A strain of Lactococcus lactis HC3519, its inoculant and its application

Abstract

This invention relates to a strain of *Lactococcus lactis* HC3519, its inoculum, and its applications, belonging to the technical field of microbiology. *Lactococcus lactis* (… Lactococcus lactis HC3519 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30765, deposited on May 24, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This invention also provides the application of *Lactococcus lactis* HC3519 in the preparation of products for inhibiting and preventing *Mycoplasma pneumoniae*, and in the preparation of antioxidant products. The *Lactococcus lactis* HC3519 provided by this invention can alleviate oxidative stress damage to human alveolar basal epithelial cells (A549) induced by *Mycoplasma pneumoniae*, and reduce the inflammatory damage caused by *Mycoplasma pneumoniae* to the body.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Lactococcus lactis HC3519, its inoculum, and its applications. Background Technology

[0002] Mycoplasma pneumoniae (MP) is the smallest pathogenic microorganism between bacteria and viruses, capable of surviving independently on cell-free culture media, and is a common pathogen causing respiratory infections. Mycoplasma pneumoniae often causes significant lung damage and acute and chronic airway-related inflammation, such as mycoplasma pneumoniae pneumonia (MPP), asthma, and chronic obstructive pulmonary disease (COPD). In addition, Mycoplasma pneumoniae can also cause other extrapulmonary related diseases, such as nephritis, myocarditis, meningoencephalitis, skin and mucous membrane damage, and atherosclerosis.

[0003] The macrolide antibiotics used in the conventional treatment of Mycoplasma pneumoniae pneumonia can disrupt the gut microbiota, leading to a further reduction in beneficial bacteria such as Bifidobacteria and causing secondary damage. Supplementing with probiotics can regulate the gut microbiota structure and reduce the pathogenic bacteria load. Therefore, regulating the gut microbiota and reconstructing the gut-lung axis balance has become an important strategy for the prevention and control of Mycoplasma pneumoniae pneumonia, and also provides a theoretical basis and practical evidence for the development of probiotics with heat-clearing and lung-protecting functions.

[0004] Lactococcus lactis, a member of the Lactococcus genus, is a probiotic with various benefits for the human body. It exhibits particularly excellent activity in regulating intestinal flora and protecting the mucosal barrier. However, in existing published technical literature and patents, there are no reports of using Lactococcus lactis as an active ingredient to antagonize Mycoplasma pneumoniae pneumonia and thus alleviate lung diseases. Its application value in the prevention and treatment of Mycoplasma pneumoniae pneumonia needs further exploration. Summary of the Invention

[0005] To address the issue that existing technologies do not have the application of Lactococcus lactis in inhibiting or preventing Mycoplasma pneumoniae, this invention provides a strain of Lactococcus lactis HC3519, its bacterial agent, and its applications to solve the aforementioned problem.

[0006] In a first aspect, the present invention provides a strain of *Lactococcus lactis* HC3519, wherein *Lactococcus lactis* ( Lactococcus lactis HC3519 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30765, on May 24, 2024. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] Furthermore, the 16S rDNA sequence of the lactococcus HC3519 is shown in SEQ ID NO.3.

[0008] In a second aspect, the present invention provides a microbial agent containing fermentation broth of Lactococcus lactis HC3519, bacterial lysate of Lactococcus lactis HC3519, or fermentation supernatant of Lactococcus lactis HC3519; the bacterial lysate is obtained by ultrasonically disrupting the bacterial fermentation broth using an ultrasonic disruptor.

[0009] Furthermore, the concentration of Lactococcus lactis HC3519 in the fermentation broth is ≥1×10⁻⁶. 9 CFU / mL.

[0010] Furthermore, the fermentation broth is prepared by culturing Lactococcus lactis HC3519 in MRS liquid medium at 37°C for 24-28 hours.

[0011] Furthermore, the fermentation supernatant is obtained by centrifuging the fermentation broth of the strain.

[0012] Furthermore, after culturing in MRS liquid medium at 37°C for 24 hours, the fermentation broth was collected and ultrasonically broken up for 20 minutes using an ultrasonic disruptor to obtain a lysate containing Lactococcus lactis HC3519.

[0013] 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.

[0014] Thirdly, the present invention provides the application of Lactococcus lactis HC3519 in the preparation of products for inhibiting and preventing Mycoplasma pneumoniae.

[0015] Fourthly, the present invention provides an application of Lactococcus lactis HC3519 in the preparation of antioxidant products.

[0016] The beneficial effects of this invention are as follows: (1) The Lactococcus lactis HC3519 provided by the present invention does not produce hemolysin and cannot lyse blood cells. It is sensitive to antibiotics such as erythromycin, gentamicin, streptomycin, tetracycline, clindamycin and ampicillin, and has good biosafety.

[0017] (2) The Lactococcus lactis HC3519 provided by the present invention has a strong tolerance to gastric acid and bile salts, can reach the human gastrointestinal tract in a living state, and can adhere to intestinal epithelial cells HT-29, colonize the intestinal epithelial cells and exert the probiotic properties of lowering cholesterol.

[0018] (3) The Lactococcus lactis HC3519 provided by the present invention has strong antioxidant function, which helps to improve the body's antioxidant capacity.

[0019] (4) The *Lactococcus lactis* HC3519 provided by this invention can alleviate oxidative stress damage induced by *Mycoplasma pneumoniae* in human alveolar basal epithelial cells A549, improve A549 cell activity, reduce A549 cell apoptosis rate, and reduce A549 cell reactive oxygen species levels. After treatment with fermentation lysate and supernatant of *Lactococcus lactis* HC3519, the expression levels of antioxidant-related proteins Nrf2, Keap1, HO-1, and NQO1 in A549 cells increased, indicating improved antioxidant capacity.

[0020] (5) The *Lactococcus lactis* HC3519 provided by this invention can regulate the oxidative stress response in mice with *Mycoplasma pneumoniae* pneumonia, reduce the inflammatory damage caused by *Mycoplasma pneumoniae*, including reducing the content of *Mycoplasma pneumoniae* in lung tissue, and reducing NOS, NO, and MDA produced by oxidative stress, while increasing the body's SOD level. Furthermore, the combined use of azithromycin and *Lactococcus lactis* HC3519 in treating *Mycoplasma pneumoniae* pneumonia is more effective in reducing the body's oxidative stress level. Attached Figure Description

[0021] 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.

[0022] Figure 1 This is a diagram showing the antibacterial experiment results in Example 1 of the present invention.

[0023] Figure 2 This is a microscope image of Lactococcus lactis HC3519 in Example 1 of the present invention.

[0024] Figure 3 This is an optical microscope image of Lactococcus lactis HC3519 in Example 1 of the present invention.

[0025] Figure 4 This is the RAPD fingerprint of Lactococcus lactis HC3519 in Example 1 of the present invention.

[0026] Figure 5 This is the rep-PCR fingerprint of Lactococcus lactis HC3519 in Example 1 of this invention.

[0027] Figure 6These are graphs showing the results of cell viability, apoptosis, and intracellular reactive oxygen species (ROS) levels in each group in Example 8 of this invention. (a) shows a comparison of cell viability results among the groups; (b) shows a comparison of apoptosis rates among the groups; and (c) shows a comparison of ROS fluorescence intensity results among the groups.

[0028] Figure 7 This is a graph showing the changes in the relative expression levels of various proteins in Example 9 of the present invention. Among them, (a) is a comparison of the relative expression levels of Nrf2 protein in each group of cells; (b) is a comparison of the relative expression levels of Keap1 protein in each group of cells; (c) is a comparison of the relative expression levels of HO-1 protein in each group of cells; and (d) is a comparison of the relative expression levels of NQO1 protein in each group of cells.

[0029] Figure 8 This is a graph showing the changes in the levels of various components in mouse serum during Example 9 of the present invention. (a) shows a comparison of the changes in NOS levels in the serum of each group of mice; (b) shows a comparison of the changes in NO levels in the serum of each group of mice; (c) shows a comparison of the changes in MDA levels in the serum of each group of mice; and (d) shows a comparison of the changes in SOD levels in the serum of each group of mice. Detailed Implementation

[0030] 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.

[0031] The components of the various culture media used in the following examples are as follows: MRS solid culture medium: Weigh out MRS (Man Rogosa Sharpe, Haibo) ® 66.2g of solid culture medium was added to 1L of pure water and stirred evenly. The mixture was then autoclaved at 121℃ for 15min. After cooling, the mixture was poured into sterile plates to obtain MRS solid culture medium.

[0032] MRS liquid culture medium: peptone 10 g / L, beef extract 8 g / L, yeast extract 4 g / L, sodium acetate 5 g / L, glucose 20 g / L, K₂HPO₄ 2 g / L, Tween 80 1.0 mL / L, diamine citrate 2.0 g / L, MgSO₄·7H₂O 0.02 g / L, MnSO₄·7H₂O 0.04 g / L, with pure water to a final volume of 1 L, and pH adjusted to 5.7 ± 0.2. Brain and heart broth culture medium (containing 5% fetal bovine serum): 10.0g peptone, 12.5g dehydrated calf brain extract powder, 5.0g dehydrated bovine heart extract powder, 5.0g NaCl, 2.0g glucose, 2.5g Na2HPO4, and 1L of purified water, pH 7.4±0.2. Autoclave at 121℃ for 15 minutes. After sterilization, allow the culture medium to cool to approximately 50℃, then add 50mL of fetal bovine serum.

[0033] Semi-solid culture medium containing 5% fetal bovine serum (FBS): 4.0 g / L bovine brain extract, 4.0 g / L bovine heart extract, 5.0 g / L peptone, 16.0 g / L casein peptone, 5.0 g / L sodium chloride, 2.0 g / L glucose, 2.5 g / L Na₂HPO₄, 13.5 g / L agar, pH 7.4 ± 0.2. Autoclave at 121℃ for 15 minutes. After sterilization, allow the medium to cool to approximately 50℃, then add 50 mL of FBS.

[0034] Example 1 Isolation, screening and identification of Lactococcus lactis HC3519 1. Sampling: Homemade fermented cheese collected from a herder family in Xilingol League, Inner Mongolia Autonomous Region on September 18, 2022.

[0035] 2. Isolation: Take 10g of fermented cheese collected in step (1) and place it in a sampling bag containing 1L of sterile physiological saline. Repeatedly tap the bag for 5 minutes. Spread 50μL of the sample solution onto MRS solid medium and incubate at 37℃ for 48 hours. After single colonies grow on the plate, pick a single clone and continue streaking. After three purification cultures, pick a single clone for rapid MALDI-TOF-MS identification. The rapid MALDI-TOF-MS identification was performed according to the kit instructions. Based on the MALDI-TOF-MS identification results, a total of 20 strains were screened from the sample.

[0036] 3. Screening (1) Activation of strain: Single colonies of the 20 selected strains were streaked onto MRS solid medium and incubated at 37°C for 24–48 h. After single colonies grew on the MRS solid medium, they were aseptically inoculated into MRS liquid medium and incubated overnight at 37°C until the cell density reached 10⁻⁶. 9 Stop culturing when the concentration of CFU / mL reaches a certain level, and collect the fresh bacterial culture for later use.

[0037] Take a frozen Streptococcus pneumoniae ATCC49619 glycerol tube and inoculate Streptococcus pneumoniae ATCC49619 into brain heart broth medium (containing 5% fetal bovine serum) at an inoculation rate of 1%. Incubate at 37°C for 16-24 hours.

[0038] (2) Oxford cup antibacterial test First, prepare the lower layer of culture medium for the Oxford cup experiment. After autoclaving and cooling, pour the nutrient agar medium into plates, ensuring the plates are fully covered. Once the agar has solidified, evenly place four sterile Oxford cups on each plate. Next, prepare the upper layer of culture medium. Inoculate Streptococcus pneumoniae ATCC49619 bacterial suspension at a rate of 0.2% (v / v) into brain and heart infusion semi-solid medium (containing 5% fetal bovine serum), and evenly spread an appropriate amount onto the lower layer. After the upper layer has solidified, remove the Oxford cups and inoculate 150 μL of the fresh bacterial suspension to be tested into three wells. One well is left uninoculated as a blank control. Incubate the medium at 37°C for 24 hours, observing and measuring the size of the inhibition zone.

[0039] The results showed that 5 out of the 20 strains obtained in the initial screening exhibited inhibitory activity against Streptococcus pneumoniae ATCC49619, with inhibition zone diameters ranging from 10.41±0.22 mm to 21.30±0.37 mm. Among them, strain HC3519 produced the largest inhibition zone, reaching 21.30±0.37 mm, and its inhibition zone was as follows... Figure 1 As shown.

[0040] 4. Identification (1) Identification of colony morphology Strain strain HC3519 was inoculated onto MRS solid medium and incubated at 37°C for 24 hours. Single colonies of HC3519 were observed to be off-white, 1.0–2.0 mm in size, with neat edges and a glossy, raised surface. The colonies of strain HC3519 appeared as follows: Figure 2 As shown in the image. Under an optical microscope, strain HC3519 appears as spherical cells of roughly uniform size, arranged in clusters. The optical microscope image is shown below. Figure 3 As shown.

[0041] (2) Identification of physiological and biochemical characteristics The inoculum for strain HC3519 was prepared as follows: Activated strain HC3519 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 as the inoculum.

[0042] (2.1) Temperature growth range experiment The HC3519 inoculum was inoculated into 10 ml LMR liquid medium at a rate of 10%. 10 ml LMR liquid medium without inoculation was used as a control. The medium was placed in constant temperature incubators at 0℃, 10℃, 25℃, 37℃, 45℃ and 60℃ and incubated for 48 h. The turbidity of the culture medium was observed.

[0043] The results showed that after 48 hours of incubation at 0℃, 10℃, and 60℃, the culture medium for strain HC3519 remained clear; it became slightly turbid at 25℃ and 45℃; and a large number of cells were produced after 48 hours of incubation at 37℃, with the highest turbidity observed. Therefore, the optimal growth temperature for strain HC3519 is 37℃.

[0044] (2.2) Salinity tolerance test Under aseptic conditions, HC3519 inoculum was inoculated at a rate of 10% into 5 mL of MRS liquid medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%, respectively. 5 mL of MRS liquid medium without 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.

[0045] The results showed that the culture medium for strain HC3519 became turbid at salt concentrations of 1%–3%, but cleared at salt concentrations above 3%. Therefore, the maximum salt concentration that strain HC3519 can tolerate is 3%.

[0046] (2.3) Antibiotic resistance and hemolytic test (2.3.1) Antibiotic tolerance test The minimum inhibitory concentration (MIC) of antibiotics against strain HC3519 was determined using the microbroth dilution method. Antibiotic preparation: 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 prepare 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 (1–1024 μg / mL) to each of the eleven wells in columns 2–12, and then inoculate each well with 10 μL of inoculum. Each experiment was repeated four times, with wells without inoculation serving 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 ​​for different strains were calculated. Specific results are shown in Table 1.

[0048] Table 1 - MIC values ​​of antibiotic resistance test for strain HC3519

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

[0050] As can be seen from the results in Table 1, strain HC3519 is sensitive to common antibiotics such as erythromycin, gentamicin, streptomycin, tetracycline, clindamycin, and ampicillin, and has good biosafety.

[0051] (2.3.2) Hemolytic test Prepare TBS basal medium, comprising the following components: 1L distilled water, 17.0g tryptone, 5.0g NaCl, 3.0g soybean peptone, 2.5g KH2PO4, and 2.5g glucose. After dissolving, autoclave at 121°C for 15min. When the TBS medium has cooled to 50°C, add 5% sterile defibrinated sheep blood, mix well, and pour into plates to obtain blood cell plates.

[0052] Strawberry strain HC3519 was streaked onto prepared blood cell plates and incubated at 37°C for 24-48 hours. The presence of hemolysis was then observed.

[0053] The results showed that strain HC3519 could not grow and there were no changes on the blood cell plate, indicating that strain HC3519 does not produce hemolysin and cannot lyse blood cells, thus exhibiting good biosafety.

[0054] 5. Carbon source fermentation experiment (1) Carbon source metabolism experiment Prepare the basic culture medium: 1.5g peptone, 0.6g yeast extract, 0.1g Tween 80, 0.5mL salt solution, 18mg phenol red, and 100mL distilled water. Adjust the pH to 7.4±0.2.

[0055] The salt solution composition is as follows: 11.5 g MgSO4·7H2O, 8 g MnSO4·4H2O, and 100 mL distilled water; prepare a 10 g / mL solution of sugars, alcohols, and glycosides, and filter it through a 0.22 μm sterile filter.

[0056] Under aseptic conditions, 20 μL of sterile carbohydrate solution was added to each well of a 96-well plate, with four replicates for each carbohydrate. Then, 170 μL of sterile basal medium containing phenol red was added, followed by 10 μL of inoculum. Wells without inoculation 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. The results are shown in Table 2.

[0057] Table 2 - Results of carbon source metabolism experiments for strain HC3519

[0058] Note: + indicates positive; - indicates negative.

[0059] (2) Glucose acid and gas production test Preparation of acid- and gas-producing culture medium: peptone 0.5g, yeast extract 0.3g, Tween 80 0.1mL, salt solution A 0.5mL, salt solution B 0.5mL, sodium acetate 0.5g, glucose 2.5g, 2% bromocresol green ( w / v 0.05 mL of distilled water and 100 mL of water; pH = 6.8~7.0. Dispense the prepared culture medium into large test tubes containing inverted small test tubes, 3 mL / tube, and autoclave at 121℃ for 15 min.

[0060] Salt solution A consists of 10.0g KH2PO4 and 1.0g K2HPO4, dissolved in distilled water and brought to a final volume of 100mL.

[0061] Salt solution B consists of: 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 was inoculated into the culture medium at a rate of 2%, with an uninoculated culture medium serving as a control. The top was then sealed with 2 mL of sterile liquid paraffin and incubated at 37°C for 48 hours. The color of the culture medium was then observed for any changes.

[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 the HC3519 strain produced acid but not gas during glucose fermentation.

[0064] 6. Molecular biological identification A single colony of strain HC3519 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] (1) Identification of 16S rDNA gene sequence Using TIANGEN ® The 2×Taq PCR premix kit was used to amplify the 16S rDNA gene of strain HC3519. The reaction system and reaction cycle settings were performed according to the kit instructions.

[0066] The upstream primer was 27F: AGAGTTTGATCCTGGCTCA (SEQ ID NO.1); The downstream primer was 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO.2).

[0067] Electrophoresis confirmed that the PCR amplification product was approximately 1500 bp in size, which met the requirements. 16S rDNA sequencing results showed that the 16S rDNA sequence of strain HC3519 is as shown in SEQ ID NO.3: The sequence was compared with BALST on the EzBioCloud website and it matched that of Lactococcus lactis ( Lactococcus lactis The strain showed the highest similarity to *Lactococcus lactis*. Therefore, strain HC3519 was identified as *Lactococcus lactis*. Lactococcus lactis Lactococcus lactis HC3519 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30765, deposited on May 24, 2024. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0068] (2) RAPD fingerprint identification Using TIANGEN ® The 2×Taq PCR premix kit was used to amplify strain HC3519. The reaction system and reaction cycle settings were performed according to the kit instructions. The M13 primer sequence was: 5'-GAGGGTGGCGGTTCT-3' (SEQ ID NO.4).

[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 Lactococcus lactis HC3519 is shown below. Figure 4 As shown. A comparison revealed no similarity in existing publicly available reports. Figure 4 The matching RAPD fingerprint profiles indicate that Lactococcus lactis HC3519 is a novel Lactococcus lactis strain.

[0070] (3) Rep-PCR fingerprint identification Using TIANGEN ® The 2×Taq PCR premix kit was used to amplify Lactococcus lactis HC3519. The reaction system and reaction cycle settings were performed according to the kit instructions. The rep-PCR primer was: 5'-GTGGTGGTGGTGGTG-3' (SEQ ID NO. 5).

[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 *Lactococcus lactis* HC3519 is shown below. Figure 5 As shown. A comparison revealed no similarity in existing publicly available reports. Figure 5 The matching rep-PCR fingerprint pattern indicates that the selected Lactococcus lactis HC3519 is a new Lactococcus lactis strain.

[0072] Based on the results of MALDI-TOF-MS identification, molecular biology experiments, carbon source metabolism experiments, and glucose acid and gas production experiments, it can be concluded that Lactococcus lactis HC3519 is different from all currently reported Lactococcus lactis strains and is a new strain of Lactococcus lactis.

[0073] Example 2 Preparation of fermentation supernatant of Lactococcus lactis HC3519: Take Lactococcus lactis HC3519 after 3 generations of activation, culture it in MRS liquid medium at 37℃ for 24 hours, centrifuge at 6000 r / min for 10 min, and collect the supernatant as the fermentation supernatant.

[0074] Preparation of lysate containing Lactococcus lactis HC3519: After activating Lactococcus lactis HC3519 for 3 generations, the fermentation broth was collected after being cultured in MRS liquid medium at 37°C for 24 hours. The fermentation broth was then ultrasonically disrupted for 20 minutes to obtain a lysate containing Lactococcus lactis HC3519.

[0075] Example 3 Lactococcus lactis HC3519 simulated gastrointestinal tolerance test 1. Determination of the HRS ability of Lactococcus lactis HC3519 to scavenge DPPH and hydroxyl radicals. Activated Lactococcus lactis HC3519 colonies were picked and inoculated into freshly prepared MRS liquid medium. The culture was incubated at 37°C with shaking for 24 h. Then, 1% of the culture was added to the MRS liquid medium, and the culture was continued at 37°C with shaking for another 24 h. The bacterial suspension was then collected as the inoculum. The commercially available Lactococcus lactis strain ATCC9936 was used as the control strain, and its culture method was the same as that of Lactococcus lactis HC3519.

[0076] Preparation of artificial gastric juice: Add 5g peptone, 2.5g yeast extract, 1g glucose, and 2g NaCl to 1L of distilled water. Mix thoroughly and adjust the pH to 3.0. Sterilize at 115℃ for 20 minutes. After sterilization, allow the solution to cool to a suitable temperature. Before the experiment, add 3.2g of porcine mucosal pepsin powder and gently shake the container to mix the powder thoroughly.

[0077] 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 to dissolve, add 77mL NaOH solution (0.2N) to adjust the pH to 6.8±0.1. Then sterilize at 115℃ for 20 minutes. Before the experiment, add 1.0g trypsin powder to the cooled sterilized solution and gently shake to mix.

[0078] The absorbance (OD) of the activated Lactococcus lactis HC3519 bacterial solution was measured. 600 Adjust to 1.5. Take 1 mL of bacterial culture and incubate at 4℃ for 6000 × 10⁻⁶ hours. g Centrifuge for 10 min, discard the supernatant, and resuspend the bacterial culture in 1 mL of simulated gastric fluid. Incubate 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:

[0079] 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 3.

[0080] Table 3 - Tolerance results of Lactococcus lactis HC3519 to simulated gastric and intestinal fluids

[0081] Note: Viable bacteria count units (Log) 10 (CFU / mL).

[0082] Table 3 shows that *Lactococcus lactis* HC3519 exhibits high tolerance to simulated gastric and intestinal fluids. After digestion with simulated gastric fluid, the viable bacterial survival rate was 96.95% ± 0.45%, and after digestion with simulated intestinal fluid, the viable bacterial survival rate was still 94.90% ± 0.59%. This indicates that *Lactococcus lactis* HC3519 can withstand the harsh acidic environment of the stomach in the human body with minimal loss of viable bacteria, successfully entering the small intestine. *Lactococcus lactis* HC3519 can not only tolerate the harsh acidic environment of the stomach but also the bile salt environment of the intestines, which is beneficial for its survival and reaching the gastrointestinal tract to exert its probiotic function.

[0083] Example 4 Hydrophobic cell surface assay of Lactococcus lactis HC3519 Activated Lactococcus lactis HC3519 colonies were picked and inoculated into freshly prepared MRS liquid medium. The culture was incubated at 37°C with shaking for 24 hours. Then, 1% of the culture was added to the MRS liquid medium, and the culture was continued at 37°C with shaking for another 24 hours. The culture was then centrifuged at 6000×g for 10 minutes. The bacterial cells were collected, washed twice with sterile physiological saline, and resuspended in 1 mL of sterile KNO3 (0.1M) solution as the test solution. The commercially available Lactococcus lactis strain ATCC9936 was used as a control strain, and its culture method was the same as that of Lactococcus lactis HC3519.

[0084] 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. Cell hydrophobicity is calculated using the formula: Hydrophobicity = (A0 - A1) / A1 × 100%, and the average value is taken from three measurements.

[0085] Testing revealed that the surface hydrophobicity of *Lactococcus lactis* HC3519 cells was 63.35% ± 2.84%, while that of the control strain *Lactococcus lactis* ATCC9936 cells was 15.28% ± 1.05%. A positive correlation exists between surface hydrophobicity and adhesion of lactic acid bacteria; higher hydrophobicity indicates higher adhesion. The superior surface hydrophobicity of *Lactococcus lactis* HC3519 cells compared to the control strain *Lactococcus lactis* ATCC9936 suggests greater adhesion potential, which is beneficial for its adhesion to intestinal epithelial cells, thereby enabling it to colonize the intestinal environment and exert its probiotic properties.

[0086] Example 5 Adhesion of Lactococcus lactis HC3519 to intestinal epithelial cells Intestinal epithelial HT-29 cells were revived, passaged, and cultured until the number of cells reached the required amount. Subsequent experiments were conducted when the cells were observed to have reached approximately 80% confluence under an inverted microscope.

[0087] Discard the original culture medium in the cell culture flask and rinse twice with PBS. 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. Observe under a microscope to ensure the cells 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 in fresh culture medium. Count the cells using a hemocytometer. Dilute the cell suspension with PBS as needed. In a 6-well plate, the number of cells in each well should be 2 × 10-1. 6Add 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.

[0088] After activation, *Lactococcus lactis* HC3519 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 × 10⁻⁶. g Centrifuge for 10 min, collect bacterial cells, wash twice with PBS, and resuspend the bacterial cells in MRS medium to a refill of 5 × 10⁻⁶. 7 CFU / mL was prepared for later use. The adherent HT-29 monolayer in the 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.

[0089] After culturing, HT-29 cells were washed three times with PBS to remove unadhered bacteria. PBS buffer was slowly added along the cell wall to avoid upsetting 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 determine the number of adhered bacteria. The adhesion ability of the tested strain was calculated using the following formula. The commercially available strain *Lactococcus lactis* ATCC9936 was used as a control strain, and its culture method was the same as that of *Lactococcus lactis* HC3519.

[0090] Adhesion capacity (CFU / cell) = Total number of bacteria adhering in each culture well / Total number of cells in each culture well.

[0091] The adhesion ability of *Lactococcus lactis* HC3519 to intestinal epithelial cells HT-29 was tested to be 74.47±3.28, while that of the control strain *Lactococcus lactis* ATCC9936 to intestinal epithelial cells HT-29 was 31.56±5.55. This indicates that *Lactococcus lactis* HC3519 has excellent adhesion properties to intestinal epithelial cells HT-29, which is beneficial for its adhesion to intestinal epithelial mucosal cells and thus helps its colonization and survival.

[0092] Example 6 In vitro cholesterol degradation assay of Lactococcus lactis HC3519 Preparation of cholesterol solution: 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 the cholesterol solution.

[0093] Preparation of cholesterol-containing liquid culture medium: Weigh 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 2.0g diammonium citrate, 20.0g glucose, 1.0mL Tween 80, 5.0g CH3COONa, 0.1g MgSO4, 0.05g MnSO4, 2.0g K2HPO4, and 1000mL 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 the cholesterol-containing liquid culture medium.

[0094] Activated Lactococcus lactis HC3519 colonies were picked and inoculated into freshly prepared MRS liquid medium. The medium was incubated at 37°C with shaking for 24 hours. Then, 1% of the inoculum was transferred to MRS liquid medium and incubated at 37°C with shaking for another 24 hours. Finally, the culture was cultured at 6000× g Centrifuge for 10 min, collect bacterial cells, wash twice with PBS, and resuspend the bacterial cells in MRS medium to a refill of 5 × 10⁻⁶. 7 CFU / mL available for use.

[0095] Lactococcus lactis HC3519 inoculum was inoculated into cholesterol-containing liquid culture medium at an inoculation rate of 0.1% (v / v) and cultured statically at 37℃ for 48 h. Then, 0.2 mL of the bacterial culture was added to 1.8 mL of anhydrous ethanol, mixed well, and allowed to stand for 10 min before centrifugation at 3000 rpm for 5 min. 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 calculated. The commercially available strain Lactococcus lactis ATCC9936 was used as the control strain, and its culture method was the same as that of Lactococcus lactis HC3519.

[0096] Testing showed that *Lactococcus lactis* HC3519 exhibited a cholesterol degradation rate of 88.63% ± 2.78%, while the control strain, *Lactococcus lactis* ATCC9936, had a cholesterol degradation rate of 40.00% ± 3.52%. Cholesterol is an essential nutrient for the human body, and high blood cholesterol levels can easily induce cardiovascular and cerebrovascular diseases. The superior cholesterol-lowering ability of *Lactococcus lactis* HC3519 compared to the control strain helps reduce blood cholesterol levels and promotes cardiovascular and cerebrovascular health.

[0097] Example 7 Determination of antioxidant function of Lactococcus lactis HC3519 1. Determination of anti-lipid peroxidation capacity Preparation of linoleic acid emulsion: 0.1 mL linoleic acid, 0.2 mL Tween 20, and 19.7 mL deionized water. Add 1 mL of the linoleic acid emulsion and 1 mL of FeSO4 (1%) to 0.5 mL of PBS solution, then add 0.5 mL of the fermentation supernatant or lysis buffer of the test strain. Incubate at 37°C for 1.5 h. Add 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) to the mixture, incubate at 100°C for 30 min, cool rapidly, centrifuge at 4000 rpm / min for 15 min, and collect the supernatant. 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:

[0098] After testing, the anti-lipid peroxidation rates of the fermentation supernatant and lysate of *Lactococcus lactis* HC3519 were 52.58%±4.59% and 66.74%±3.60%, respectively. In contrast, the anti-lipid peroxidation rates of the supernatant and lysate of the control strain *Lactococcus lactis* ATCC9936 were 8.40%±4.18% and 12.17%±2.26%, respectively. This indicates that both the fermentation supernatant and lysate of *Lactococcus lactis* HC3519 exhibit strong anti-lipid peroxidation capabilities, superior to those of *Lactococcus lactis* ATCC9936, which is beneficial in reducing cell damage caused by lipid peroxidation in the body.

[0099] 2. Determination of DPPH and hydroxyl radical scavenging ability (HRS) (1) Preparation of test solution The fermentation supernatant containing *Lactococcus lactis* HC3519 and the lysate containing *Lactococcus lactis* HC3519 prepared in Example 2 were used as controls. The fermentation supernatant containing *Lactococcus lactis* ATCC9936 and the lysate containing *Lactococcus lactis* ATCC9936 were prepared according to the method in Example 2.

[0100] (2) Measurement of HRS capability Take 200 μL of each of the four test solutions prepared in step (1), and add 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, and 500 μL of deionized water to each test solution. After mixing, add 100 μL of hydrogen peroxide solution (3 mM) to each solution, incubate in a water bath at 37°C for 15 min, and then measure the absorbance of the sample at a wavelength of 510 nm. The HRS scavenging rate is calculated according to the following formula:

[0101] 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.

[0102] The scavenging rates of HRS by the fermentation supernatant and lysis buffer of *Lactococcus lactis* HC3519 were 67.82%±5.06% and 82.45%±7.28%, respectively. In contrast, the scavenging rates of HRS by the fermentation supernatant and lysis buffer of *Lactococcus lactis* ATCC9936 were 15.40%±3.65% and 22.48%±1.40%, respectively. This indicates that both the fermentation supernatant and lysis buffer of *Lactococcus lactis* HC3519 exhibit strong scavenging ability against HRS, demonstrating superior performance compared to the control strain.

[0103] (3) Determination of the strain's ability to scavenge DPPH free radicals Take 1 mL of each of the four test solutions prepared in step (1), add 1 mL of freshly prepared 0.4 mM DPPH free radical solution to each, mix well, and then react in the dark at room temperature for 30 min. Then, measure the OD of the four test solutions respectively. 517 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 determined. 517 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. 517 Absorbance A at point 空白 The clearance rate is calculated using the following formula:

[0104] The DPPH scavenging rates of the fermentation supernatant and lysis buffer of *Lactococcus lactis* HC3519 were 82.00%±4.63% and 80.18%±3.37%, respectively. In contrast, the DPPH scavenging rates of the control strain *Lactococcus lactis* ATCC9936 were 45.62%±5.01% and 63.92%±3.95%, respectively. This indicates that both the fermentation supernatant and lysis buffer of *Lactococcus lactis* HC3519 exhibit strong DPPH scavenging capabilities, outperforming the control strain.

[0105] Example 8 Effects of Lactococcus lactis HC3519 on Mycoplasma pneumoniae (MP)-induced oxidative stress damage in A549 cells 1. Strains and cell culture The frozen Mycoplasma pneumoniae ATCC15531 standard strain was inoculated into Mycoplasma pneumoniae liquid culture medium tubes (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., product model: HBPT079) and incubated in a 37℃, 5% CO2 saturated nitrogen anaerobic incubator. When the culture medium color changed from red to yellow, continuous subculturing was performed, and the 3rd generation was used for later use. The concentration of Mycoplasma pneumoniae ATCC15531 was diluted to 1×10⁻⁶. 7 CFU / mL is used as the working bacterial solution for later use.

[0106] Human alveolar basal epithelial cells (A549) were purchased from Beina Biotechnology and cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture at 37°C in a 5% CO2 incubator. A549 cells in the logarithmic growth phase were harvested, digested with trypsin, and resuspended into a single-cell suspension. Cell counting was performed using a cell counting chamber, and the cell concentration was adjusted to 1 × 10⁶ cells / cells. 6 per mL.

[0107] 2. Grouping and Intervention A549 cells were seeded into 6-well plates at a density of 3 × 10⁶ cells per well. 5 Each group was divided into three replicates. The infection group received 200 μL of Mycoplasma pneumoniae ATCC15531 bacterial suspension; the supernatant group received 200 μL of Mycoplasma pneumoniae ATCC15531 bacterial suspension and 200 μL of Lactococcus lactis HC3519 fermentation supernatant prepared in Example 2; the lysis buffer group received 200 μL of Mycoplasma pneumoniae ATCC15531 bacterial suspension and 200 μL of Lactococcus lactis HC3519 lysis buffer prepared in Example 2; and the blank group received 400 μL of culture medium containing 10% fetal bovine serum and 1% double antibiotic RPMI-1640.

[0108] 3. Detection indicators (1) Cell viability, apoptosis and intracellular reactive oxygen species (ROS) levels Cell viability was assessed using the Cell Counting Kit-8 (CCK-8 assay). Cells in each group were cultured at 37°C and 5% CO2 for 48 hours. The culture medium in each well was then discarded, and the cells were washed once with PBS buffer and digested with trypsin. The CCK-8 solution was diluted 10-fold with serum-free essential basal medium, and 100 μL of the diluted CCK-8 solution was added to each well. After sample addition, the 6-well plates were incubated at 37°C and 5% CO2 for 1 hour. The absorbance (OD) of each well was measured at 450 nm using a microplate reader. 450 (Value).

[0109] The apoptosis rate was determined using the Annexin V-FITC apoptosis detection kit and a double staining method with propidium iodide (PI) staining solution. Cells from each group were cultured at 37℃ and 5% CO2 for 24 h. After incubation, the cell culture medium in each well was discarded, and the cells were washed once with PBS buffer and digested with an appropriate amount of trypsin. After digestion, the cells were collected in flow cytometry tubes and cultured at 1000× g Centrifuge for 5 min, discard the supernatant, resuspend in 195 μL Annexin V-FITC binding solution, then add 5 μL Annexin V-FITC and mix gently. Incubate at 4°C in the dark for 15 min, then add 5 μL PI staining solution, mix gently, and incubate at 4°C in the dark for 5 min. Use a negative control without Annexin V-FITC and PI. Immediately afterwards, perform flow cytometry to detect the cell apoptosis rate.

[0110] ROS levels in A549 cells were detected using a ROS detection kit (DCFH-DA fluorescence method). Cells in each group were cultured at 37℃ and 5% CO2 for 24 h. The cell culture medium in each well was discarded, and the cells were washed twice with PBS buffer. 1 mL of dye working solution (10 μM) was added, and the cells were incubated at room temperature for 30 min, followed by incubation at 400× [missing value]. g After centrifugation for 3 min, the supernatant was discarded. The cells were washed twice with PBS, resuspended in 1 mL of PBS, and then the fluorescence intensity was detected by flow cytometry.

[0111] Cell viability results for each group are as follows Figure 6 As shown in (a). From Figure 6 As shown in Figure (a), compared with the control group, the cell viability of A549 cells decreased by 56.19% after infection with Mycoplasma pneumoniae, with a highly significant difference (***, P < 0.001). Compared with the infection group, the cell viability of A549 cells in the supernatant group and the lysis buffer group increased by 79.60% and 89.05%, respectively, and the differences were also significant (**, P < 0.01). The results indicate that the fermentation supernatant and lysis buffer of Lactococcus lactis HC3519 can alleviate the activity damage of A549 cells caused by Mycoplasma pneumoniae, thereby reducing lung injury.

[0112] The apoptosis rate results for each group are as follows: Figure 6 As shown in (b). From Figure 6As shown in Figure (b), compared with the control group, the apoptosis rate of A549 cells after infection with Mycoplasma pneumoniae significantly increased, with the infection group showing a 7.3-fold increase in apoptosis rate, which was highly significant (***, P < 0.001). Compared with the infection group, the apoptosis rates of A549 cells in the supernatant group and the lysis buffer group decreased by 48.25% and 55.13%, respectively, and the differences were also significant (**, P < 0.01). These results indicate that the fermentation supernatant and lysis buffer of Lactococcus lactis HC3519 can alleviate the apoptosis induced by Mycoplasma pneumoniae in A549 cells, helping to improve cell viability and thus reduce lung injury.

[0113] The ROS fluorescence intensity results for each group of cells are shown in Figure 6(c). Figure 6 As shown in Figure (c), compared with the control group, the apoptosis rate of A549 cells after infection with Mycoplasma pneumoniae was significantly increased, and the ROS level of the infected cells increased by 105.59%, which was highly significant (***, P < 0.001). Compared with the infected group, the ROS levels of A549 cells in the supernatant group and the lysis buffer group decreased by 26.00% and 26.44%, respectively, and the differences were also highly significant (***, P < 0.001). The results indicate that the fermentation supernatant and lysis buffer of Lactococcus lactis HC3519 can reduce the oxidative stress level induced by Mycoplasma pneumoniae in A549 cells, which helps to alleviate pneumonia damage.

[0114] (2) Intracellular oxidative stress-related protein levels After culturing cells in each group at 37℃ and 5% CO2 for 3 days, the cell culture medium in each well was discarded, cells were collected, and total cellular protein was extracted. The concentration of total cellular protein was determined by the BCA method. The expression levels of nuclear transcription factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), cytoplasmic chaperone molecule (Keap1), and quinone oxidoreductase (NQO1) were detected by Western blot. 25 μg of protein was loaded into each well and separated by 10% polyacrylamide gel electrophoresis (stacking gel 80V, 20 min; separating gel 120V, 60 min). The separated proteins were electrotransferred to a PVDF membrane. Blocking buffer of 5% skim milk was added, and the membrane was blocked on a shaker at room temperature for 1 h. Wash the membrane three times with TBST for 5 min each time. Add the corresponding Nrf2 antibody (1:1000 dilution), Keap1 antibody (1:1000 dilution), HO-1 antibody (1:1000 dilution), NQO1 antibody (1:1000 dilution), and GAPDH antibody (1:5000 dilution) respectively, and incubate overnight at 4°C. The next day, wash the membrane three times with TBST for 5 min each time, then add HRP-labeled goat anti-rabbit IgG (volume dilution 1:1000), and react at room temperature for 1 h. Wash the membrane three more times with TBST for 5 min each time, and then develop according to the chromogenic kit instructions. Analyze the gray values ​​of each band using a gel imaging analysis system and ImageJ software. The relative protein expression level is calculated as: target protein band gray value / internal control band gray value.

[0115] Studies have found that oxidative stress plays a crucial role in the pathogenesis of lung injury caused by Mycoplasma pneumoniae infection, and changes in the body's redox state affect the development and prognosis of Mycoplasma pneumoniae pneumonia. Nrf2 is currently recognized as a key target with antioxidant activity. Under normal physiological conditions, Nrf2 binds to Keap1 in the cytoplasm and is rapidly degraded by proteases. When Nrf2 is stimulated by oxidative stress, it dissociates from Keap1, translocates to the nucleus, binds to ARE, and initiates the expression of downstream phase II detoxification enzymes of ARE (such as HO-1 and NQO1). Targeting Nrf2 can maintain intracellular redox balance, enabling cells to achieve antioxidant and anti-inflammatory activities.

[0116] The relative protein expression levels of Nrf2 in each group of cells are as follows: Figure 7 As shown in (a). From Figure 7 As shown in (a), compared with the blank group, the relative expression level of Nrf2 protein in A549 cells decreased by 63.67% after infection with Mycoplasma pneumoniae, and the difference was extremely significant (***, P < 0.001); compared with the infection group, the relative expression level of Nrf2 protein in the supernatant group and the lysate group increased by 59.63% and 65.14% respectively, and the differences were also significant (*, P < 0.05).

[0117] The relative expression levels of Keap1 protein in each group of cells are as follows: Figure 7 As shown in (b). From Figure 7 As shown in Figure (b), compared with the control group, the relative expression level of Keap1 protein in A549 cells decreased by 45.00% after infection with Mycoplasma pneumoniae, and the difference was highly significant (***, P < 0.001); compared with the infection group, the relative expression level of Keap1 protein in the supernatant group and the lysate group increased by 32.73% and 49.09% respectively, and the differences were also significant (*, P < 0.05; **, P < 0.01).

[0118] The relative expression levels of HO-1 protein in each group of cells are as follows: Figure 7 As shown in (c). From Figure 7 As shown in (c), compared with the blank group, the relative expression level of HO-1 protein in A549 cells decreased by 46.33% after infection with Mycoplasma pneumoniae, and the difference was extremely significant (***, P < 0.001); compared with the infection group, the relative expression level of HO-1 protein in the supernatant group and the lysate group increased by 52.80% and 56.52% respectively, and the differences were also significant and extremely significant (**, P < 0.01; ***, P < 0.001).

[0119] The relative protein expression levels of NQO1 in each group of cells are as follows: Figure 7 As shown in (d). From Figure 7 As shown in Figure (d), compared with the control group, the relative expression level of NQO1 protein in A549 cells decreased by 64.67% after infection with Mycoplasma pneumoniae, and the difference was extremely significant (***, P < 0.001). Compared with the infection group, the relative expression level of NQO1 protein in the supernatant group and the lysate group increased by 103.77% and 133.96%, respectively, and the differences were also extremely significant (***, P < 0.001).

[0120] The results showed that the expression levels of proteins related to oxidative stress regulation in A549 cells decreased significantly after stimulation by Mycoplasma pneumoniae. However, after treatment with fermentation lysate and supernatant of Lactococcus lactis HC3519, the expression levels of antioxidant-related proteins Nrf2, Keap1, HO-1, and NQO1 in A549 cells increased, indicating improved antioxidant capacity.

[0121] Example 9 Evaluation of the effect of Lactococcus lactis HC3519 on oxidative stress levels in Mycoplasma pneumoniae pneumonia (MPP) mice In this embodiment, the preparation method of Lactococcus lactis HC3519 bacterial liquid is as follows: Inoculate strain HC3519 in MRS liquid medium and culture overnight. After the culture is completed, collect the bacterial liquid and adjust the cell concentration to 1×10 9 CFU / mL for standby, and prepare it freshly each time. The culture method and concentration of Mycoplasma pneumoniae ATCC15531 standard strain are the same as those in Example 8.

[0122] 1. Experimental animals and grouping Select 30 male BALB / c mice at 3 weeks old with a body weight of 18±2 g. The experimental mice are purchased from Shanghai Xipu-Bikai Laboratory Animal Co., Ltd., license number: SCXK (Shanghai) 2013-0016, certificate number: 20130016010285. The experimental mice are randomly grouped as follows: (1) blank group, 6 mice; (2) modeling group, 6 mice; (3) azithromycin group, 6 mice; (4) probiotic group, 6 mice; (5) mixed group, 6 mice. The mice are raised in a SPF-level experimental animal room for 1 week of adaptive feeding. The room temperature is maintained at 22±2°C, the humidity is controlled at 40% - 50%, the light and dark alternate once every 12 h, and standard diet is provided, allowing the mice to eat and drink freely. After 1 week of adaptive feeding, the experiment is started.

[0123] 2. Experimental plan (1) Modeling From the first day of modeling, except for the blank group, mice in all groups are instilled with Mycoplasma pneumoniae ATCC15531 (50 μL / time) once in the morning and once in the evening every day, and the Mycoplasma pneumoniae working bacterial liquid is slowly instilled into the nasal cavity of the mice to enter the tracheobronchus, and it is stopped after 3 consecutive days. From the 8th day, except for the blank group, mice in all groups are instilled with Mycoplasma pneumoniae ATCC15531 (50 μL / time) once in the morning and once in the evening every day until the 10th day. Mice in the blank group are instilled with normal saline (50 μL / time) during the same period. Dissolve 1 mg of azithromycin in 10 mL to prepare a working solution as a positive control.

[0124] During the same period, starting from the first day of modeling, mice in the blank group and the modeling group are gavaged with 200 μL of normal saline once a day for 10 days; mice in the azithromycin group are gavaged with 200 μL of azithromycin solution once a day on the 1st - 3rd days and the 8th - 10th days; mice in the probiotic group are gavaged with 200 μL of Lactococcus lactis HC3519 bacterial liquid once a day for 10 days; mice in the mixed group are gavaged with 200 μL of azithromycin solution once a day on the 1st - 3rd days and the 8th - 10th days, and are gavaged with 200 μL of Lactococcus lactis HC3519 bacterial liquid once a day during the same period for 10 days.

[0125] (2) Detection plan On day 11 of the experiment, all mice in all groups were euthanized, and blood was collected by enucleation. The blood was centrifuged at 8000 r / min, and the supernatant was stored at -80℃ for later analysis. A 3mm × 3mm × 3mm tissue sample from the right lung was taken and stored in 10% formalin for later use.

[0126] The expression levels of nitric oxide synthase (NOS, Solarbio, Cat: SEKH-0159), nitric oxide (NO, Solarbio, Cat: BC1475), malondialdehyde (MDA, Solarbio, Cat: BC6415), and superoxide dismutase (SOD, Solarbio, Cat: BC5165) in mouse serum were detected by biochemical methods. The detection procedures and calculation methods were performed in accordance with the instructions of the detection kit.

[0127] The DNA content of Mycoplasma pneumoniae ATCC15531 in mouse lung tissue homogenate was detected by real-time PCR. 50 mg of mouse lung tissue was collected, homogenized, and lysed to obtain a tissue homogenate. DNA extraction was performed according to the instructions of the kit (Tiangen, DP340). Real-time PCR was performed according to the instructions of the Mycoplasma pneumoniae nucleic acid detection kit (DaAn Gene, Cat: DA-D064).

[0128] 3. Data Statistics and Analysis All data are expressed as mean ± standard error (mean ± sd). Comparisons among multiple groups were performed using one-way ANOVA. P <0.05 is considered statistically significant.

[0129] 4. Experimental Results (1) Mycoplasma pneumoniae DNA content in lung tissue The Mycoplasma pneumoniae DNA content in the lung tissue of mice in each group is shown in Table 4.

[0130] Table 4 - Mycoplasma pneumoniae DNA content in mouse lung tissue

[0131] Note: Compared with the blank group, different letters represent significant differences (P < 0.05). Table 4 shows that, compared with the modeling group, the levels of Mycoplasma pneumoniae DNA in the lung tissue of mice in the azithromycin group, probiotic group, and mixed group were significantly lower (P < 0.05), indicating that both azithromycin and Lactococcus lactis HC3519 can reduce the Mycoplasma pneumoniae load in mouse lung tissue. Furthermore, compared with the azithromycin group, the levels of Mycoplasma pneumoniae DNA in the lung tissue of mice in the mixed group were significantly lower (P < 0.05), indicating that the use of azithromycin combined with gavage administration of Lactococcus lactis HC3519 was more effective in inhibiting Mycoplasma pneumoniae proliferation and further reducing its load. Therefore, Lactococcus lactis HC3519 alone can reduce the Mycoplasma pneumoniae load in the lungs of mice with Mycoplasma pneumoniae pneumonia, and the combination of azithromycin and Lactococcus lactis HC3519 is more effective in reducing the Mycoplasma pneumoniae load in the lungs of mice with Mycoplasma pneumoniae pneumonia.

[0132] (2) Mycoplasma pneumoniae can attach to the cell membrane, insert microtubules into the cell and release hydrogen peroxide and superoxide free radicals. Neutrophils and other cells can activate NOS and produce excessive NO during the inflammatory response. Therefore, lung injury caused by oxidative stress is closely related to the occurrence and development of Mycoplasma pneumoniae pneumonia.

[0133] Changes in serum NOS levels in each group of mice are as follows: Figure 8 As shown in (a). By Figure 8 As shown in (a), compared with the blank group, the NOS level of mice in the modeling group increased by 2.32 times and the difference was statistically significant (P < 0.05); compared with the modeling group, the NOS level of mice in the azithromycin group, probiotic group and mixed group decreased and the differences were statistically significant (P < 0.05); compared with the azithromycin group, the NOS level of mice in the mixed group decreased and the difference was statistically significant (P < 0.05).

[0134] Changes in serum NO levels in each group of mice are as follows: Figure 8 As shown in (b). From Figure 8 As can be seen in (b), its level change shows the same trend as NOS.

[0135] Oxidative stress releases excessive amounts of reactive oxygen species (ROS), which bind to lipids, proteins, and DNA, causing peroxidation. This leads to apoptosis and even necrosis of cells through cellular oxidative stress, ultimately resulting in the destruction of tissue structure and function. In vivo, free radicals act on lipids to cause peroxidation, with MDA as the final product. MDA causes cross-linking and polymerization of biomolecules such as proteins and nucleic acids, and it is cytotoxic, making it an important indicator for assessing the degree of oxidative damage in the body.

[0136] Changes in serum MDA levels in each group of mice are as follows: Figure 8 As shown in (c). From Figure 8 As can be seen from (c), its level change shows the same trend as the changes in NOS and NO.

[0137] Studies have shown that peripheral blood mononuclear cells in patients with Mycoplasma pneumoniae pneumonia exhibit oxidative stress. When the body is attacked by external pathogens, neutrophils, as the body's main source of reactive oxygen species (ROS), are activated and produce large amounts of ROS, while superoxide dismutase (SOD) is widely involved in the acute inflammatory response, and its content decreases significantly. When ROS production exceeds the body's antioxidant reserves, the impact of oxygen free radicals on lung tissue increases significantly. Therefore, SOD can be used to evaluate the body's antioxidant capacity.

[0138] Changes in serum SOD levels in each group of mice are as follows: Figure 8 As shown in Figure (d), compared with the blank group, the SOD level of mice in the modeling group decreased by 63.24% and the difference was statistically significant (P < 0.05); compared with the modeling group, the SOD level of mice in the azithromycin group, probiotic group and mixed group increased and the differences were statistically significant (P < 0.05); compared with the azithromycin group, the SOD level of mice in the mixed group increased and the difference was statistically significant (P < 0.05).

[0139] The above results indicate that *Lactococcus lactis* HC3519 can regulate oxidative responses in mice, effectively reducing NOS, NO, and MDA levels, increasing SOD levels, and decreasing *Mycoplasma pneumoniae* DNA levels, thereby alleviating oxidative stress caused by *Mycoplasma pneumoniae* and reducing the viral load of *Mycoplasma pneumoniae* in lung tissue. Furthermore, the combined use of *Lactococcus lactis* HC3519 with azithromycin in the treatment of *Mycoplasma pneumoniae* pneumonia demonstrates a more significant effect in regulating oxidative stress and reducing lung inflammation.

[0140] 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 Lactococcus lactis strain HC3519, characterized in that, The lactococcus lactis ( Lactococcus lactis HC3519 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30765, on May 24, 2024. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The Lactococcus lactis HC3519 of claim 1, characterized in that, The 16S rDNA sequence of the lactococcus HC3519 is shown in SEQ ID NO.

3.

3. An inoculant characterized in that, The bacterial agent contains the fermentation broth of Lactococcus lactis HC3519 as described in claim 1, the strain lysate of Lactococcus lactis HC3519 as described in claim 1, or the fermentation supernatant of Lactococcus lactis HC3519 as described in claim 1; the strain lysate is obtained by ultrasonically disrupting the strain fermentation broth using an ultrasonic disruptor.

4. The bacterial agent as claimed in claim 3, characterized by The concentration of Lactococcus lactis HC3519 in the fermentation liquor is ≥ 1 × 10 9 CFU / mL.

5. The bacterial agent of claim 3, wherein The fermentation supernatant was obtained by centrifuging the fermentation broth of Lactococcus lactis HC3519.

6. The bacterial agent of claim 4 or 5, wherein The fermentation broth of the strain is prepared by culturing Lactococcus lactis HC3519 in MRS liquid medium at 37°C for 24-28 hours.

7. The bacterial agent as claimed in claim 6, characterized by 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, and 1 L of pure water to adjust the pH to 5.7 ± 0.

2.

8. The use of Lactococcus lactis HC3519 as described in claim 1 in the preparation of products for inhibiting and preventing Mycoplasma pneumoniae.

9. The use of Lactococcus lactis HC3519 as described in claim 1 in the preparation of antioxidant products.

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