Space breeding saliva combined lactobacillus ZK-88-S and application thereof

Abstract

The present application relates to the technical field of microorganisms, and in particular to a space breeding lactobacillus salivarius ZK-88-S and application thereof; the lactobacillus salivarius ZK-88-S has been preserved in the China General Microbiological Culture Collection Center on December 24, 2024, and the preservation number is CGMCC No.33181; the lactobacillus salivarius ZK-88-S can inhibit escherichia coli, staphylococcus aureus, candida albicans, pseudomonas aeruginosa, fusobacterium nucleatum, porphyromonas gingivalis, streptococcus mutans, streptococcus salivarius, streptococcus gordonii or actinomyces naeslundii, and can be applied to the preparation of drugs for oral treatment and oral cleaning, daily necessities.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a space-bred Lactobacillus saliva-associated strain ZK-88-S and its applications. Background Technology

[0002] In the field of microbial research and application, the development and utilization of probiotics has always been a focus of attention. As an important member of the probiotic family, *Lactobacillus salivarius* has demonstrated unique value in regulating the balance of the human microecology and enhancing immunity, with related research results constantly emerging. *Lactobacillus salivarius* ZK-88 has been proven to have significant inhibitory effects on a variety of Gram-negative and Gram-positive pathogenic bacteria and can be used to prepare antibacterial drugs, providing a new direction for oral health and the prevention and treatment of related diseases. However, existing *Lactobacillus salivarius* strains still face many challenges in practical applications.

[0003] From an environmental adaptability perspective, the colonization ability and survival persistence of common *Lactobacillus salivarius* in the complex and ever-changing human microecological environment need improvement. For example, in the oral environment, facing food residue, pH changes, and competition from various microorganisms, some strains struggle to maintain stable efficacy. In the gastrointestinal environment, digestive fluids such as gastric acid and bile can also inhibit its activity, leading to a reduction in the number of effective bacteria reaching the intestines and exerting their effects, thus limiting its application in the prevention and treatment of gastrointestinal diseases.

[0004] Regarding efficacy enhancement, although *Lactobacillus salivarius* ZK-88 has demonstrated some antibacterial ability, its antibacterial spectrum and inhibitory strength still need further expansion and enhancement in the face of constantly mutating pathogens and increasingly complex infection situations. For some drug-resistant pathogens, the existing inhibitory effects of *Lactobacillus salivarius* are insufficient to meet clinical needs. In terms of immune function regulation, although it can induce immune responses, its adaptability to different individual immune states and the precision of its regulatory effects are insufficient, failing to fully realize its potential in the prevention and treatment of immune-related diseases. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention aims to provide a space-bred Lactobacillus salivarius ZK-88-S and its application, thereby enhancing the antibacterial effect and stability of Lactobacillus salivarius ZK-88.

[0006] To solve the above problems, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a space-bred *Ligilactobacillus salivarius* ZK-88-S, which was deposited on December 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33181.

[0008] Secondly, the present invention provides the application of the aforementioned space-bred Ligilactobacillus salivarius ZK-88-S in the preparation of oral treatment, oral cleaning drugs or daily necessities.

[0009] Thirdly, the present invention provides the application of the aforementioned space-bred *Ligilactobacillus salivarius* ZK-88-S in the preparation of inhibitors or removers of biofilm colonization on the surface of oral medical devices.

[0010] As one possible implementation method, the space-bred *Ligilactobacillus salivarius* ZK-88-S inhibits *Escherichia coli*, *Staphylococcus aureus*, *Candida albicans*, *Pseudomonas aeruginosa*, *Fusobacterium nucleatum*, or *Porphyromonas gingivalis*.

[0011] As one possible implementation, the space-bred *Ligilactobacillus salivarius* ZK-88-S inhibits *Streptococcus mutans*, *Streptococcus gordonii*, *Streptococcus salivarius*, or *Actinomyces negrius*.

[0012] Fourthly, the present invention provides a medicine or daily product comprising the space-bred *Ligilactobacillus salivarius* ZK-88-S and / or its metabolites.

[0013] The beneficial effects of this invention are as follows: The unique conditions of the space environment, such as microgravity, high radiation, and weak geomagnetism, can induce genetic mutations and changes in physiological characteristics in microorganisms. Microorganisms cultured in space exhibit significant changes in growth rate, metabolite synthesis, and environmental adaptability. The ZK-88-S strain, obtained by space-culturing *Lactobacillus salivarius* ZK-88, utilizes the special space environment to optimize its performance, overcoming the limitations of traditional strains and achieving breakthroughs in antibacterial activity, immune regulation, and environmental adaptability, thus opening new avenues for the development of novel probiotic products and the treatment of related diseases. The present invention relates to *Lactobacillus salivarius* ZK-88-S, which was deposited on December 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33181. *Lactobacillus salivarius* ZK-88-S inhibits *Escherichia coli*, *Staphylococcus aureus*, *Candida albicans*, *Pseudomonas aeruginosa*, *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, *Streptococcus mutans*, *Streptococcus salivarius*, *Streptococcus Gordonii*, or *Actinomyces negrius*. It can be applied to the preparation of oral treatment and oral hygiene drugs and daily necessities, providing a better application pathway for *Lactobacillus salivarius*. Attached Figure Description

[0014] Figure 1 This is a microscopic image of the ZK-88-S strain of the present invention at passage 0.

[0015] Figure 2 This is a microscopic image of the second generation of the ZK-88-S strain of this invention.

[0016] Figure 3 The image shows the acid resistance test results of the ZK-88-S strain and the ZK-88 strain of this invention.

[0017] Figure 4 The figure shows the HPLC detection results of small molecule acids produced by the ZK-88-S strain and the ZK-88 strain of this invention.

[0018] Figure 5 The image shows the GC detection results of small molecule acids produced by the ZK-88-S strain and the ZK-88 strain of this invention.

[0019] Figure 6 This is a comparison chart of the antibiotic MIC values ​​of the ZK-88-S strain and the ZK-88 strain of the present invention.

[0020] Figure 7 This is a comparison diagram of the diameter of the inhibition zone of oral pathogens between the ZK-88-S strain and the ZK-88 strain of this invention.

[0021] Figure 8 This is a comparison chart of the MIC values ​​of oral pathogens between the ZK-88-S strain and the ZK-88 strain of this invention.

[0022] Figure 9 This is a comparison chart of biofilm amounts between the ZK-88-S strain and the ZK-88 strain of this invention.

[0023] Figure 10 This is a comparison chart of the viable cell counts of the ZK-88-S strain and the ZK-88 strain of the present invention. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments.

[0025] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention. Example

[0026] The present invention relates to a space-bred *Ligilactobacillus salivarius* ZK-88-S, which was deposited on December 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33181, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0027] The space-bred *Ligilactobacillus salivarius* ZK-88-S of this invention was obtained by space breeding of *Ligilactobacillus salivarius* ZK-88 via the Shijian-19 satellite. Subculture details are available in the table below. Figures 1-2 .

[0028] I. Isolation and Identification of Strains

[0029] Lactobacillus salivarius strain ZK-88 and the space-transmitted mutant strain ZK-88-S were respectively streaked onto plates to obtain ZK-88 plates and ZK-88-S plates. These plates were then incubated in anaerobic culture bags at 37°C for 36 hours to obtain plates containing ZK-88 and S-88 colonies. Using a pipette tip, multiple ZK-88 single colonies and multiple S-88 single colonies were picked from each plate and placed in 20 µL of deionized water, mixed thoroughly, and used as DNA templates.

[0030] Prepare the PCR reaction system: Add 2 µL of DNA template, 1 µL of 27F (5-AGAGTTTGATCCTGGCTCAG-3) forward primer, 1 µL of 1492R (5-GGTTACCTTGTTACGACTT-3) reverse primer, 10 µL of PCR-mix (2× DNA polymerase + NTPs), and 6 µL of distilled water to an EP tube to prepare a 20 µL PCR reaction system. In addition, prepare a separate PCR reaction system for DNA standards in another EP tube.

[0031] PCR reaction conditions: 95°C for 10 min pre-denaturation; 30 cycles: 95°C for 30 s denaturation, 55°C for 30 s annealing, 72°C for 1.5 min extension; final extension at 72°C for 5 min, and storage at 4°C.

[0032] PCR product analysis: submitted for testing.

[0033] The 16S rRNA gene amplification product was sent to a sequencing company for sequencing. The sequencing results were analyzed using BLAST on the website of the National Center for Biotechnology Information (NCBI). Sequence alignment results showed that the most similar strain to ZK-88-S was *Lactobacillus salivarius*, with a similarity of 99%. The sequencing results of ZK-88-S are shown in SEQ ID No. 1:

[0034]

[0035] II. Evaluation of the genetic stability of Lactobacillus salivarius ZK-88-S

[0036] 1. Method

[0037] MRS medium (for bacterial culture): 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 1mL Tween 80, 5g sodium acetate, 2g diammonium citrate, 2g K2HPO4, 0.2g MgSO4·7H2O, 0.05g MnSO4·4H2O, 0.1% cysteine ​​hydrochloride, 1000mL distilled water, pH 6.5, sterilized at 121℃ for 20min.

[0038] Culture conditions: static culture at 37℃, transfer once every 12 hours, and subculture for 50 generations.

[0039] (1) Detection indicators:

[0040] Phenotypic stability: Colony morphology (diameter, edge, transparency) and Gram staining characteristics were observed every 5 generations.

[0041] (2) Molecular stability:

[0042] Whole genome sequencing: The strains of the 1st, 25th and 50th generations were sequenced using Illumina NovaSeq and genomic variations (SNPs / InDel) were compared.

[0043] (3) Oxygen tolerance test:

[0044] Aerobic culture conditions: MRS liquid medium, cultured at 37℃ with shaking at 150 rpm, with dissolved oxygen concentration maintained at 50% saturation.

[0045] (4) Detection indicators:

[0046] Growth curve: OD600 was measured every 2 hours, and the specific growth rate (μ) was calculated.

[0047] Survival rate: After 24 hours of incubation, the number of viable bacteria (CFU / mL) was determined by plate counting.

[0048] Antioxidant enzyme activity: The activities of superoxide dismutase (SOD) and catalase (CAT) were determined by spectrophotometry.

[0049] 2. Results

[0050] Comparison of generational stability:

[0051] (1) Phenotypic consistency:

[0052] ZK-88-S: The colony morphology (2-3 mm in diameter, with neat edges and semi-transparent) and Gram staining results are stable within 50 generations.

[0053] ZK-88: After the 30th generation, the colony diameter shrinks to 1.5-2 mm, the edges become serrated, and Gram-negative cells are occasionally seen.

[0054] (2) Molecular level:

[0055] Genomic variation:

[0056] ZK-88-S: No significant SNP or InDel mutations within 50 generations, and the sequences of core functional genes (such as the gene encoding the antimicrobial peptide) are consistent.

[0057] ZK-88: Five SNP mutations were detected in the 25th generation, and one InDel appeared in the 50th generation, resulting in a partial deletion of the antimicrobial peptide gene.

[0058] (3) Comparison of oxygen tolerance:

[0059] Growth characteristics:

[0060] ZK-88-S: μ was 0.35±0.02h⁻¹, significantly higher than that of the original strain (0.28±0.01h⁻¹).

[0061] ZK-88: Slow growth under aerobic conditions, with a growth rate of only 0.15±0.01h⁻¹.

[0062] Survival rate:

[0063] ZK-88-S: 24-hour viable count reaches 1.2 × 10⁻⁶. 9 CFU / mL, compared to the original strain (3.5 × 10⁻⁶). 8 The CFU / mL level increased by 3.4 times.

[0064] ZK-88: After 24 hours, the viable bacterial count decreased to 5.0 × 10⁻⁶. 7 CFU / mL.

[0065] Antioxidant enzyme activity:

[0066] ZK-88-S: SOD activity was 125±10 U / mg protein, and CAT activity was 85±8 U / mg protein, which were 2.3 times and 1.8 times that of the original strain, respectively.

[0067] ZK-88: SOD activity was 54±5 U / mg protein, and CAT activity was 47±4 U / mg protein, both of which showed a decreasing trend with increasing passage number.

[0068] After 50 passages, *Lactobacillus salivarius* ZK-88-S maintained stable phenotype, physiological characteristics, and genome sequence, with significantly improved oxygen tolerance (survival rate, metabolic activity, and antioxidant enzyme activity were all superior to the original strain). In contrast, the original strain ZK-88 exhibited phenotypic degeneration, decreased antibacterial activity, and genomic variation during passage, and showed insufficient oxygen tolerance.

[0069] III. Evaluation of the genetic stability of acid resistance in Lactobacillus salivarius ZK-88-S

[0070] 1. Method

[0071] Simulating gastric acid environment: The strain was inoculated into MRS liquid medium (containing 0.3% pepsin) at pH 2.5 and incubated at 37°C for 3 hours.

[0072] Testing indicators:

[0073] Survival rate: The number of viable bacteria (CFU / mL) was determined by plate counting after culture, and the survival rate was calculated as (number of viable bacteria / initial number of bacteria × 100%).

[0074] Cell membrane integrity: Flow cytometry was used to detect the positive rate of propidium iodide (PI) staining.

[0075] Metabolites: Lactic acid production was detected by high performance liquid chromatography (HPLC).

[0076] Expression of acid tolerance-related genes:

[0077] Real-time quantitative PCR (qPCR): Detects the expression levels of glutamate decarboxylase gene (gadB), ATPase gene (atpA), and catalase gene (katA).

[0078] Whole genome sequencing: The sequence stability of acid tolerance-related genes (such as the gad operon and cydAB) was analyzed by taking strains from the 1st, 25th and 50th generations.

[0079] 2. Results

[0080] Comparison of acid resistance:

[0081] Survival rate:

[0082] ZK-88-S: After culturing in a pH 2.5 environment for 3 hours, the survival rate reached 82.5±3.2%, which was significantly higher than that of the original strain ZK-88 (21.7±2.1%).

[0083] ZK-88: The survival rate gradually decreases with the number of generations, reaching only 8.5±1.5% in the 50th generation.

[0084] Cell membrane integrity:

[0085] ZK-88-S: The positive rate of PI staining was 12.3±1.8%, indicating that the cell membrane damage was relatively small.

[0086] ZK-88: The positive rate of PI staining reached 45.6±4.2%, indicating a significant increase in cell membrane permeability.

[0087] Metabolites:

[0088] ZK-88-S: Lactic acid production was 32.1±2.5 g / L, which was 71.7% higher than that of the original strain (18.7±1.2 g / L).

[0089] ZK-88: Lactic acid production gradually decreased with increasing generation number, reaching only 10.3±1.0 g / L in the 50th generation.

[0090] Gene expression:

[0091] ZK-88-S: The expression levels of gadB, atpA, and katA were upregulated by 4.1-fold, 2.8-fold, and 3.5-fold, respectively.

[0092] ZK-88: There was no significant change in the expression levels of acid tolerance-related genes.

[0093] Comparison of generational stability:

[0094] Acid resistance survival rate:

[0095] ZK-88-S: The survival rate remains stable at over 80% within 50 generations, with a coefficient of variation of <5%.

[0096] ZK-88: Survival rate drops significantly after the 10th generation, reaching only 5.2±1.0% in the 50th generation.

[0097] Acid production rate:

[0098] ZK-88-S: Average 2.8 ± 0.2 hours (pH 4.5), coefficient of variation < 5%.

[0099] ZK-88: The acid production rate decreased significantly after the 10th generation, reaching 4.2±0.5 hours in the 50th generation.

[0100] After 50 passages, *Lactobacillus salivarius* ZK-88-S exhibited significantly better acid tolerance than the original strain ZK-88, characterized by higher survival rate (82.5% vs. 21.7%), better cell membrane integrity (PI positivity rate 12.3% vs. 45.6%), stronger metabolic activity (lactate production 32.1 g / L vs. 18.7 g / L), and stable expression of acid tolerance-related genes (gadB, atpA, katA). In contrast, the original strain ZK-88 showed phenotypic degeneration, decreased acid tolerance, and genomic variation during passage.

[0101] (1) Subculture:

[0102] ZK-88-S and ZK-88 were inoculated into MRS liquid medium and cultured anaerobically at 37°C for 24 hours to obtain the first generation of bacterial culture.

[0103] Transfer the bacterial culture to fresh MRS medium at an inoculum rate of 1%, repeat the above culture conditions, and passage continuously up to the 15th generation.

[0104] (2) Acid resistance test:

[0105] Collect the bacterial cells by centrifugation of each generation of bacterial culture, and wash them three times with sterile physiological saline.

[0106] The bacterial cells were resuspended in a hydrochloric acid solution (pH=2.5) containing 0.3% pepsin and treated at 37°C for 3 hours.

[0107] The number of viable bacteria before and after treatment was calculated using the plate colony counting method, and the survival rate was calculated.

[0108] 2. Results

[0109] Viable bacterial counts before and after treatment are shown in the figure. Figure 3 .

[0110] After 15 generations of continuous subculturing, the survival rate of ZK-88-S remained above 95% at pH 2.5, with a coefficient of variation of <1.5%, significantly higher than that of the original strain ZK-88 (whose survival rate dropped to around 75%). The experiment demonstrates that the acid-tolerant trait obtained through space breeding has high genetic stability, and no reversion mutations were observed.

[0111] IV. Detection of the ability of Lactobacillus ZK-88-S in producing small molecule fatty acids via salivary fusion

[0112] 1. Method

[0113] Culture medium: MRS liquid medium (pH 6.5), containing 2% glucose, 1% peptone, and 0.5% yeast extract.

[0114] Culture conditions: Incubate at 37℃ for 24 hours in an anaerobic environment.

[0115] Detection method:

[0116] Sample preparation: Take the fermentation supernatant, centrifuge at 12,000 rpm for 10 minutes, and filter (0.22 μm filter membrane).

[0117] HPLC detection:

[0118] Instrument: Agilent 1260 Infinity II HPLC system, equipped with a refractive index detector (RID).

[0119] Chromatographic column: Aminex HPX-87H column (300mm×7.8mm, Bio-Rad).

[0120] Mobile phase: 5 mL H₂SO₄, flow rate: 0.6 mL / min, column temperature: 65 °C.

[0121] GC detection:

[0122] Instrument: Agilent 7890B gas chromatograph equipped with flame ionization detector (FID).

[0123] Chromatographic column: DB-FFAP capillary column (30m×0.25mm×0.25μm).

[0124] Injection conditions: split ratio 10:1, injection port temperature 250℃, detector temperature 280℃.

[0125] Programmed temperature rise: Initial temperature 80℃, hold for 2 minutes; rise to 180℃ at 10℃ / min, hold for 5 minutes.

[0126] Standards: lactic acid, acetic acid, propionic acid, and butyric acid standards (purity ≥99%, Sigma-Aldrich).

[0127] 2. Results

[0128] HPLC detection results are as follows Figure 4 GC detection results are as follows: Figure 5 .

[0129] Differences in the distribution of metabolites:

[0130] ZK-88-S: Primarily composed of lactic acid (accounting for 89.7% of the total acid content), while also showing significant accumulation of acetic acid, propionic acid, and butyric acid.

[0131] ZK-88: Lactic acid accounts for as high as 95.8%, while the content of other fatty acids is extremely low.

[0132] ZK-88-S significantly outperformed the original strain ZK-88 in the production of small molecule fatty acids, particularly in the synthesis of acetic acid (+133%), propionic acid (+167%), and butyric acid (+400%). Space mutagenesis may have enhanced the strain's sugar metabolism diversion ability, enabling it to activate the synthesis pathways of acetic acid, propionic acid, and butyric acid on the basis of homolactic fermentation.

[0133] V. Antibiotic Resistance Detection of Lactobacillus saliva ZK-88-S

[0134] 1. Method

[0135] Antibiotic selection:

[0136] Tetracycline, chloramphenicol, erythromycin, ampicillin, neomycin, rifampicin.

[0137] Detection method:

[0138] Agar dilution method:

[0139] Culture medium preparation: After melting the MRS agar medium (pH 6.5), add the antibiotic stock solution respectively to prepare plates containing antibiotics at different concentrations (the gradient concentration refers to the CLSI standard).

[0140] Inoculation: Inoculate the strain at a concentration of 10 6 CFU / mL onto the plates containing antibiotics, and repeat 3 times for each concentration.

[0141] Cultivation conditions: Anaerobic cultivation at 37°C for 48 hours.

[0142] Result determination: Observe the growth of colonies, and record the minimum inhibitory concentration (MIC), that is, the lowest antibiotic concentration that completely inhibits the growth of colonies.

[0143] Drug resistance phenotype classification:

[0144] Sensitive (S): MIC ≤ sensitive breakpoint value.

[0145] Intermediate (I): Sensitive breakpoint value < MIC < resistant breakpoint value.

[0146] Resistant (R): MIC ≥ resistant breakpoint value.

[0147] Quality control strains:

[0148] Enterococcus faecalis ATCC29212: Used to verify the antibiotic activity.

[0149] Staphylococcus aureus ATCC25923: Used to verify the sensitivity of ampicillin and rifampicin.

[0150] 2. Results

[0151] The comparison of MIC values is shown in Figure 6 .

[0152] Analysis of resistance trend:

[0153] Tetracycline: The MIC of ZK-88-S is 8 μg / mL (intermediate), which is significantly lower than 32 μg / mL (resistant) of ZK-88, indicating that space mutagenesis may reduce tetracycline resistance.

[0154] Chloramphenicol: The MIC of ZK-88-S was 4 μg / mL (sensitive), while that of ZK-88 was 16 μg / mL (intermediate), indicating that space-induced mutagenesis enhanced the sensitivity of chloramphenicol.

[0155] Erythromycin: The MIC of ZK-88-S is 2 μg / mL (sensitive), while that of ZK-88 is 8 μg / mL (intermediate), indicating that space-induced mutagenesis improves the sensitivity of erythromycin.

[0156] Ampicillin: Both strains showed susceptibility (MIC≤8μg / mL), but ZK-88-S had a lower MIC (0.5μg / mL), suggesting that it was slightly more sensitive to β-lactam antibiotics than the original strain.

[0157] Neomycin: The MIC of ZK-88-S is 8 μg / mL (sensitive), while that of ZK-88 is 32 μg / mL (resistant), indicating that space-induced mutagenesis significantly reduced neomycin resistance.

[0158] Rifampin: The MIC of ZK-88-S was 4 μg / mL (sensitive), while that of ZK-88 was 16 μg / mL (intermediate), indicating that space-induced mutagenesis enhanced the sensitivity of rifampin.

[0159] ZK-88-S exhibited significantly superior resistance phenotypes to tetracycline, chloramphenicol, erythromycin, neomycin, and rifampin compared to the original strain ZK-88, demonstrating lower MIC values ​​and better sensitivity classifications. Space-induced mutagenesis may have reduced the strain's inherent antibiotic resistance through genomic rearrangement or metabolic regulation, making it more compliant with the safety requirements for probiotics.

[0160] VI. In vitro inhibitory effect of Lactobacillus saliva-associated with oral pathogens ZK-88-S on oral pathogens

[0161] 1. Method

[0162] Test strain:

[0163] Gram-positive bacteria: Staphylococcus aureus (ATCC6538), Fusobacterium nucleatum (ATCC25586).

[0164] Gram-negative bacteria: Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Porphyromonas gingivalis (ATCC33277).

[0165] Fungus: Candida albicans (ATCC10231).

[0166] Other oral pathogens: Streptococcus mutans (ATCC25175), Streptococcus salivarius (ATCC13813).

[0167] Culture medium:

[0168] MRS medium: used for lactic acid bacteria culture.

[0169] BHI medium: used for the culture of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa.

[0170] Brain-Heart Infusion (BHI) + 0.5% yeast extract: used for anaerobic culture of Fusobacterium nucleatum and Porphyromonas gingivalis.

[0171] Sabouraud dextrose agar (SDA): used for Candida albicans culture.

[0172] Selective medium for Streptococcus mutans (MSB): containing 20% ​​sucrose, 0.2% peptone, and 0.1% yeast extract.

[0173] Test method:

[0174] Oxford Cup Method:

[0175] Preparation of bacterial suspension: The test bacteria were cultured to the logarithmic growth phase and the concentration was adjusted to 10. 6 CFU / mL.

[0176] Double-layer plate preparation: The bottom layer is a solid culture medium containing bacteria (15 mL), and the top layer is a semi-solid culture medium containing bacteria (5 mL).

[0177] Sample loading: Place an Oxford cup on the surface of the plate and add 200 μL of cell-free fermentation supernatant (CFS) or live bacterial suspension (10 μL). 8 (CFU / mL).

[0178] Cultivation conditions:

[0179] Aerobic bacteria: Incubate at 37℃ for 24 hours under aerobic conditions.

[0180] Anaerobic bacteria: anaerobic culture at 37℃ for 48 hours (using anaerobic bags or anaerobic incubator).

[0181] Fungi: Aerobic culture at 30℃ for 48 hours.

[0182] Results determination: Measure the diameter of the inhibition zone (mm), and repeat 3 times for each group.

[0183] Positive controls: Ampicillin (10 μg / mL) for bacteria, nystatin (100 U / mL) for fungi.

[0184] Negative control: sterile MRS medium.

[0185] 2. Results

[0186] Comparison of inhibition zone diameters, see Figure 7 .

[0187] Activity trend analysis:

[0188] Gram-positive bacteria: ZK-88-S showed a 47%-45% increase in the diameter of the inhibition zone against Staphylococcus aureus and Fusobacterium nucleatum compared to ZK-88, indicating stronger inhibitory ability.

[0189] Gram-negative bacteria: The inhibition zone against Escherichia coli, Pseudomonas aeruginosa, and Porphyromonas gingivalis was increased by 50%-40%, which may be related to the increased membrane permeability of metabolites.

[0190] Fungi: ZK-88-S showed a 66% greater inhibition zone against Candida albicans compared to ZK-88, which is presumably related to the activation of antifungal metabolic pathways by space-induced mutagenesis.

[0191] Oral pathogens: The inhibition zone against Streptococcus mutans was increased by 50%, and the inhibition zone against Streptococcus salivarius was increased by 57%, demonstrating the ability to target and regulate the oral flora.

[0192] ZK-88-S exhibited superior antibacterial activity compared to the original strain ZK-88 in both cell-free fermentation supernatant (CFS) and live bacterial suspension, particularly showing significantly enhanced inhibitory effects against anaerobic bacteria such as *Fusobacterium nucleatum* and *Porphyromonas gingivalis*, as well as *Candida albicans*. Space-induced mutagenesis may have increased the production of antibacterial metabolites (such as organic acids) in the strain through metabolic regulation.

[0193] Minimum inhibitory concentration (MIC) comparison

[0194] 1. Method

[0195] Detection method:

[0196] Micro-broth dilution method:

[0197] Preparation of bacterial suspension: The concentration of test bacteria was adjusted to 10. 6 CFU / mL.

[0198] Dilution gradient: Perform 2-fold serial dilutions of CFS or live bacterial suspension (100%, 50%, 25%, 12.5%, 6.25%).

[0199] Cultivation conditions: Same as the Oxford Cup method.

[0200] Result determination: The lowest concentration that completely inhibits bacterial growth is defined as MIC (%).

[0201] Quality control standards: Refer to the CLSI (Clinical and Laboratory Standards Institute) guidelines.

[0202] 2. Results

[0203] For a comparison of MIC values, see Figure 8 .

[0204] Sensitivity analysis:

[0205] Gram-positive bacteria: The MIC of CFS of ZK-88-S against Staphylococcus aureus is 6.25%, which is only 1 / 4 of that of ZK-88.

[0206] Anaerobic bacteria: The MIC for Fusobacterium nucleatum and Porphyromonas gingivalis was 3.12%, which is 4 times higher than that of ZK-88.

[0207] Fungi: The MIC of CFS of ZK-88-S against Candida albicans is 25%, while the CFS of ZK-88 cannot completely inhibit it even at 100% concentration.

[0208] Oral pathogens: The MIC against Streptococcus mutans was 6.25%, which is 4 times higher than that of ZK-88, showing strong inhibition against cariogenic bacteria.

[0209] The MIC values ​​of cell-free fermentation supernatant and live bacterial suspension of ZK-88-S against oral pathogens were significantly lower than those of the original strain ZK-88, especially showing a significant increase in inhibitory activity against anaerobic bacteria and fungi. Space mutagenesis may have enhanced the strain's ability to secrete antimicrobial metabolites.

[0210] VII. Verification of the targeted removal effect of saliva combined with Lactobacillus ZK-88-S metabolites on dental plaque biofilm.

[0211] 1. Method

[0212] Test strain:

[0213] Streptococcus mutans (ATCC25175): a cariogenic bacterium and a major component of biofilms.

[0214] Streptococcus Gordonii (ATCC10558): an early colonizing bacterium that promotes biofilm formation.

[0215] Other related bacteria: Streptococcus salivarius (ATCC13813), Actinomyces negrius (ATCC12104).

[0216] Culture medium:

[0217] MRS medium: used for lactic acid bacteria culture.

[0218] BHI medium: used for streptococcal culture.

[0219] Brain-Heart Infusion (BHI) medium + 0.5% yeast extract: used for anaerobic culture of actinomycetes.

[0220] Preparation of metabolites:

[0221] Cellless fermentation supernatant (CFS): The strain was inoculated into MRS medium, anaerobically cultured at 37°C for 48 hours, centrifuged (10,000×g, 15 minutes) and then filtered to remove bacteria (0.22μm filter membrane).

[0222] Biomembrane model construction:

[0223] Single-strain biofilm: The test bacteria were placed in a 10... 6 Inoculate CFU / mL into 96-well plates and incubate at 37°C for 24 hours (aerobic bacteria) or 48 hours (anaerobic bacteria).

[0224] Multi-species biofilm: Inoculate Streptococcus mutans, Streptococcus Gordonii, Streptococcus salivarius, and Actinomyces nellie in a 1:1:1:1 ratio, and culture under the same conditions as above.

[0225] Cleanup Experiment:

[0226] Treatment group: Add 200 μL CFS (100% concentration) to the biofilm and incubate at 37°C for 2 hours.

[0227] Control group: Add an equal volume of sterile MRS culture medium.

[0228] Quantitative analysis:

[0229] Crystal violet staining: Discard the culture medium, wash three times with PBS, add 0.1% crystal violet solution and stain for 15 minutes. After elution with ethanol, measure the absorbance (OD value) at 595 nm.

[0230] Viable cell count: The treated biofilm was ultrasonically disrupted, serially diluted, spread on plates, and incubated at 37°C for 48 hours. Colony forming units (CFU / mL) were then calculated.

[0231] 2. Results

[0232] For a comparison of biofilm amounts, see Figure 9 For comparison of viable cell counts, see [link to viable cell count]. Figure 10 .

[0233] Activity trend analysis:

[0234] Single-strain biofilm: ZK-88-S improves the clearance rate of Streptococcus mutans and Streptococcus Gordonii by 62%-90% compared to ZK-88, and improves the clearance rate of Streptococcus salivarius and Actinomyces neuri by 129%-130%.

[0235] Multi-species biofilm: ZK-88-S achieved a clearance rate of 91.7% for complex biofilms, significantly higher than ZK-88's 63.8%, demonstrating its broad-spectrum inhibitory ability on oral flora.

[0236] Cell-free fermentation supernatant (CFS) of ZK-88-S showed significantly better removal of dental plaque biofilm than the original strain ZK-88, especially with a removal rate of over 60% against Streptococcus mutans and Streptococcus Gordonii. Space mutagenesis may disrupt biofilm structure and inhibit bacterial survival by enhancing the secretion of metabolites (such as organic acids).

[0237] Verification of biomembrane structure disruption

[0238] 1. Method

[0239] Sample preparation:

[0240] Biofilm culture: Mix Streptococcus mutans and Streptococcus Gordonii and inoculate onto a glass slide and incubate at 37°C for 24 hours.

[0241] Treatment: Treat with CFS of ZK-88-S or ZK-88 for 2 hours, wash with PBS and fix with 2.5% glutaraldehyde.

[0242] Scanning electron microscopy (SEM) observation:

[0243] Dehydration: Gradient ethanol (30%, 50%, 70%, 90%, 100%) dehydration, critical point drying.

[0244] Coating: Ion sputtering gold plating, accelerating voltage 10kV.

[0245] Imaging: Observe the surface structure of biofilms and the distribution of bacteria.

[0246] 2. Results

[0247] ZK-88-S treatment group: The biofilm structure was loose, the bacteria were scattered, and the extracellular polysaccharide (EPS) matrix was significantly reduced. The cell walls of Streptococcus mutans and Streptococcus Gordonii were damaged, and some cells were lysed.

[0248] ZK-88 processing group:

[0249] The biofilm retains its dense network structure, with bacteria densely packed together, and the EPS matrix remains intact. The bacteria are morphologically complete, with only a few cells having blurred edges.

[0250] ZK-88-S metabolites significantly reduce the structural integrity of biofilms by disrupting the biofilm matrix and damaging bacterial cell walls. Space-induced mutagenesis may have enhanced the targeting of the metabolites to EPS and bacterial cell membranes.

[0251] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A space-bred *Ligilactobacillus salivarius* ZK-88-S, which was deposited on December 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33181.

2. The use of the space-bred *Ligilactobacillus salivarius* ZK-88-S as described in claim 1 in the preparation of drugs for treating oral diseases or in the preparation of daily necessities for cleaning oral pathogens. The oral diseases mentioned are caused by Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, Fusobacterium nucleatum, or Porphyromonas gingivalis. The oral pathogens mentioned are Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, Fusobacterium nucleatum, or Porphyromonas gingivalis.

3. The application of the space-bred *Ligilactobacillus salivarius* ZK-88-S described in claim 1 in the preparation of an inhibitor or remover for biofilm colonization on the surface of oral medical devices. The biofilm colonization is produced by Streptococcus mutans, Streptococcus Gordonii, Streptococcus salivarius, or Actinomyces neuridis.

4. A medicine or daily necessities, characterized in that, The medicine or daily product described herein contains the space-bred *Ligilactobacillus salivarius* ZK-88-S as described in claim 1.

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