Lactobacillus gasseri, polypeptide, and saliva-promoting, throat-protecting, cough-relieving, and lung-moistening fermentation liquor, and preparation method and application thereof

Abstract

This invention provides *Lactobacillus gasseri*, polypeptides, and a fermented liquid for promoting saliva production, soothing the throat, relieving cough, and moisturizing the lungs, along with their preparation methods and applications. Specifically, the strain number of *Lactobacillus gasseri* is YS-LG03, and its preservation number is CGMCC No. 37762. This invention also provides antimicrobial polypeptides produced by the *Lactobacillus gasseri*. Furthermore, this invention discloses a fermentation broth obtained by fermenting raw materials such as *Ophiopogon japonicus* and *Polygonatum sibiricum* using this strain. The abundance of metabolites such as acetylcholine in the fermentation broth is significantly increased, with the relative abundance of acetylcholine increasing by 14.39 times compared to before fermentation. This fermentation broth also contains antimicrobial polypeptides and other active metabolite components. The fermentation broth provided by this invention can reduce the frequency of citric acid-induced coughs, promote saliva secretion, and alleviate lung inflammation caused by smoking. It can be used to prepare medicines with antibacterial, saliva-promoting, throat-soothing, cough-relieving, lung-moisturizing, and anti-inflammatory effects.

Description

Technical Field

[0001] This invention belongs to the field of fermentation of probiotics, microbial metabolites and medicinal and edible raw materials, specifically involving Lactobacillus gasseri, polypeptides and fermentation liquids that promote saliva production, protect the throat, relieve cough and moisten the lungs, as well as their preparation methods and applications. Background Technology

[0002] Dry throat, itchy throat, throat discomfort, and irritating cough can be caused by various factors such as dry environment, overuse of the voice, upper respiratory tract microecological imbalance, or airway inflammation. Existing related products mostly use plant extracts, syrups, lozenges, or antibacterial drugs. Plant extracts often suffer from insufficient release of active ingredients, significant batch-to-batch variations, and unclear target points; antibacterial drugs are unsuitable for long-term use without indication. Developing a safe product that simultaneously inhibits common throat pathogens, regulates inflammatory responses, and improves mucosal moisture is of practical significance.

[0003] Probiotics, as a safe microbial resource, can produce active substances such as organic acids, peptides, and short-chain fatty acids through metabolism, showing potential applications in regulating the microecology, inhibiting pathogenic bacteria, and alleviating inflammation. Bacteriocins, as protein or peptide antibacterial substances synthesized by probiotic ribosomes, possess characteristics such as being degradable by proteases, having a low risk of in vivo residue, and exhibiting clear antibacterial activity, making them an important direction for replacing traditional antibacterial agents. However, currently reported lactic acid bacteria bacteriocins are mostly developed targeting foodborne spoilage bacteria or intestinal pathogens, and strains with high antibacterial activity against common throat pathogens such as Streptococcus pyogenes remain limited.

[0004] Lactobacillus gasseri is a human-native probiotic that has been widely used in the regulation of intestinal, gastric, and reproductive tract microecologies. For example, Chinese patent CN117025479B discloses Lactobacillus gasseri SHMB 0001 and its application in alleviating acute colitis, which exerts intestinal protective effects by protecting the intestinal mucosal barrier and upregulating tight junction protein expression; Chinese patent application CN117801994A discloses Lactobacillus gasseri HP2C12, mainly used for vaginal microecological regulation, with both antibacterial and antioxidant properties; and Chinese patent CN114561313B discloses Lactobacillus gasseri LG-7, which can inhibit Helicobacter pylori and colonize the stomach. All of these studies focus on intestinal, gastric, or vaginal settings. There are currently no reports of Lactobacillus gasseri exhibiting significant inhibitory activity against common pathogenic bacteria in the throat, or being used in the fermentation of food and medicinal materials to achieve the effects of promoting saliva production, protecting the throat, relieving cough, and moisturizing the lungs.

[0005] In summary, current technologies lack *Lactobacillus gasseri* strains that possess multiple functions, including inhibiting throat pathogens, regulating airway inflammation, and promoting glandular secretion. Furthermore, there is a lack of products prepared using fermented medicinal and edible raw materials from these strains, demonstrating a clear mechanism and synergistic effects for relieving sore throat, cough, and lung dryness. Therefore, screening for highly active *Lactobacillus gasseri* strains that target throat pathogens and developing its peptides and medicinal / edible fermented products is of significant practical importance for enriching the technological pathways for throat-protecting and lung-drying products and enhancing their efficacy. Summary of the Invention

[0006] The first objective of this invention is to provide Lactobacillus gasseri YS-LG03, which has an inhibitory effect on common pathogenic bacteria in the throat and has good anti-inflammatory activity.

[0007] A second objective of the present invention is to provide the polypeptide YS-LG03-MAX produced by the Lactobacillus gasseri YS-LG03.

[0008] A third objective of this invention is to provide a method for preparing the polypeptide YS-LG03-MAX.

[0009] The fourth objective of this invention is to provide a fermented liquid that promotes saliva production, protects the throat, relieves cough, and moisturizes the lungs, obtained by fermenting the medicinal and edible raw material from Lactobacillus gasseri YS-LG03.

[0010] The fifth objective of the invention is to provide a method for preparing and applying a fermented liquid that promotes saliva production, protects the throat, relieves cough, and moisturizes the lungs, obtained by fermenting medicinal and edible raw materials from Lactobacillus gasseri YS-LG03.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A strain of *Lactobacillus gasseri* YS-LG03, with accession number CGMCC No. 37762, was deposited on February 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. *Lactobacillus gasseri* YS-LG03 was viable at the time of deposit.

[0012] This invention also discloses a polypeptide produced by the aforementioned Lactobacillus gasseri YS-LG03, the amino acid sequence of which is: AYGLLNIAVAGGKIDASMKG.

[0013] This polypeptide is present in Lactobacillus gasseri YS The antibacterial active component produced by LG03 is named YS. LG03 MAX exhibits good thermal stability and a wide range of pH stability.

[0014] The polypeptide YS-LG03-MAX is a linear peptide, and its chemical structure is as follows: .

[0015] A method for preparing the aforementioned polypeptide includes the following steps: The Lactobacillus gasseri YS-LG03 was cultured to obtain the fermentation supernatant; Salt was added to the fermentation supernatant to carry out a salting-out reaction. After centrifugation, the precipitate was collected, reconstituted, and dialyzed to obtain a crude polypeptide extract. The crude extract of the polypeptide was filtered through a micromembrane and then purified by gel filtration chromatography, gel chromatography, and high-performance liquid chromatography to obtain the polypeptide.

[0016] The salt includes ammonium sulfate; The solution used for reconstitution is a phosphate buffer solution with a pH of 5.5-6.5 and a concentration of 0.05-0.1 mol / L. The gel filtration chromatography includes the steps of loading the filtered crude peptide extract onto a Sephadex G-25 chromatography column and eluting with a pH of 5.5-6.5 and a 0.05-0.1 mol / L phosphate buffer. The elution flow rate for the gel filtration chromatography is 0.5-1.5 mL / min; The pore diameter of the micromembrane is 0.2-0.3 μm.

[0017] The gel chromatography column used was Sephadex LH-20, and the eluent was a methanol solution with a volume fraction of 75-85%. The high-performance liquid chromatography separation and purification includes the step of chromatographic purification using a Venusil XBP C18 semi-preparative chromatographic column; The mobile phase A used for high-performance liquid chromatography separation and purification is a pure aqueous solution; the mobile phase B is an acetonitrile solution. The elution program for high performance liquid chromatography separation and purification is as follows: 0-20 min, 0%-60% linear gradient elution of phase B; 20-25 min, 60%-100% of phase B; 25-30 min, phase B maintained at 100%.

[0018] A fermented liquid that promotes saliva production, protects the throat, relieves cough, and moisturizes the lungs, wherein the fermented liquid is obtained by fermenting food-medicine homologous substrates with Lactobacillus gasseri YS-LG03; The medicinal and edible substrates include Ophiopogon japonicus, Polygonatum sibiricum, Polygonatum odoratum, Siraitia grosvenorii, Citrus reticulata peel, and Mentha haplocalyx in a weight ratio of 14-16∶8-10∶8-10∶5-7∶5-7∶2-4.

[0019] The method for preparing the fermentation broth includes the following steps: The food-medicine homology substrate is sterilized with a solvent to obtain the fermentation substrate; The Lactobacillus gasseri YS-LG03 was inoculated into the fermentation substrate for fermentation. After fermentation, sterilize and centrifuge, and collect the supernatant to obtain the final product.

[0020] The solvent includes water; The ratio of the medicinal and edible substrate to water is 1:9-11 (w / v). The sterilization conditions for the fermentation substrate are 110-121℃ and 10-20min.

[0021] The inoculation amount of Lactobacillus gasseri YS-LG03 is 1.5-2.5% (v / v). The fermentation temperature is 30-35℃, and the time is 42-54 hours; The sterilization conditions for the fermentation broth are 100-110℃ and 5-30 min. The centrifugation conditions are 3000-5000 rpm for 4-6 min.

[0022] The fermentation broth is used to prepare medicines that relieve dry throat, reduce coughing frequency, or alleviate airway inflammation; or to prepare medicines with anti-inflammatory functions.

[0023] Compared with the prior art, the beneficial effects of the present invention are: (1) The Lactobacillus gasseri YS-LG03 screened in this invention has a strong antibacterial effect against Streptococcus pyogenes, and its effect on improving macrophage inflammation caused by Streptococcus pyogenes is better than that of commercially available Lactobacillus gasseri.

[0024] (2) A novel polypeptide YS was identified from the antibacterial active component produced by Lactobacillus gasseri YS-LG03. LG03 MAX, this peptide is tolerant to a wide range of temperatures and pH levels, has a strong antibacterial ability against Streptococcus pyogenes, and can improve macrophage inflammation caused by Streptococcus pyogenes.

[0025] (3) Fermentation of Ophiopogon japonicus, Polygonatum sibiricum, Polygonatum odoratum, Siraitia grosvenorii, Citrus reticulatae and Mentha haplocalyx using Lactobacillus gasseri YS-LG03 can significantly reshape the substrate metabolite profile and increase the relative abundance of metabolites such as acetylcholine, spermidine, phenyllactic acid, hydroxyphenyllactic acid and α-linolenic acid.

[0026] (4) Animal experiments showed that the fermented liquid of the medicine and food homology could reduce the number of coughs in citric acid-induced cough guinea pigs by 58.11% within 5 minutes compared with the model group, increase the amount of saliva secretion in smoke-smoked and atropine-induced dry mouth mice by 50.37% compared with the model group, and significantly reduce the levels of inflammatory cells, TNF-α and IL-6 in the bronchoalveolar lavage fluid of smoke-smoked mice. Attached Figure Description

[0027] Figure 1 This is a microscopic image of Lactobacillus gasseri YS-LG03 in one embodiment of the present invention; Figure 2 This is a diagram illustrating the antibacterial effects of different strains against Streptococcus pyogenes ATCC 19615 in one embodiment of the present invention. Figure 3 The image shows the antibacterial effect of Lactobacillus gasseri YS-LG03 and a commercial strain against Streptococcus pyogenes ATCC19615 in one embodiment of the present invention. Figure 4 The thermal stability and pH stability of the antibacterial substances in the fermentation supernatant of Lactobacillus gasseri YS-LG03 in one embodiment of the present invention; Figure 5 This is a diagram illustrating the antibacterial effect of polypeptide YS-LG03-MAX against Streptococcus pyogenes ATCC 19615 in one embodiment of the present invention. Figure 6 This is a principal component analysis (PCA) score diagram of the medicinal and edible homology composition before and after fermentation in one embodiment of the present invention; Figure 7 This is a volcano diagram of the differential metabolites of the food-medicine homology composition before and after fermentation in one embodiment of the present invention; Figure 8 This is a distribution diagram of the different metabolites before and after fermentation of the food-medicine homology composition in one embodiment of the present invention. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0029] Example 1 This embodiment provides a process for screening strains with antibacterial function.

[0030] Specifically, the strains used in this screening process were sourced from the laboratory strain bank of Hunan Nutrition Tree Biotechnology Co., Ltd. The specific screening process is as follows: A batch of highly active Gram-positive bacterial strains, totaling 10 strains, were screened from the laboratory strain library. These strains were YS-LP01, YS-LP15, and... YS-LM07, YS-LM10, YS-LM13, YS-LG03, YS-LG11, YS-LG18, YS-LC05, and YS-LC09. See Table 1 for details.

[0031] The strain was streaked on MRS solid medium, and a single colony was picked and inoculated into MRS liquid medium. The culture was carried out at 37°C for 24 hours, and activated for two generations before use.

[0032] Streptococcus pyogenes ATCC 19615 was streaked on BHI solid medium, and single colonies were picked and concentrated in BHI liquid medium. The culture was carried out at 37°C for 24 hours, and activated for two generations before use.

[0033] The antibacterial effect was tested using the Oxford cup perforation method. Streptococcus pyogenes ATCC 19615 was selected as the indicator bacterium. The concentrations of the bacterial suspensions of the indicator bacterium and the test bacterium were adjusted to 10⁻⁶ using sterile physiological saline. 6 -10 7 CFU / mL, spread on BHI solid medium and incubated at 37℃ for 48h.

[0034] Culture medium used: MRS solid culture medium: Product No.: 027319; Manufacturer: Guangdong Huankai Biotechnology Co., Ltd. MRS liquid culture medium: Product number: 027312; Manufacturer: Guangdong Huankai Biotechnology Co., Ltd. BHI liquid culture medium: Product number: HB8297-1; Manufacturer: Qingdao Haibo Biotechnology Co., Ltd.; BHI solid culture medium: Add 1.5% agar powder to the liquid culture medium before use.

[0035] Table 1. Inhibitory ability of different strains against indicator bacteria (inhibition zone: mm) As shown in the table above, the 10 candidate strains exhibited varying degrees of antibacterial activity against Streptococcus pyogenes ATCC 19615. Among them, YS-LG03 showed an inhibition zone diameter of 21.46 mm against Streptococcus pyogenes ATCC 19615, demonstrating superior antibacterial activity compared to the other strains. YS-LM07, YS-LC05, and YS-LC09 also showed strong antibacterial activity, and therefore these four strains were selected for further screening.

[0036] The specific process for strain rescreening is as follows: To eliminate false positives caused by bacterial colony obstruction, four initially screened strains were inoculated at a 2% inoculum into MRS liquid medium and cultured at 37°C and 60 rpm for 24 h. After culture, the fermentation supernatant was collected by centrifugation at 8000 rpm for 5 min, and a second screening was conducted using the Oxford cup perforation method. The inhibition zones of the supernatant of each strain against Streptococcus pyogenes ATCC 19615 were observed. Figure 2 .

[0037] Table 2. Rescreening of the antibacterial ability of strain supernatant (inhibition zone: mm) The results are shown in Table 2. The inhibition zone of YS-LG03 fermentation supernatant against Streptococcus pyogenes ATCC 19615 was 20.62 mm (the size of the inhibition zone varied among different batches), indicating the best antibacterial effect. Therefore, YS-LG03 was selected as the strain with the best antibacterial performance for subsequent experimental studies.

[0038] Based on this, Shenzhen BGI Genomics Co., Ltd. was commissioned to conduct biological identification of the strain. The assembled sequence was BLAST-aligned in GenBank, and the results showed that the strain was identified as *Lactobacillus gasseri*, and it was deposited on February 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37762. Microscopic images of the strain are shown below. Figure 1 Its 16S rRNA gene sequence is shown in SEQ ID NO. 1.

[0039] SEQ ID NO. 1: To verify that Lactobacillus gasseri YS-LG03 has a significant advantage over commercially available Lactobacillus gasseri, three comparative groups were set up. Except for the different samples, the diameter of the inhibition zone of each strain's fermentation supernatant against Streptococcus pyogenes ATCC 19615 was measured under identical conditions.

[0040] Comparative Example 1: Commercially available Lactobacillus gasseri JCM 5344 was used; Comparative Example 2: Commercially available Lactobacillus gasseri LA39 was used; Comparative Example 3: Commercially available Lactobacillus gasseri ATCC 33323 was used.

[0041] Table 3. Antibacterial effects of fermentation supernatant from different bacterial strains (inhibition zone: mm) Note: "-" indicates that no inhibition zone was detected.

[0042] As shown in the table above, *Lactobacillus gasseri* JCM 5344 (Comparative Example 1) has no inhibitory effect on *Streptococcus pyogenes* ATCC 19615. In the prior art, *Lactobacillus gasseri* LA39 (Comparative Example 2) can secrete the bacteriocin Gasseri A, but it only has inhibitory effects against foodborne pathogens and has no inhibitory activity against *Streptococcus pyogenes* ATCC 19615; *Lactobacillus gasseri* ATCC 33323 (Comparative Example 3) has some inhibitory ability against *Streptococcus pyogenes* ATCC 19615, but the inhibitory effect is far less than that of YS-LG03 (the size of the inhibition zone varies between different batches; see the specific inhibition zone image for details). Figure 3 ).

[0043] In summary, the YS-LG03 strain of this invention exhibits strong antibacterial activity against Streptococcus pyogenes ATCC 19615, and its antibacterial performance is superior to that of existing commercially available Lactobacillus gasseri standard strains, demonstrating excellent application advantages.

[0044] Example 2 This embodiment identifies and analyzes the antibacterial substances produced by Lactobacillus gasseri YS-LG03.

[0045] In Example 1, this invention verified the inhibitory effect of YS-LG03 supernatant on pathogenic bacteria. In order to analyze the chemical nature of the antibacterial active substances in YS-LG03 fermentation supernatant, this example preliminarily identified the antibacterial substances by excluding interfering factors such as organic acids and hydrogen peroxide, and by combining protease sensitivity test.

[0046] Sample preparation: The fermentation broth of Lactobacillus gasseri YS-LG03 was prepared according to the method in Example 1. The supernatant was collected by centrifugation to obtain the fermentation supernatant of Lactobacillus gasseri YS-LG03, which was then stored at 4°C for later use.

[0047] To eliminate interference from organic acids: Take the fermentation supernatant and adjust the pH to 6.5 (to eliminate the antibacterial activity of organic acids). Use the unadjusted fermentation supernatant as a control and use Streptococcus pyogenes ATCC 19615 as an indicator bacterium. Determine the diameter of the inhibition zone using the Oxford cup method.

[0048] To eliminate the interference of hydrogen peroxide: Take the fermentation supernatant, add catalase to a final concentration of 5 mg / mL, and treat in a 37°C water bath for 2 h. Use the untreated fermentation supernatant as a control to determine the diameter of the inhibition zone against Streptococcus pyogenes ATCC 19615.

[0049] Protease sensitivity test: Fermentation supernatant was taken and proteinase K, trypsin, and pepsin were added to a final concentration of 1 mg / mL. Each treatment group was incubated in a 37℃ water bath (the pepsin group was incubated at 37℃ and pH 2.0) for 2 h. The diameter of the inhibition zone against Streptococcus pyogenes ATCC 19615 was measured. Fermentation supernatant without enzymes (incubated under the same conditions) was used as a blank control.

[0050] Thermal stability test: Fermentation supernatant was treated in water baths at 40℃, 60℃, 80℃, and 100℃ for 30 min, and then autoclaved at 121℃ for 15 min. After cooling to room temperature, the diameter of the inhibition zone against Streptococcus pyogenes ATCC 19615 was measured.

[0051] pH stability test: Take the fermentation supernatant and adjust the pH to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. After standing at 4℃ for 4 hours, adjust the pH back to 6.5 and measure the diameter of the inhibition zone against Streptococcus pyogenes ATCC 19615.

[0052] Table 4. Antibacterial results of fermentation supernatant of strain YS-LG03 after different treatments. Table 4 shows that the YS-LG03 fermentation supernatant retained over 90% of its activity after pH adjustment to 6.5 and catalase treatment, indicating that organic acids and hydrogen peroxide were not the primary antibacterial substances. Treatment with proteinase K, trypsin, and pepsin completely destroyed the antibacterial activity, and no inhibition zone was observed. These results indicate that the antibacterial active substances produced by YS-LG03 are sensitive to proteases and possess protein or polypeptide properties, consistent with typical characteristics of proteins or polypeptides.

[0053] The results of thermal and pH stability of YS-LG03 fermentation supernatant are shown in the table below. Figure 4 ,like Figure 4As shown, the antibacterial substance retains ≥90% activity after treatment at 40-100℃ for 30 min and ≥70% activity after autoclaving at 121℃ for 15 min, indicating good thermal stability. After treatment at pH 2.0-10.0 for 4 h, its activity remains ≥85%, indicating broad pH stability.

[0054] Based on the above results, the antibacterial active substance produced by YS-LG03 has the characteristics of being degradable by proteases, thermal stability, and pH stability, and can be preliminarily identified as a protein or polypeptide active substance.

[0055] The isolation and purification process of this antibacterial substance is as follows: Take the fermentation broth of YS-LG03, centrifuge at 10000×g for 30 min at 4℃, collect the supernatant, add ammonium sulfate to the supernatant until 55% saturation, let stand overnight at 4℃, and centrifuge to remove the precipitate. Continue to add ammonium sulfate to the supernatant until 80% saturation, let stand overnight at 4℃, and centrifuge to obtain the precipitate. Dissolve the precipitate in 1 / 40 of the original volume of 0.05mol / L, pH 6.0 phosphate buffer.

[0056] The above samples were placed in dialysis bags with a molecular weight cutoff of 1000~2000 Da and dialyzed at 4℃ for 24 h, with the solution changed 3~4 times during the process to remove the influence of salt ions.

[0057] Take 0.5 mL of the dialysis sample, filter it through a 0.22 μm micromembrane, and load it onto a Sephadex G-25 column (1.6 × 60 cm). Use 0.05 mol / L, pH 6.0 phosphate buffer as the eluent, and collect each fraction automatically at a flow rate of 1 mL / min. Use Streptococcus pyogenes ATCC 19615 as the indicator bacterium and determine the antibacterial activity of each fraction using the Oxford cup method.

[0058] The antibacterial components were combined, evaporated to dryness, and then reconstituted with an equal volume of 80% methanol. The samples were then loaded onto a Sephadex LH-20 chromatography column (1.6×120cm) and further purified using 80% chromatographic grade methanol as the mobile phase. Each fraction was automatically collected, and the antibacterial activity was verified according to the above method.

[0059] The active component obtained after purification using Sephadex LH-20 was filtered through a 0.22 μm micromembrane and further purified using semi-preparative high-performance liquid chromatography (HPLC): the chromatographic column was Venusil XBP C18 (5 μm, 4.6 × 150 mm), mobile phase A was pure aqueous solution, mobile phase B was acetonitrile solution, the detection wavelength was 215 nm, the flow rate was 1 mL / min, the sample loading volume was 0.1 mL, and the elution program was: 0–20 min, 0–60% linear gradient of phase B; 20–25 min, 60%–100% of phase B; 25–30 min, phase B held at 100%. The eluent fraction with antibacterial activity was collected, and the obtained active eluent fraction was re-loaded into HPLC for purity detection, and then freeze-dried and stored at -80℃.

[0060] The antibacterial active component obtained by semi-preparative high-performance liquid chromatography (HPLC) was purified by HPLC. The sample was filtered through a 0.22 μm micromembrane and analyzed using HPLC: the column was a ZORBAX Eclipse Plus C18 (5 μm, 4.6 × 250 mm); mobile phase A was a pure aqueous solution; mobile phase B was an acetonitrile solution; the detection wavelength was 215 nm; the flow rate was 1 mL / min; the sample loading volume was 0.1 mL; and the elution program was: 0–20 min, B phase 0–60%; 20–25 min, B phase 60%–100%; 25–30 min, B phase maintained at 100%. The results showed that the antibacterial active component purified by semi-preparative HPLC had a purity of over 95% as determined by HPLC. This purified antibacterial active component was named YS-LG03-MAX.

[0061] The N-terminal amino acid sequence was determined using the Edman degradation method, with 15 cycles yielding the N-terminal 15 amino acid residue sequence AYGLLNIAVAGGKID. To further confirm the full-length sequence, the purified sample underwent de novo LC-MS / MS mass spectrometry sequencing. The resulting MS / MS spectra were analyzed using PEAKS Studio software, showing a peptide fragment ion coverage of over 90%, and the obtained N-terminal sequence was in perfect agreement with the Edman degradation results. Sequencing results indicated that YS-LG03-MAX is a polypeptide with the full-length amino acid sequence H2N-AYGLLNIAVAGGKIDASMKG-OH (SEQ ID NO. 2). BLASTp alignment of this full-length sequence with known polypeptide sequences in the NCBI non-redundant protein database (nr) showed no known polypeptide sequences that were either completely identical or highly homologous to this sequence.

[0062] The molecular weight of the peptide was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in linear positive ion mode. The matrix used was α-cyano-4-hydroxycinnamic acid (CHCA), and a matrix solution (50% acetonitrile-0.1% trifluoroacetic acid aqueous solution) was prepared. The sample and matrix were mixed in equal volumes and applied to the target. Mass calibration was performed using the external standard method. The molecular weight was determined to be 1949.06 Da. Based on the amino acid sequence shown in SEQ ID NO.2, the theoretical molecular formula of the peptide was calculated to be C0. 86 H 145 N 23 O 26 The theoretical molecular weight of YS-LG03-MAX is 1948.05 Da, and the measured value deviates from the theoretical value by 1.01 Da (approximately 0.05%), which is within the normal allowable error range for MALDI-TOF mass spectrometry (usually ≤0.1%–0.2%). In summary, YS-LG03-MAX is a linear peptide with a free amino group at the N-terminus and a free carboxyl group at the C-terminus. No significant post-translational modifications were detected in the mass spectrometry results, and its primary amino acid sequence has been determined. Database comparisons did not yield any previously reported peptides with high homology, suggesting that YS-LG03-MAX is a novel peptide.

[0063] Using the Oxford Cup method to evaluate YS LG03 MAX was used to verify the reproducibility of its antibacterial activity at a concentration of 0.1 mg / mL, with sterile pure water as a negative control. The results are shown in Table 5. LG03 MAX peptides exhibit good antibacterial activity against Streptococcus pyogenes ATCC 19615 (see image for inhibition zone). Figure 5 ).

[0064] Table 5. Antibacterial effect of peptide YS-LG03-MAX against Streptococcus pyogenes ATCC 19615 (inhibition zone: mm) The references for the method in this embodiment are: Zhang Manmin, Zeng Xiangyi, Fang Limin, et al. Isolation, purification and physicochemical stability analysis of bacteriocin produced by Lactobacillus plantarum C010 [J]. Food and Fermentation Industries, 2023, 49(3):31-37. Li Yaling. Isolation, purification and physicochemical properties of bacteriocins from Pyrococcus lactis [D]. Tianjin University, 2007.

[0065] Example 3 Protective effect of Lactobacillus gasseri YS-LG03 and its polypeptide YS-LG03-MAX against macrophage inflammatory damage Macrophages, as important professional immune cells in the body, play a crucial role in clearing pyogenic streptococcal infections and regulating inflammatory responses. Therefore, this embodiment establishes a RAW264.7 macrophage infection model to investigate whether this peptide can inhibit the excessive inflammatory response caused by infection, reduce cell damage, and further elucidate the anti-infection mechanism of YS-LG03-MAX, providing cellular-level experimental evidence for its subsequent in vivo application.

[0066] Sample preparation: The activated YS-LG03 and commercially available control strains (comparative examples 1-3) were inoculated into MRS liquid medium and fermented at 37°C with shaking. The fermentation supernatant was obtained by centrifugation and filtered through a 0.22μm filter membrane for sterilization. The YS-LG03-MAX peptide was isolated and purified from the above examples and dissolved in sterile PBS to prepare the stock solution.

[0067] Cell model construction: RAW264.7 macrophages were resuscitated and cultured in DMEM high-glucose medium (containing 10% FBS and 1% penicillin-dextrose antibody) at 37°C and 5% CO2. Once the cells reached a suitable growth state, they were seeded and plated. Streptococcus pyogenes ATCC 19615 was added at a multiplicity of infection (MOI) of 10 and incubated for 2 hours to construct a cell infection and inflammation model. After infection, the supernatant was discarded, and the cells were washed three times with PBS to remove free bacteria before being divided into groups for drug administration.

[0068] The groups are as follows: Blank control group (Group C, normal cells without infection, no drug treatment), Model group (Group M, Streptococcus pyogenes infection, no drug treatment), YS-LG03 supernatant group (LG03 group, drug volume fraction 5%, v / v), YS LG03 MAX treatment group (MAX group, final drug concentration 0.1 mg / mL). Each group was added with the corresponding sample, and the blank group and model group were added with an equal volume of blank MSR medium. They were incubated at 37℃ and 5% CO2 for 12 h.

[0069] Indicator detection: After incubation, cells and cell culture supernatant from each group were collected, and the viability of macrophages in each group was detected by MTT assay. The secretion levels of pro-inflammatory factors (TNF-α, IL-6, IL-1β) and anti-inflammatory factors (IL-10) in the cell supernatant were detected according to the instructions of the corresponding ELISA kit.

[0070] Comparative Example 4: Unlike the YS-LG03 supernatant group, the fermentation supernatant of commercially available Lactobacillus gasseri JCM-5344 was used; Comparative Example 5: Unlike the YS-LG03 supernatant group, this one uses the fermentation supernatant of commercially available Lactobacillus gasseri LA-39; Comparative Example 6: Unlike the YS-LG03 supernatant group, this example uses the fermentation supernatant of commercially available Lactobacillus gasseri ATCC 33323.

[0071] Table 6 Results of the improvement effect of RAW264.7 macrophage infection model The results are shown in Table 6. After infection with Streptococcus pyogenes, the viability of RAW264.7 macrophages in group M was significantly reduced, and the pro-inflammatory factor TNF was elevated. α, IL 6. IL Massive release of 1β, anti-inflammatory factor IL The decrease in secretion levels (10) indicates the successful establishment of the macrophage infection model. Compared with group M, the fermentation supernatant of group LG03 can improve cell viability to a certain extent, inhibit the secretion of pro-inflammatory factors, and upregulate the anti-inflammatory factor IL-1. Level 10; the MAX purified peptide treatment group showed the most significant improvement, with cell viability restored to the level of the blank control group, and its TNF level... α, IL 6. IL 1β secretion levels were significantly reduced, while the anti-inflammatory factor IL-1 was effectively increased. 10% secretion. The three control Lactobacillus gasseri samples showed only weak protective and anti-inflammatory effects, with overall efficacy weaker than YS. LG03 fermentation supernatant and purified peptide YS LG03 MAX. The results show that YS LG03 MAX peptides can alleviate macrophage damage caused by Streptococcus pyogenes infection, inhibit excessive inflammatory response in the body, and have good immunomodulatory activity.

[0072] The references for the method in this embodiment are: Niu Junbo. Study on the regulatory effect and mechanism of purple sweet potato polysaccharide PSPP-1 on lipopolysaccharide-induced inflammatory response of RAW264.7 macrophages [D]. Harbin University of Commerce, 2023. Kurosawa M, Oda M, Domon H, et al. Streptococcus pyogenes CAMP factorattenuates phagocytic activity of RAW 264.7 cells[J]. Microbes and Infection, 2016, 18(2): 118-127. Example 4 This embodiment provides a method for screening medicinal and edible raw materials, preparing compositions and fermentation broths.

[0073] The foregoing examples have confirmed that *Lactobacillus gasseri* YS-LG03 can secrete a novel antibacterial and anti-inflammatory polypeptide, YS-LG03-MAX. This polypeptide exhibits outstanding thermostability, retaining over 90% of its antibacterial activity even after treatment at 100°C for 30 minutes, and can improve macrophage inflammatory damage caused by *Streptococcus pyogenes* infection. In this example, this strain is used to ferment a medicinal and edible composition. In a traditional Chinese medicine fermentation substrate, the strain can synthesize and secrete the polypeptide YS-LG03-MAX. The subsequent 100°C, 30-minute sterilization process only causes a slight loss of polypeptide activity, with the active polypeptide remaining in the fermentation supernatant. The resulting *Ophiopogon japonicus* lung-moistening fermented liquid simultaneously contains: the YS-LG03-MAX polypeptide produced by the strain, metabolites generated through probiotic biotransformation, and inherent active ingredients released from the medicinal and edible raw materials. These multiple components synergistically exert antibacterial, anti-inflammatory, throat-protecting, cough-relieving, and lung-moistening effects. This embodiment selects 12 raw materials from the list of medicinal and edible homologous ingredients, including dried plum, Solomon's seal, green plum, Dendrobium officinale, American ginseng, Ophiopogon japonicus, lily bulb, monk fruit, Malva nut, Polygonatum sibiricum, peppermint, and Citrus reticulata peel. Single raw material screening and compound ratio optimization are carried out to explore the synergistic anti-infective effect of polypeptide YS-LG03-MAX and medicinal and edible homologous transformation products in the fermentation system, so as to prepare fermentation broth that promotes body fluid production, moistens the lungs, relieves cough and soothes the throat, and alleviates bacterial pharyngitis or pneumonia.

[0074] Single raw material screening Sample preparation: All raw materials were pulverized and passed through a 60-mesh sieve. Appropriate amounts of the raw powder were weighed and added to distilled water at a material-to-liquid ratio of 1:10 (w / v). The mixture was sterilized at 115℃ for 15 min, cooled, and used as a single-raw-material screening medium. Lactobacillus gasseri YS-LG03 was activated and inoculated into each single-raw-material medium at a 2% (v / v) inoculation rate. Fermentation was carried out at 30℃ and 60 rpm for 48 h. The fermentation broth was sterilized at 100℃ for 30 min, centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane for sterilization and stored at 4℃ for later use.

[0075] Cell modeling and grouping: RAW264.7 macrophages were resuscitated and cultured in DMEM high-glucose medium (containing 10% FBS and 1% penicillin antibiotics) at 37°C and 5% CO2. Once the cells reached a suitable growth stage, they were seeded and plated. The experiment included a blank control group (Group C), a model group (Group M), and treatment groups for each raw material fermentation broth. Except for the blank control group, all other groups were incubated with lipopolysaccharide (LPS) at a final concentration of 1 μg / mL for 2 h to construct a cell inflammation model. After modeling, the LPS-containing culture medium was discarded, and the cells were washed twice with PBS. Each raw material fermentation broth treatment group was incubated with fresh DMEM complete medium containing 5% (v / v) of the corresponding raw material fermentation broth; the blank control group and model group were incubated with an equal volume of blank medium for another 24 h.

[0076] Indicator determination: After incubation, cells and cell culture supernatant were collected from each group. Cell viability was detected by MTT assay, NO inhibition rate was detected by Griess reagent assay, and macrophage phagocytosis rate was detected by neutral red phagocytosis assay.

[0077] Table 7. Cell viability, NO inhibition rate, and phagocytic activity of Lactobacillus gasseri YS-LG03 after fermentation with a single raw material. Note: The overall score = 40 × (NO inhibition rate / highest NO inhibition rate within the group) + 40 × (phagocytic activity / highest phagocytic activity within the group) + 20 × (cell survival rate / 100). The highest NO inhibition rate and highest phagocytic activity within the group were only obtained from the fermentation treatment groups of 12 medicinal and edible raw materials, excluding the blank control group C and the model group M. The weighting of the indicators is as follows: NO inhibition rate weighted at 40 points, phagocytic activity weighted at 40 points, reflecting the in vitro efficacy of the sample; cell survival rate weighted at 20 points, reflecting the cell safety properties of the sample. The theoretical maximum score for the overall score is 100 points. This overall score is an evaluation system based on empirical values ​​assigned in this study and is only used for ranking the relative merits of samples within the same batch of experiments.

[0078] As shown in Table 7, compared with the blank control group, the cell viability of group M decreased significantly after LPS induction, and the cell survival rate of each raw material fermentation broth group was >90%, indicating that none of the 12 fermentation broths at a concentration of 5% (v / v) had cytotoxicity.

[0079] The core pathological basis of dry, sore throat, itchy throat, and cough due to lung heat is low-grade inflammation of the airway mucosa. Noxillin (NO) is a key pro-inflammatory mediator released by airway epithelial cells and macrophages. Excessive NO can lead to mucosal congestion, edema, dryness, damage, and exacerbated throat pain. Inhibiting excessive NO secretion can reduce inflammation and damage to the throat and airways, creating a foundation for mucosal repair and saliva production. In this experiment, group M showed increased NO release. All 12 fermentation broths inhibited LPS-induced NO release to varying degrees. Ophiopogon japonicus showed the highest NO inhibition rate at 68.52%, higher than the other groups; Polygonatum sibiricum and Polygonatum odoratum were next, with no significant difference between the two.

[0080] Respiratory macrophages are the core of the airway immune barrier. Enhanced phagocytic capacity can effectively clear pathogenic bacteria colonizing the throat, metabolic waste, and inflammatory necrotic tissue from the airway, reducing persistent inflammatory stimulation and improving the airway microenvironment. Simultaneously, restored immune homeostasis can promote normal salivary gland secretion, relieving dry mouth and throat at their root. LPS is a major component of the outer membrane of Gram-negative bacterial cell walls and a classic agonist of Toll-like receptor 4 (TLR4). LPS stimulation can activate signaling pathways such as TLR4 / NF-κB in RAW264.7 macrophages, leading to morphological and functional activation such as cytoskeleton remodeling (increased pseudopodia formation). This results in a compensatory enhancement of phagocytic activity; therefore, the phagocytic activity in the model group (111.87%) was higher than that in the blank control group (100.00%), which is a normal stress response of macrophages to strong stimuli. Unlike the model group which simply showed increased stress response, the fermentation broth groups not only further enhanced phagocytic activity (promoting pathogen clearance), but also significantly improved cell survival and inhibited excessive NO release. This means that the fermentation broth did not simply enhance the immune response, but rather guided macrophages from an inflammatory stress state to a homeostatic immune regulatory state through the synergistic regulation of anti-inflammation, repair, and clearance.

[0081] Based on the comprehensive score, the 12 raw materials are ranked as follows: Ophiopogon japonicus > Polygonatum sibiricum > Polygonatum odoratum > Siraitia grosvenorii > Citrus reticulata peel > Mentha haplocalyx > Dendrobium officinale > Lilium brownii > Panax quinquefolius > Terminalia chebula > Prunus mume > Malva nut.

[0082] The basis for the pairing of monarch, minister, assistant and envoy Based on the comprehensive scoring ranking and the traditional efficacy and modern pharmacological activity of each ingredient, Ophiopogon japonicus was selected as the principal ingredient, Polygonatum sibiricum and Polygonatum odoratum as the assistant ingredients, Siraitia grosvenorii and Citrus reticulata as the adjuvant ingredients, and Mentha haplocalyx as the guiding ingredient. Ophiopogon japonicus: Nourishes Yin and generates fluids, moistens the lungs and clears the heart; Polygonatum: Tonifies Qi and nourishes Yin, strengthens the spleen and moistens the lungs. Polygonatum odoratum: Nourishes Yin and moistens dryness, promotes body fluid production and quenches thirst Monk fruit: Clears heat and moistens the lungs, soothes the throat and improves voice; Huajuhong (Citrus reticulata peel): Regulates Qi and relieves chest congestion, dries dampness Peppermint: It disperses wind-heat, clears the head and eyes, soothes the throat, promotes rash eruption, and soothes the liver and regulates qi.

[0083] Composition blending ratio screening Using the top six herbs in the comprehensive score—Ophiopogon japonicus (principal), Polygonatum sibiricum and Polygonatum odoratum (assistant), Siraitia grosvenorii and Citrus reticulata (adjuvant), and Mentha haplocalyx (guide)—as the main ingredients, and following the principle that the dosage of the principal herb is greater than that of the assistant herbs, the assistant herbs are greater than those of the adjuvant herbs, and the adjuvant herbs are greater than those of the guide, four candidate ratios (by mass) were designed to investigate the effects of the principal-assistant-adjuvant-guide ratio on the anti-inflammatory and immunomodulatory activities of the compound fermentation broth. Raw materials for each group were weighed and mixed according to the corresponding mass ratio, pulverized through a 60-mesh sieve, and an appropriate amount of raw powder was weighed. Distilled water was added at a material-to-liquid ratio of 1:10 (w / v), sterilized at 115℃ for 15 min, and cooled for later use as the compound raw material culture medium. Lactobacillus gasseri YS-LG03 was activated and inoculated into each compound raw material culture medium at a 2% (v / v) inoculation rate, and fermented at 30℃ and 60 rpm for 48 h. The fermentation broth was sterilized at 100℃ for 30 min, centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane for sterilization and stored at 4℃ for later use. The same cell models and indicator measurement methods as those used in single-raw material screening were employed for evaluation.

[0084] The four candidate pairings are as follows: F1: Ophiopogon japonicus: Polygonatum sibiricum: Polygonatum odoratum: Siraitia grosvenorii: Citrus reticulata peel: Mentha haplocalyx = 6:3:3:2:2:1; F2: Ophiopogon japonicus: Polygonatum sibiricum: Polygonatum odoratum: Siraitia grosvenorii: Citrus reticulata peel: Mentha haplocalyx = 5:3:3:2:2:1; F3: Ophiopogon japonicus: Polygonatum sibiricum: Polygonatum odoratum: Siraitia grosvenorii: Citrus reticulata peel: Mentha haplocalyx = 5:3:3:1.5:1.5:0.5; F4: Ophiopogon japonicus: Polygonatum sibiricum: Polygonatum odoratum: Siraitia grosvenorii: Citrus reticulata peel: Mentha haplocalyx = 5:4:4:2:2:1.

[0085] Table 8. Cell viability, NO inhibition rate, and phagocytic activity of fermentation broths with different ratios of principal, assistant, adjuvant, and guide herbs. As shown in Table 8, the cell survival rate of all four compound fermentation broths was >95%, with no significant difference compared to the blank control group, and none showed cytotoxicity. Group F2 (ratio 5:3:3:2:2:1) showed the best overall effect, with a NO inhibition rate of 78.52% and a phagocytic activity of 135.83%, both superior to other ratios and single Ophiopogon japonicus fermentation broth, and was determined to be the optimal compound ratio.

[0086] In summary, the fermented liquid of Ophiopogon japonicus can significantly enhance the phagocytic function of RAW264.7 macrophages, which helps the body clear invading streptococci. At the same time, it exerts an anti-inflammatory effect by inhibiting excessive NO release, thereby achieving immune homeostasis regulation. It can be used as a core ingredient for subsequent functions such as promoting body fluid production, moistening the lungs, relieving cough and sore throat, and alleviating bacterial pharyngitis / pneumonia.

[0087] Fermentation broth preparation By weight, 15 parts of Ophiopogon japonicus, 9 parts of Polygonatum sibiricum, 9 parts of Polygonatum odoratum, 6 parts of Siraitia grosvenorii, 6 parts of Citrus reticulata peel, and 3 parts of Mentha haplocalyx were weighed, mixed, and pulverized through a 60-mesh sieve. An appropriate amount of the original powder of the composition was weighed and added to distilled water at a material-to-liquid ratio of 1:10 (w / v). The mixture was sterilized at 115℃ for 15 min. Lactobacillus gasseri YS-LG03 was activated and inoculated into the extract of the composition at an inoculum of 2% (v / v). The mixture was fermented at 30℃ and 60 r / min for 48 h. After fermentation, the mixture was sterilized at 100℃ for 30 min and centrifuged at 4℃ and 4000 rpm for 5 min. The supernatant was collected to obtain the Ophiopogon japonicus lung-moistening fermentation broth.

[0088] Example 5 Non-targeted metabolomics analysis of Ophiopogon japonicus lung-moistening fermentation broth The non-target metabolomics analysis of the fermentation broth is as follows: The fermented Ophiopogon japonicus extract prepared in Example 4 was used as the sample group (MF group), and the unfermented extract was used as the control group (M group). The samples were entrusted to Shanghai Baipu Biotechnology Co., Ltd. for non-targeted metabolomics analysis, and the results were analyzed on the Baipu Omichub cloud platform.

[0089] Results Analysis Principal Component Analysis (PCA) score plot results show ( Figure 6 The fermented group and the unfermented control group were clearly separated along the first principal component, indicating that probiotic fermentation remodeled the metabolite profile. Using VIP>1 and P<0.05 as screening criteria, a total of 550 significantly different metabolites were identified in the samples before and after fermentation. Among them, 162 metabolites showed a significant increase in abundance after fermentation, and 388 metabolites showed a significant decrease in abundance. Figure 7 The differentially metabolites covered categories including carboxylic acids and their derivatives (19.01%), acyl groups (9.14%), organic oxygenated compounds (7.90%), terpene lipids (6.91%), organic nitrogen-containing compounds (6.67%), flavonoids (5.43%), and benzene and its substituted derivatives (5.19%). Figure 8 The distribution of differentially metabolite categories indicates that fermentation treatment had a wide range of effects on the strain's amino acid metabolism, lipid metabolism, and phenylpropanoid metabolic pathways.

[0090] A systematic literature review and pharmacological activity analysis were conducted on metabolites whose abundance increased significantly after fermentation. Key metabolites closely related to the effects of promoting salivation, protecting the throat, relieving cough, and moisturizing the lungs were screened out. The results are shown in Table 9.

[0091] Table 9 Key functional metabolites with significantly increased abundance after fermentation Note: Mean.M is the mean relative abundance of the unfermented group, Mean.MF is the mean relative abundance of the fermented group; FC is the ratio of the mean abundance of the fermented group to the unfermented group; P.value is the significance level.

[0092] Table 9 shows that among the functional metabolites whose abundance significantly increased after fermentation, the substances that play a key role in promoting saliva production, protecting the throat, relieving cough, and moisturizing the lungs include: alpha-linolenic acid, hydroxyphenyllactic acid, diosgenin, L-3-phenyllactic acid, palmitoylethanolamide, acetylcholine, and spermidine. These substances cover the core dimensions of promoting saliva secretion, mucosal repair, cough relief, anti-inflammation, and lung moisturizing.

[0093] Among them, the relative abundance of acetylcholine after fermentation was 14.39 times that before fermentation, ranking high among differential metabolites in terms of VIP value. Acetylcholine is a key end-effect molecule in the cholinergic metabolic network, mainly regulating the secretion of salivary glands and airway mucosal glands through M3 receptors, and is an important mediator for achieving the physiological effects of saliva production, throat moistening, and lung moistening. Based on its biosynthetic pathway, the abundance of upstream substances such as phosphatidylcholine and choline was analyzed. The results show that the Ophiopogon japonicus lung-moistening fermentation broth prepared in this invention may efficiently promote the metabolic flow towards the end product acetylcholine by activating the complete metabolic pathway of phosphatidylcholine → glycerophosphatidylcholine → choline → acetylcholine, thus achieving a large accumulation of acetylcholine.

[0094] Table 10. Relative abundance changes of key metabolites and their derivatives before and after fermentation. Table 10 shows the abundance differences of core metabolites and their derivatives in the acetylcholine biosynthesis pathway. The abundance changes of phosphatidylcholine, glycerophosphatidylcholine, free choline, and acetylcholine confirm that the cholinergic metabolic network was significantly activated during the fermentation of the food-medicine homology composition by the strain. The results show that the relative abundance of acetylcholine in the system after fermentation was 14.39 times that before fermentation; the relative abundance of its direct synthetic precursor, free choline, was 0.67 times that before fermentation; the relative abundance of its upstream reserve metabolite, phosphatidylcholine, was 0.70 times that before fermentation; and the relative abundance of the pathway hydrolysis intermediate, glycerophosphatidylcholine, was 3.17 times that before fermentation. The above metabolite abundance change pattern indicates that Lactobacillus gasseri YS-LG03 fermentation of the food-medicine homology composition may mobilize the hydrolytic metabolism of phosphatidylcholine, generating glycerophosphatidylcholine intermediate, which is further converted into free choline. The free choline is then catalyzed by acetyltransferase active substances derived from the strain to generate acetylcholine. As the end product of the conversion pathway, the large accumulation of acetylcholine in the fermentation broth is consistent with the characteristics of the activation of the glycerophospholipid metabolic pathway.

[0095] The biosynthesis of acetylcholine in fermentation systems can be divided into two core stages: the upstream pathway of choline formation and the downstream pathway of acetylcholine synthesis. The upstream pathway is centered on choline synthesis: phosphatidylcholine. Phosphatidylcholine is a major component of cell membrane phospholipids and the main storage form of intracellular choline. It can release free choline through two key pathways. The first pathway is a direct hydrolysis pathway mediated by phospholipase D, where phosphatidylcholine is directly broken down under the catalysis of phospholipase D to generate free choline and phosphatidic acid. The second pathway is a stepwise hydrolysis pathway, where phosphatidylcholine first undergoes deacylation by phospholipase A1 / A2 to generate glycerophosphatidylcholine. Then, free choline is released through hydrolysis by glycerol phosphate diesterase. The downstream pathway is the acetylcholine conversion stage, where free choline generated in the system can be converted into acetylcholine under the action of acetyltransferase active substances. The comprehensive metabolomic profile results show that fermentation with Lactobacillus gasseri YS-LG03 can efficiently activate the upstream pathway of choline synthesis, significantly increase the accumulation of acetylcholine precursors, and provide a sufficient material basis for the efficient synthesis of downstream acetylcholine, thus achieving synergistic activation of upstream and downstream metabolic pathways. Related existing technologies, such as Li Z, Vance DE. Phosphatidylcholine and choline homeostasis[J]. Journal of Lipid Research, 2008, 49(6): 1187-1194, elucidate the cyclic pathway of phosphatidylcholine and choline homeostasis, providing the basic synthetic background of phosphatidylcholine → glycerophosphatidylcholine → choline → acetylcholine.

[0096] To further verify the regulatory ability of *Lactobacillus gasseri* YS-LG03 on the acetylcholine synthesis pathway during fermentation, the activity of key transferases mediated by YS-LG03 in acetylcholine synthesis was detected. The procedure was performed according to the instructions of the choline acetyltransferase activity assay kit (catalog number: SLBB929Hu, manufacturer: Lamborghini Biotechnology), used only to characterize the choline acetylation activity of the crude bacterial lysate. 0.1 mL of *Lactobacillus gasseri* YS-LG03 bacterial culture was centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the bacterial sludge precipitate (approximately 1 × 10⁻⁶) was retained. 7 Add 1 mL of extraction buffer to each live bacterial cell, and sonicate the cells on ice (200 W, 3 s, 10 s interval, repeated 30 times). Then centrifuge at 12000 rpm, 4 °C for 10 min, and collect the supernatant on ice for testing. Perform the specific detection according to the kit instructions, adding the reaction reagents sequentially to complete the detection. The choline acetylation activity of the strain in this experiment is expressed as enzyme activity units (U / mL) per milliliter of bacterial lysate extract.

[0097] Existing techniques (Deguang Song, Brianna Duncan-Lowey. Commensal-derived acetylcholine enhances mucosal immune education[J]. Nature, 2026, Jul;655(8124):1027-1036.) have shown that intestinal commensal bacteria such as *Bifidobacterium breve* and *Pediococcus pentosaceus* can directly convert dietary choline into acetylcholine, and enzymes mediating acetylcholine synthesis have been identified in these strains. These enzymes belong to the acetyltransferase family. The results of this study indicate that the choline acetylation activity in the crude cell lysate of *Lactobacillus gasseri* YS-LG03 was 13.06 U / mL, while this activity was not detected in the control *Lactobacillus gasseri* ATCC 33323. This suggests that *Lactobacillus gasseri* YS-LG03 may possess the ability to catalyze the production of acetylcholine from choline substrates, and this activity is one of the important potential molecular mechanisms for the significant increase in acetylcholine abundance in fermentation systems.

[0098] Comparative Example 7: Commercially available Lactobacillus gasseri ATCC 33323 was used, and the choline acetylation activity of the bacterial cell lysate was determined according to the same sample pretreatment and detection methods as described in this example.

[0099] Table 11 Results of choline acetylation activity assay in bacterial cell lysates Note: "-" indicates that the activity was below the detection limit of the kit and no detectable activity was detected.

[0100] Example 6 This embodiment verifies the effects of Ophiopogon japonicus lung-moistening fermented liquid on promoting saliva production, protecting the throat, relieving cough, and moisturizing the lungs through animal experiments.

[0101] Sample preparation: Unfermented extract: The sterilized but uninoculated extract of the composition prepared according to the method of Example 4; Ophiopogon japonicus lung-moistening fermented liquid: Ophiopogon japonicus lung-moistening fermented liquid prepared according to the method in Example 4; Peptide YS-LG03-MAX solution: The lyophilized YS-LG03-MAX was purified according to the method in Example 2, dissolved and diluted with sterile physiological saline to 200 μg / mL.

[0102] Cough-relieving efficacy verification Male Hartley guinea pigs (250-300g) were housed in an SPF-grade animal facility at a temperature of 22±2℃ and humidity of 50±10%, with a 12h light / 12h dark cycle and free access to food and water. After 7 days of acclimatization, the experiments were conducted. The guinea pigs were randomly divided into 5 groups of 15 each, with the grouping and treatments as follows: Normal control group (CK): No modeling treatment was performed; the same volume of physiological saline was administered by gavage. Model group (MD): Modeling was performed by gavage with an equal volume of physiological saline; Unfermented extract group (UMF): Modeling treatment was performed, and unfermented extract was administered by gavage; Ophiopogon japonicus lung-moistening fermentation liquid group (MF): Modeling treatment was performed, and fermentation liquid was administered by gavage; The YS-LG03-MAX group (YS-LG03-MAX) was subjected to modeling treatment and YS-LG03-MAX solution was administered by gavage. The gavage volume for each group was 1 mL / (kg·d), once daily for 7 consecutive days. One hour after the last administration, the guinea pigs to be modeled were placed in a closed nebulizer and coughed for 1 minute with 17.5% citric acid solution (airflow rate 15 L / min). Immediately after the nebulization ended, the time was set for 5 minutes, and the number of coughs was manually counted based on the standard of severe abdominal contraction accompanied by mouth opening and short exhalation.

[0103] Results analysis: Table 12 Effect of fermentation broth on the number of coughs in citric acid-induced coughing guinea pigs within 5 minutes As shown in the table above, the model group had an average of 14.80 coughs in 5 minutes, indicating that nebulized citric acid can stably induce an acute cough model. Compared with the model group, the number of coughs decreased by 24.32% and 32.43% in the unfermented extract group and the peptide group, respectively, indicating that both the unfermented extract and the peptide have certain antitussive effects, with the peptide being slightly more effective than the unfermented extract. The Ophiopogon japonicus lung-moistening fermented liquid group showed the best effect with a reduction of 58.11%, which was better than the other groups. This indicates that there is a synergistic effect between the YS-LG03-MAX peptide and other food-medicine homologous metabolites such as acetylcholine and spermidine in the fermented liquid system, and that multiple active ingredients work together to exert an antitussive effect.

[0104] Verification of its effects in promoting body fluid production and moistening the lungs Considering that atropine (an anticholinergic drug) can significantly interfere with airway gland secretion and inflammatory cell infiltration, this section uses two independent batches of mice for parallel experiments: batch I was used for saliva secretion measurement (intraperitoneal injection of atropine), and batch II was used for bronchoalveolar lavage fluid (BALF) inflammatory marker detection (without atropine injection to exclude the direct interference of the drug on lung inflammation).

[0105] SPF-grade ICR male mice (18-22g) were used under the same feeding conditions as before, and were acclimatized for 7 days. Except for the normal control group (C), all other mice underwent smoke-induced modeling: mice were placed in a static exposure chamber and exposed to smoke twice daily (30 min each time, 4 h apart) for 30 consecutive days to establish a chronic bronchitis model. Administration of medication was started 7 days before modeling and continued once daily for 37 days during the modeling period. Grouping was the same as in the cough suppression experiment (CK group, MD group, UMF group, MF group, YS-LG03-MAX group), with 15 mice in each group. For batch I, 6 mice were taken from each group; for batch II, the remaining 9 mice were taken from each group. Group C did not undergo smoke-induced modeling; its orbital blood collection, atropine treatment, and sampling procedures were the same as for the other modeling groups.

[0106] Batch I treatment: 30 minutes after the last administration, atropine (5 mg / kg) was injected intraperitoneally to inhibit saliva secretion. 30 minutes after the injection, a pre-weighed sterile cotton ball was placed in the cheek of the mouse's mouth. After 5 minutes, the cotton ball was removed and weighed to calculate the amount of saliva secreted.

[0107] Batch II treatment: One hour after the last administration (without atropine), mice were sacrificed, and bronchoalveolar lavage (BALF) was performed aseptically. The lavage fluid was collected. The total number of inflammatory cells in the BALF was counted using a hemocytometer; the proportion of neutrophils was classified and counted using Wright-Giemsa staining smears; and the levels of pro-inflammatory factors TNF-α and IL-6 in the supernatant were quantitatively detected using an ELISA kit.

[0108] Table 13 Effects of fermentation broth on saliva secretion in smoke-smoked and atropine-induced dry mouth mice As shown in the table above, after smoke fumigation modeling, saliva secretion in the model group decreased by 54.84% compared to the control group, indicating that chronic inflammation inhibited salivary gland secretion function, and the dry mouth model was successfully established. The unfermented extract group showed a limited increase in saliva secretion of 17.56%; the polypeptide group showed an increase of 29.02%; while the fermented Ophiopogon japonicus lung-moistening extract group showed an increase of 50.37%, significantly better than the other groups. In this experiment, saliva secretion was measured in the presence of the M receptor antagonist atropine. The fermented extract still increased saliva secretion by 50.37% compared to the model group, indicating that its saliva-promoting effect is not solely dependent on the M receptor / acetylcholine pathway, but mainly the result of multi-target synergistic regulation of glandular secretory function by multiple active ingredients.

[0109] Table 14 Effects of fermentation broth on inflammatory cells and inflammatory factors in the lungs of smoked mice. As shown in the table above, after 30 days of smoke inhalation, all inflammatory markers in the model group were significantly higher than those in the control group, indicating that the chronic bronchitis model was successfully established. The unfermented extract group showed a 23.29% decrease in total cell count and a 24.40% decrease in TNF-α level, indicating that the unfermented raw material possesses basic anti-inflammatory activity. The polypeptide group showed a 25.57% decrease in total cell count and a 22.85% decrease in TNF-α level. Although the polypeptides may be partially degraded by digestive enzymes after oral administration, the polypeptide group still exhibited certain antitussive and anti-inflammatory effects in this experiment, suggesting that its degradation products may exert their activity indirectly through intestinal immune regulation. This is consistent with the literature report that some small molecule polypeptides still have immunomodulatory effects after oral administration. The Ophiopogon japonicus lung-moistening fermented liquid group showed the best performance: a 55.25% decrease in total cell count, a 48.29% decrease in neutrophil proportion, a 49.20% decrease in TNF-α level, and a 49.60% decrease in IL-6 level, all of which were significantly better than those in the unfermented extract group and the polypeptide group.

[0110] Based on the above experimental results, the fermentation broth of this invention is a multi-component system of polypeptides and food-grade metabolites, which can simultaneously exert the in vivo effects of promoting saliva production, protecting the throat, relieving cough, and moisturizing the lungs. Specifically, the polypeptide YS-LG03-MAX is mainly responsible for inhibiting pathogenic bacteria in the throat and regulating excessive inflammatory responses of macrophages; acetylcholine and spermidine in the fermentation metabolites focus on promoting salivary gland secretion and repairing airway mucosa; the two work synergistically through multiple pathways and targets to jointly achieve the in vivo effects of promoting saliva production, protecting the throat, relieving cough, and moisturizing the lungs. After administration by gavage, the polypeptide may exert indirect regulatory effects through incompletely degraded active ingredients, degradation products, active substances produced by intestinal flora metabolism, or through the intestinal mucosal immune network. The fermentation broth of this invention has good application potential in the fields of soothing the throat and protecting the respiratory tract.

Claims

1. A Lactobacillus gasseri strain YS-LG03, characterized in that: The preservation number of Lactobacillus gasseri YS-LG03 is CGMCC No. 37762.

2. A polypeptide produced by Lactobacillus gasseri YS-LG03 as described in claim 1, characterized in that: The amino acid sequence of the polypeptide is as follows: AYGLLNIAVAGGKIDASMKG.

3. A method for preparing the polypeptide as described in claim 2, characterized in that: Includes the following steps: The Lactobacillus gasseri YS-LG03 was cultured to obtain the fermentation supernatant; Salt was added to the fermentation supernatant to carry out a salting-out reaction. After centrifugation, the precipitate was collected, reconstituted, and dialyzed to obtain a crude polypeptide extract. The crude extract of the polypeptide was filtered through a micromembrane and then purified by gel filtration chromatography, gel chromatography, and high-performance liquid chromatography to obtain the polypeptide.

4. The method for preparing the polypeptide as described in claim 3, characterized in that: The salt includes ammonium sulfate; The solution used for reconstitution is a phosphate buffer solution with a pH of 5.5-6.5 and a concentration of 0.05-0.1 mol / L. The gel filtration chromatography includes the steps of loading the filtered crude peptide extract onto a Sephadex G-25 chromatography column and eluting with a pH of 5.5-6.5 and a 0.05-0.1 mol / L phosphate buffer. The elution flow rate for the gel filtration chromatography is 0.5-1.5 mL / min; The pore diameter of the micromembrane is 0.2-0.3 μm.

5. The method for preparing the polypeptide as described in claim 3, characterized in that: The gel chromatography used a Sephadex LH-20 column and the eluent was a methanol solution with a volume fraction of 75-85%; the high performance liquid chromatography separation and purification included a step of chromatographic purification using a Venusil XBP C18 semi-preparative chromatographic column. The mobile phase A used for high-performance liquid chromatography separation and purification is a pure aqueous solution; the mobile phase B is an acetonitrile solution. The elution program for high performance liquid chromatography separation and purification is as follows: 0-20 min, 0%-60% linear gradient elution of phase B; 20-25 min, 60%-100% of phase B; 25-30 min, phase B maintained at 100%.

6. A fermented liquid for promoting saliva production, protecting the throat, relieving cough, and moisturizing the lungs, characterized in that: The fermentation broth was obtained by fermenting food and medicine homologous substrates with Lactobacillus gasseri YS-LG03; The medicinal and edible substrates include Ophiopogon japonicus, Polygonatum sibiricum, Polygonatum odoratum, Siraitia grosvenorii, Citrus reticulata peel, and Mentha haplocalyx in a weight ratio of 14-16∶8-10∶8-10∶5-7∶5-7∶2-4.

7. The method for preparing the fermented liquid for promoting saliva production, protecting the throat, relieving cough, and moisturizing the lungs as described in claim 6, characterized in that, Includes the following steps: The food-medicine homology substrate is sterilized with a solvent to obtain the fermentation substrate; The Lactobacillus gasseri YS-LG03 was inoculated into the fermentation substrate for fermentation. After fermentation, sterilize and centrifuge, and collect the supernatant to obtain the final product.

8. The preparation method of the fermented liquid for promoting saliva production, protecting the throat, relieving cough, and moisturizing the lungs as described in claim 7, characterized in that: The solvent includes water; The ratio of the medicinal and edible substrate to water by weight-volume is 1:9-11 (w / v). The sterilization conditions for the fermentation substrate are 110-121℃ and 10-20min.

9. The preparation method of the fermented liquid for promoting saliva production, protecting the throat, relieving cough, and moisturizing the lungs as described in claim 7, characterized in that: The inoculum amount of *Lactobacillus gasseri* YS-LG03, by volume, is 1.5-2.5%. The fermentation temperature is 30-35℃, and the time is 42-54 hours; The sterilization conditions for the fermentation broth are 100-110℃ and 5-30 min. The centrifugation conditions are 3000-5000 rpm for 4-6 min.

10. The application of the fermented liquid for promoting saliva production, protecting the throat, relieving cough, and moisturizing the lungs as described in claim 6, characterized in that: Used in the preparation of medicines to relieve dry throat, reduce coughing frequency, or alleviate airway inflammation; or It is used in the preparation of drugs with anti-inflammatory functions.

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