 6000 rpm for 10 min. The supernatant was collected as the fermentation supernatant. After culturing *Lactobacillus fermentans* HC1183 in MRS liquid medium at 37℃ for 24 h, the fermentation broth, along with the bacterial cells, was collected and ultrasonically disrupted for 20 min to obtain the lysis buffer of *Lactobacillus fermentans* HC1183.

[0095] 1. Determination of the HRS ability of fermenting *Lactobacillus mucilaginosus* HC1183 to scavenge DPPH and hydroxyl radicals. 1.1 Determination of the ability of fermented Lactobacillus mucilaginosus HC1183 to scavenge DPPH free radicals Take 1 mL of the fermentation supernatant and lysis buffer of *Lactobacillus mucinus* HC1183 to be tested, add 1 mL of 0.4 mM freshly prepared DPPH free radical solution to each, mix well, and then incubate at room temperature in the dark for 30 min. Then, measure the OD value of the fermentation supernatant or lysis buffer sample. 517nm Absorbance A at point 样本 The measurements were performed in triplicate. The control group samples were prepared with an equal volume of PBS solution and a DPPH-ethanol mixture, and their OD values ​​were measured. 517nm Absorbance A at point 对照 Using an equal volume of supernatant or lysis buffer mixed with ethanol as a blank control, the OD value was measured. 517nm Absorbance A at point 空白 The clearance rate is calculated using the following formula: .

[0096] The DPPH removal rates of the fermentation supernatant and lysis buffer of *Lactobacillus myxoides* HC1183 were 75.48% ± 3.15% and 92.36% ± 4.20%, respectively. This indicates that both the fermentation supernatant and lysis buffer of *Lactobacillus myxoides* HC1183 have a strong ability to remove DPPH.

[0097] 1.2 Measurement of HRS Capability Mix 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate solution, 500 μL of deionized water, and 200 μL of fermentation supernatant or lysis buffer of *Lactobacillus hygroscopicus* HC1183. Add 100 μL of 3 mM hydrogen peroxide solution. Incubate at 37°C for 15 min, then measure the absorbance (A) of the sample at 510 nm. 样品 The HRS clearance rate is calculated using the following formula: .

[0098] Among them: A 控制A was used as a substitute for deionized water in the sample. 空白 Deionized water was used to replace the sample and H2O2.

[0099] The scavenging rates of HRS by the fermentation supernatant and lysis buffer of *Lactobacillus myxoides* HC1183 were 50.15% ± 6.37% and 72.11% ± 3.63%, respectively. This indicates that both the fermentation supernatant and lysis buffer of *Lactobacillus myxoides* HC1183 have a strong scavenging ability against HRS.

[0100] 2. Determination of the strain's resistance to lipid peroxidation The linoleic acid emulsion consisted of 0.1 mL linoleic acid, 0.2 mL Tween 20, and 19.7 mL deionized water. 1 mL of the linoleic acid emulsion and 1 mL of FeSO4 (1%) were added to 0.5 mL of PBS solution, followed by 0.5 mL of the fermentation supernatant or lysis buffer of the test strain. The mixture was incubated at 37°C for 1.5 h. Then, 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) were added to the mixture. The mixture was incubated at 100°C for 30 min, rapidly cooled, and centrifuged at 4000 rpm for 15 min. The supernatant was collected and analyzed at OD00. 532nm The absorbance measured is A; the control group, represented by 0.5 mL of distilled water instead of the sample, is A0. The inhibition rate is calculated using the following formula: .

[0101] The anti-lipid peroxidation rates of the fermentation supernatant and lysate of *Lactobacillus myxoides* HC1183 provided by this invention are 75.58%±6.02% and 85.00%±4.61%, respectively. This indicates that both the fermentation supernatant and lysate of *Lactobacillus myxoides* HC1183 have strong anti-lipid peroxidation capabilities.

[0102] Lipid peroxidation involves the oxidation of biological membranes by reactive oxygen species (ROS). ROS react with macromolecules such as phospholipids, enzymes, membrane receptor-associated polyunsaturated fatty acid side chains, and nucleic acids in biological membranes, resulting in lipid peroxidation products such as malondialdehyde and 4-hydroxynonenoic acid. This alters cell membrane fluidity and permeability, ultimately leading to damage and changes in cell structure and function. Fermentation with *Lactobacillus mucilaginosus* HC1183 can help mitigate cell damage caused by lipid peroxidation in the body.

[0103] Example 8 Evaluation of the efficacy of fermented Lactobacillus mucinus HC1183 in improving blood pressure in SHR rats Using the *Lactobacillus fermentans* HC1183 bacterial suspension prepared in Example 2, the viable count in the *Lactobacillus fermentans* HC1183 bacterial suspension was controlled to be 1 × 10⁻⁶. 9 CFU / mL available for use.

[0104] 1. Laboratory animals and grouping Twenty-four spontaneously hypertensive rats (SHR) weighing 250-300g and six WKY rats (WistarKyoto, WKY) were selected. The experimental rats were provided by Qinglongshan Animal Breeding Center, license number SCXK(Su)2017-0001. The experimental rats were randomly divided into the following groups: (1) Normal control group: 6 WKY rats; (2) Model control group: 6 SHR rats; (3) Positive control group: 6 SHR rats; (4) HC1183 prevention group: 6 SHR rats; (5) HC118 treatment group: 6 SHR rats. The rats were housed in an SPF (Specific-pathogen-free, SPF) grade experimental animal room for 1 week of acclimatization. The room temperature was maintained at 22±2℃ and the humidity was controlled at 40%~50%. The light and dark were alternated every 12 hours. A standard diet was provided, and the mice were allowed to eat and drink freely. The experiment was started after 1 week of acclimatization. The specific experimental protocol is as follows: Positive group: Starting from day 1 after the end of the acclimatization period, the rats in the positive group were intraperitoneally injected with the antihypertensive drug perindopril at a standard dose of 0.4 mg / kg / day until the end of the experiment in week 6.

[0105] HC1183 Prevention Group: Starting from the acclimatization period, rats in the HC1183 prevention group were administered a single-dose fermented Lactobacillus mucinus HC1183 bacterial suspension via gavage at a standard rate of 0.2 mL / 10 g / day. After the acclimatization period ended, starting from day 1, rats in the HC1183 prevention group were intraperitoneally injected with the antihypertensive drug perindopril at a standard rate of 0.4 mg / kg / day until the end of the experiment in week 6.

[0106] HC1183 treatment group: Starting from day 1 after the end of the acclimatization period, rats in the HC1183 treatment group were intraperitoneally injected with the antihypertensive drug perindopril at a standard dose of 0.4 mg / kg / day, and simultaneously administered a suspension of fermented Lactobacillus mucinus HC1183 by gavage at a standard dose of 0.2 mL / 10 g / day, until the end of the experiment in week 6.

[0107] Normal group and model group: Rats were given the same amount of physiological saline. Rats were weighed every 3 days, and the dosage of medication and the amount of bacterial solution administered by gavage were adjusted in a timely manner.

[0108] 2. Detection indicators 2.1 Measurement of rat tail artery blood pressure Blood pressure in rats was measured using the tail artery method after drug administration. First, the tail artery measuring device was preheated for 30 minutes, and simultaneously heated by an electric fan for 20 minutes to dilate the tail artery. The rats were then placed in a cylindrical cage with their tails exposed. A pressure occlusion sleeve was gently placed on the proximal end of the rat's tail, ensuring the pulse sensor was in close contact with the tail artery. After the rats were allowed to rest for 2 minutes until the pulse signal stabilized, measurements were taken. Systolic and diastolic blood pressure values ​​were determined at the moment the pulse was blocked and at the moment the pulse just began to appear. Each rat was measured three times, and the average blood pressure value was recorded.

[0109] 2.2 Detection of rat colonic mucosal permeability After the 6-week intervention experiment, the rats were fasted for 12 hours on the day of treatment and then treated with luciferin isothiocyanate (Sigma). ® FITC-D (MW:4000) labeled with 5 mg FITC-D / 100 g was administered to rats via gavage. Four hours after gavage, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 mL / 100 g). 5 mL of blood was collected from the portal vein using a vacuum blood collection tube containing heparin sodium and EDTA. The blood was centrifuged at 3500 rpm for 15 min at 4°C, and the serum was separated. The supernatant was then stored at -20°C. The serum was analyzed using a multi-functional microplate reader (Thermo Fisher Scientific). ® FITC-D was measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

[0110] The azo matrix chromogenic method was used with the chromogenic matrix Limulus amebocyte lysate (LAL) kit (Boersen). ® The instructions specify the determination of plasma lipopolysaccharide (LPS) content.

[0111] 2.3 Measurement of plasma-related protein indicators Blood sample collection was performed according to section 2.2. Serum diamine oxidase (DAO) and D-lactate were measured using enzyme-linked immunosorbent assay (ELISA), and the procedures were performed according to the instructions of the DAO ELISA kit (Jiangsu Enzyme Immunosorbent Assay Biotechnology) and the D-lactate ELISA kit (Jiangsu Enzyme Immunosorbent Assay Biotechnology). Angiotensin II (Ang-II) and endothelin-1 (ET-1) were measured using ELISA, and the procedures were performed according to the instructions of the Ang-II ELISA kit (Shanghai Ailesa Biotechnology) and the ET-1 ELISA kit (Shanghai Ailesa Biotechnology).

[0112] 2.4 Detection of mRNA expression of vascular remodeling-related proteins Collect the thoracic aorta, locate the rat thoracic aorta, cut it off and place it in pre-cooled KH solution (KH solution formula: NaCl 119mM, MgCl2 1mM, KCl 4.7mM, KH2PO4 1.2mM, NaHCO3 25mM, CaCl2 2.5mM, D-glucose 11.1mM, pH=7.4), and separate the blood and surrounding fat and other tissues from the lumen. Then cut it into two small segments of about 1cm, place them in sterile bottles and immerse them in liquid nitrogen for flash freezing, and store them at -80℃ for later use.

[0113] Total RNA was extracted from thoracic aortic tissue using Tiangen. ® The TRNzol Universal Total RNA Extraction Kit (DP424) was used. RNA concentration was calculated by detecting absorption peaks at 260 nm and 280 nm using UV absorbance, and RNA purity was determined by the 260 / 280 nm ratio. Tiangen [a specific brand / product name] was employed. ® The FastKing One-Step RT-PCR Kit (KR123) was used for reverse transcription and PCR of target genes ET-1, Col-1, α-SMA, and the internal reference gene GADPH. The reaction system and PCR procedure were performed according to the kit instructions. Primers are detailed in Table 6. The relative expression levels of the target genes were calculated using the ΔΔCt method. △Ct (experimental group) = Ct (target gene) - Ct (internal reference gene); △Ct (control group) = Ct (target gene) - Ct (internal reference gene); △△Ct = △Ct (experimental group) - △Ct (control group); The relative expression level of the target gene = 2^ -△△Ct .

[0114] Table 6 - Primer sequences for target and internal reference genes of vascular remodeling-related proteins

[0115] 3. Data Statistics and Analysis All data are expressed as mean ± standard error (mean ± SEM). Comparisons among multiple groups were performed using one-way ANOVA. P A value <0.05 is considered statistically significant.

[0116] 4. Experimental Results 4.1. Blood pressure in the rat tail artery SHR rats are currently recognized internationally as the animal model that most closely resembles the pathogenesis of essential hypertension in humans. For example... Figure 5 As shown in (a), compared with the normal control group, the systolic blood pressure of the rats in the model control group was significantly increased ( P<0.05; Compared with the model control group, the systolic blood pressure of the positive control group, the HC1183 prevention group, and the HC1183 treatment group decreased significantly ( P <0.05; Compared with the positive control group, the systolic blood pressure in the HC1183 prevention group was lower, and the difference was statistically significant. P <0.05, systolic blood pressure decreased in the HC1183 treatment group, but the difference was not statistically significant. P >0.05); there was no significant difference in systolic blood pressure between the HC1183 prevention group and the HC1183 treatment group and the normal control group. P >0.05).

[0117] like Figure 5 As shown in (b), compared with the normal control group, the diastolic blood pressure of the rats in the model control group was significantly increased ( P <0.05; compared with the model control group, the diastolic blood pressure of the positive control group, the HC1183 prevention group and the HC1183 treatment group decreased significantly ( P <0.05; Compared with the positive control group, diastolic blood pressure decreased in both the HC1183 prevention group and the HC1183 treatment group, but the difference was not statistically significant. P >0.05); the diastolic blood pressure in the positive control group was significantly different from that in the normal control group ( P <0.05, there was no significant difference in diastolic blood pressure between the HC1183 prevention group and the HC1183 treatment group and the normal control group. P >0.05). See Table 7 for details of systolic and diastolic blood pressure levels in each group of rats.

[0118] Table 7 - Results of systolic and diastolic blood pressure tests in rats

[0119] As shown in Table 7, in lowering systolic blood pressure in rats, the combination of perindopril and *Lactobacillus fermentum* HC1183 was more effective than perindopril alone. *Lactobacillus fermentum* HC1183 restored the systolic blood pressure of SHR rats to the level of healthy rats. Furthermore, the effect of lowering systolic blood pressure was more significant after prior administration of *Lactobacillus fermentum* HC1183. Therefore, *Lactobacillus fermentum* HC1183 can help reduce excessively high systolic blood pressure in SHR rats, and its effect is more significant when administered beforehand. Similarly, in lowering diastolic blood pressure in rats, the combination of perindopril and *Lactobacillus fermentum* HC1183 was more effective than perindopril alone. *Lactobacillus fermentum* HC1183 also restored the diastolic blood pressure of SHR rats to the level of healthy rats, and its effect was more significant when administered beforehand.

[0120] 4.2 Effect of Improving the Intestinal Mucosal Barrier in Rats The permeability of the rat intestinal mucosal barrier can be reflected by detecting the concentration of FITC-D in the portal vein blood after gavage administration of fluorescein isothiocyanate-labeled dextran. DAO is an intracellular enzyme in intestinal mucosal epithelial cells. When the intestinal mucosal epithelium is damaged, DAO can enter the bloodstream, and elevated serum DAO levels can indirectly reflect the degree of damage to intestinal mucosal epithelial cells. D-lactic acid is a metabolic product of bacterial fermentation. Most of the D-lactic acid in the blood originates from the lamina propria flora of the gastrointestinal tract and enters the circulation through the intestinal mucosa. Detecting peripheral blood levels can reflect the degree of intestinal mucosal damage and changes in permeability. Endotoxin LPS is a component of the cell wall of Gram-negative bacteria. It can be presented to Toll-like receptor 4 via CD14 molecules to form a complex, activating an immune response and leading to the production of pro-inflammatory cytokines interleukin-1 (IL-1) and IL-18, thereby inducing hypertension.

[0121] The results of FITC-D levels are compared in Figure 6(a); the results of LPS levels are compared in... Figure 6 As shown in (b). Compared with the model control group, the FITC-D level in the positive control group decreased, but the difference was not statistically significant (P>0.05). Compared with the model control group, the FITC-D levels in the HC1183 prevention group and the HC1183 treatment group decreased, and the differences were statistically significant (P<0.05). Furthermore, the FITC-D levels in the HC1183 prevention group and the HC1183 treatment group were lower than those in the positive control group, and the differences were statistically significant (P>0.05). P <0.05, P <0.05. Serum LPS levels in each rat group showed the same trend as FITC-D, with significantly lower LPS levels in both the HC1183 prevention and treatment groups compared to the model control group ( ). P <0.05, P <0.05, which was also significantly lower than the positive control group ( P <0.05, P <0.05).

[0122] The results of serum DAO levels in each group of rats were compared to, for example... Figure 7 As shown in (a); D-lactic acid level results are compared to, for example Figure 7 As shown in (b), serum DAO and D-lactic acid levels in the HC1183 prevention group were significantly lower than those in the model control group. P <0.05, P The value was <0.05, and it was also lower than that of the positive control group, with a statistically significant difference. P <0.05, P <0.05). Serum DAO and D-lactic acid levels in the HC1183 treatment group showed the same trend. Detailed results of FITC-D, LPS, DAO, and D-lactic acid levels in each group of rats are shown in Table 8.

[0123] Table 8 - Results of FITC-D, LPS, DAO, and D-lactate Level Tests in Rats

[0124] The results indicate that the use of fermented Lactobacillus mucosa HC1183 can reduce intestinal mucosal barrier damage and increased intestinal wall barrier permeability, improve the integrity of the intestinal wall barrier, help reduce the risk of hypertension caused by increased levels of endotoxins such as LPS, and help lower blood pressure.

[0125] 4.3 Expression levels of vascular remodeling-related proteins and mRNAs One of the common pathological changes in hypertension and its complications is the disruption of vascular structure and function, ultimately leading to vascular remodeling. Vascular remodeling often accompanies the development and progression of hypertension, and the two influence each other, forming a vicious cycle. Studies have shown that the main factors promoting vascular remodeling in hypertension are angiotensin II (Ang II) and endothelin (ET). The renin-angiotensin system has a complex network connection between hypertension and vascular remodeling. Ang II plays a key regulatory role, stimulating vascular smooth muscle cell proliferation and increased collagen secretion through inducing vascular inflammation and oxidative stress, leading to vascular fibrosis and vascular remodeling. ET is an important and potent vasoconstrictor, with ET-1 being the predominant factor. The transformation of adventitia fibroblasts into myofibroblasts is a key step and main characteristic of the vascular remodeling process. This transformation can enhance cell proliferation and migration activity, eliminate contractile structures, and increase extracellular matrix such as collagen and fibronectin, thus exacerbating vascular remodeling. Among these, α-smooth muscle actin (α-SMA) is a characteristic marker of this transformation process. Collagen is a major component of the extracellular matrix (ECM), forming its framework structure and accounting for 80%-90% of its content, primarily composed of type I collagen (Col-1). When the blood vessel wall is subjected to various forms of damage or stimulation, ECM metabolism can become disordered, altering its composition or quantity and leading to excessive collagen deposition. Furthermore, the degradation of the ECM can also cause the proliferation and migration of vascular smooth muscle cells, exacerbating abnormal vascular conformation.

[0126] like Figure 8 As shown in (a), the expression levels of Ang II protein in the HC1183 prevention group and the HC1183 treatment group were significantly lower than those in the model control group. P <0.05, P <0.05. Compared with the positive control group, the expression level of AngII protein in the HC1183 prevention group and the HC1183 treatment group decreased and returned to the level of the normal control group, but only the HC1183 prevention group showed a significant difference (P <0.05).

[0127] like Figure 8 As shown in (b), the ET-1 protein expression level in the HC1183 prevention group and the HC1183 treatment group was significantly lower than that in the model control group. P <0.05, P <0.05. Compared with the positive control group, the expression level of ET-1 protein decreased in both the HC1183 prevention group and the HC1183 treatment group, and the difference was statistically significant. P <0.05, P <0.05), and the HC1183 preventive group showed a decrease in protein expression levels to the normal control group level. The results of Ang II and ET-1 protein expression levels in each group of rats are shown in Table 9.

[0128] Table 9 - Expression levels of rat vascular remodeling-related proteins

[0129] The results indicate that *Lactobacillus fermentum* HC1183 can reduce the expression levels of Ang II and ET-1 proteins, and the effect is more pronounced when administered beforehand, restoring Ang II and ET-1 protein expression levels to those of healthy rats. Positive control drugs are less effective than *Lactobacillus fermentum* HC1183 in reducing Ang II and ET-1 protein expression levels. Therefore, *Lactobacillus fermentum* HC1183 can alleviate vascular remodeling and thus help alleviate hypertension symptoms.

[0130] like Figure 9 As shown in (a), the relative expression levels of ET-1 mRNA in the HC1183 prevention group and the HC1183 treatment group were significantly lower than those in the model control group. P <0.05, P <0.05. Compared with the positive control group, the relative expression level of ET-1 mRNA in the HC1183 prevention group was decreased, and the difference was statistically significant. P <0.05), the ET-1 mRNA expression level decreased in the HC1183 treatment group, but the difference was not statistically significant; Figure 9 As shown in (b), the relative expression levels of Col-1 mRNA in the HC1183 prevention group and the HC1183 treatment group were significantly lower than those in the model control group. P <0.05, P <0.05. Compared with the positive control group, the relative expression level of Col-I mRNA in the HC1183 prevention group and the HC1183 treatment group decreased significantly. P <0.05); such as Figure 9As shown in (c), the relative expression levels of α-SMA mRNA in the HC1183 prevention group and the HC1183 treatment group were significantly lower than those in the model control group. P <0.05, P <0.05. Compared with the positive control group, the relative expression levels of α-SMA mRNA in the HC1183 prevention group and the HC1183 treatment group were significantly decreased. P <0.05). The expression levels of Ang II and ET-1 proteins and the relative expression levels of ET-1, Col-I and α-SMAm RNA in each group of rats are detailed in Table 10.

[0131] Table 10 - mRNA expression levels of rat vascular remodeling-related genes

[0132] The changes in the relative expression levels of ET-1, CoI-1, and α-SMA genes further validated that *Lactobacillus fermentum* HC1183 helps mitigate vascular remodeling. Therefore, *Lactobacillus fermentum* HC1183 can prevent or help alleviate hypertension by slowing down vascular remodeling.

[0133] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A strain of fermenting *Lactobacillus mucilaginosus* HC1183, characterized in that, The fermented mucinous lactobacillus ( Limosilactobacillus fermentum HC1183 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26325, on December 26, 2022. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The fermenting *Lactobacillus mucinus* HC1183 as described in claim 1, characterized in that, The 16S rDNA sequence of the fermenting Lactobacillus mucinus HC1183 is shown in SEQ ID NO.

3.

3. A liquid bacterial agent containing *Lactobacillus fermentans* HC1183 as described in claim 1.

4. The liquid bacterial agent as described in claim 3, characterized in that, The liquid bacterial agent is a bacterial suspension of Lactobacillus fermentum HC1183, a lysate of a strain containing Lactobacillus fermentum HC1183, or a fermentation supernatant containing Lactobacillus fermentum HC1183.

5. The liquid bacterial agent as described in claim 4, characterized in that, The strain lysate was obtained by ultrasonically disrupting the strain fermentation broth using an ultrasonic disruptor; the fermentation supernatant was obtained by centrifuging the strain fermentation broth.

6. The liquid bacterial agent as described in claim 5, characterized in that, The fermentation broth is prepared by culturing Lactobacillus mucinus HC1183 in MRS liquid medium at 37°C for 24-28 hours.

7. The liquid bacterial agent as described in claim 6, characterized in that, The MRS liquid culture medium comprises the following components: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 5 g / L sodium acetate, 20 g / L glucose, 2 g / L K₂HPO₄, 1.0 mL / L Tween 80, 2.0 g / L diammonium citrate, 0.02 g / L MgSO₄·7H₂O, 0.04 g / L MnSO₄·7H₂O, and 1 L of pure water to adjust the pH to 5.7 ± 0.

2.

8. The use of the fermented Lactobacillus mucinus HC1183 as described in claim 1 in the preparation of products for regulating blood pressure levels.

9. The use of the fermented Lactobacillus mucinus HC1183 as described in claim 1 in the preparation of a product that lowers blood cholesterol.

10. The use of the fermented Lactobacillus mucinus HC1183 as described in claim 1 in the preparation of antioxidant products.

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