Lactobacillus rhamnosus XB01 and application thereof in bacteriostasis, pharyngitis resistance and sleep promotion

Abstract

The invention belongs to the technical field of microorganisms, and discloses a novel lactobacillus rhamnosus XB01, the preservation number of which is CGMCC (China General Microbiological Culture Collection Center) No.34941. The invention discloses a new application of the strain or metabolites thereof in preparation of products for inhibiting specific pathogenic bacteria and intervening pharyngitis. Experiments show that XB01 has a remarkable in-vitro inhibition effect on specific pathogenic bacteria and common pathogens of pharyngitis, and can effectively relieve pharyngeal inflammation of an animal model. In addition, the invention also relates to a microbial preparation containing XB01, and the microbial preparation is combined with a specific strain bifidobacterium animalis subsp. Lactis XB02 for use, and shows an unexpected synergistic effect on enhancing the sleep promoting effect. The invention provides a new technical scheme and product form for the application of the strain in the fields of functional food, medicines or health care products, especially oral and throat health and sleep improvement.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and application, and relates to a strain of Lactobacillus rhamnosus XB01 and its application in antibacterial, anti-pharyngitis and sleep-promoting effects. In particular, it relates to a topical preparation containing XB01 for directly and effectively maintaining pharyngeal health, and its synergistic enhancement effect in sleep promotion when used in combination with Bifidobacterium animalis subsp. lactis XB02. Background Technology

[0002] Lactobacillus rhamnosus is an important member of the Lactobacillus genus. Its functions in regulating gut microbiota and enhancing immunity have been extensively studied and applied in probiotic products. Known Lactobacillus rhamnosus strains, such as ATCC 53103, possess these functions. Several Lactobacillus rhamnosus strains (CGMCC 1.78, etc.) preserved by the China General Microbiological Culture Collection Center are mainly used for basic applications such as riboflavin determination.

[0003] However, as research deepens, the limitations of existing technologies are becoming increasingly apparent. For example, regarding the antibacterial spectrum, current research on *Lactobacillus rhamnosus* largely focuses on common intestinal pathogens such as *Escherichia coli* and *Salmonella*, while its inhibitory effects on specific pathogens such as *Vibrio parahaemolyticus* and *Gardnerella vaginalis* are rarely systematically reported or protected by patents. This creates a technological gap in developing specific probiotic products targeting these pathogens. In terms of application areas, research and application of *Lactobacillus rhamnosus* are mainly focused on gastrointestinal health. Although its regulatory effects on the immune system suggest potential value in respiratory health, patents and technical solutions for its direct use in pharyngitis intervention, especially in developing topical products (such as sprays and lozenges), are extremely rare. Existing technologies largely focus on the in vivo immune regulation of lactic acid bacteria, lacking direct and rapid intervention strategies targeting local pharyngeal flora imbalance and inflammation.

[0004] Furthermore, while probiotic combination is a common strategy for synergistic effects, the effects of specific strain combinations are not simply additive. Although compound preparations containing *Lactobacillus rhamnosus* and *Bifidobacterium* have been mentioned as potentially helpful for sleep, there is a lack of conclusive experimental evidence and patent disclosures regarding whether the combination of specific strains XB01 and XB02 can produce a synergistic sleep-promoting effect of "1+1>2". The synergistic effect of such specific combinations remains unknown. Therefore, there is an urgent need in this field for a novel *Lactobacillus rhamnosus* strain with novel antibacterial properties that can be extended to new application scenarios such as pharyngeal health, and for in-depth exploration of its specific synergistic combination functions. Summary of the Invention

[0005] The primary objective of this invention is to provide a novel strain of Lactobacillus rhamnosus XB01, which expands the application of Lactobacillus rhamnosus in inhibiting specific pathogenic bacteria and intervening in pharyngitis.

[0006] Another object of the present invention is to provide a topical formulation containing XB01 for directly and effectively maintaining pharyngeal health.

[0007] Another objective of this invention is to reveal the unexpected synergistic effect in promoting sleep produced by the combined use of XB01 and Bifidobacterium animalis subsp. lactis XB02.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention is to provide a strain of Lactobacillus rhamnosus XB01, which was deposited at the China General Microbiological Culture Collection Center on June 18, 2025, with the accession number CGMCC No. 34941.

[0010] A second aspect of the present invention is to provide the use of the aforementioned *Lactobacillus rhamnosus* XB01 in the preparation of a product for inhibiting pathogenic bacteria, wherein the pathogenic bacteria are selected from at least one of *Vibrio parahaemolyticus*, *Helicobacter pylori*, *Gardnerella vaginalis*, *Streptococcus pneumoniae*, and *Staphylococcus aureus*.

[0011] A third aspect of the present invention is to provide the use of the said Lactobacillus rhamnosus XB01 in the preparation of products for the prevention or improvement of pharyngitis.

[0012] A fourth aspect of the present invention is to provide a microbial preparation comprising an effective amount of the aforementioned *Lactobacillus rhamnosus* XB01 and a pharmaceutically or food-grade acceptable carrier.

[0013] Preferably, the preparation is a dosage form suitable for local application to the pharynx, including sprays, lozenges, mouthwashes, or gels.

[0014] The fifth aspect of the present invention is to provide a microbial composition comprising the aforementioned *Lactobacillus rhamnosus* XB01 and *Bifidobacterium animalis* subsp. *lactis* XB02, wherein the preservation number of *Bifidobacterium animalis* subsp. *lactis* is CGMCC No. 34942.

[0015] Preferably, the ratio of viable bacteria of Lactobacillus rhamnosus XB01 to Bifidobacterium animalis subsp. lactis XB02 is 1:10 to 10:1.

[0016] A sixth aspect of the present invention is to provide the use of the aforementioned microbial composition in the preparation of products for improving sleep quality or prolonging sleep time.

[0017] A seventh aspect of the present invention is to provide a topical product for intervening in pharyngitis, comprising inactivated cells, cell lysates, or metabolites of the aforementioned Lactobacillus rhamnosus XB01.

[0018] The eighth aspect of this invention is to provide a method for large-scale fermentation of *Lactobacillus rhamnosus* XB01 as described above, comprising the following steps:

[0019] The optimized culture medium consisted of: 12–15% skim milk powder, 8–12% glucose, 0.5–1.5% yeast extract, 1.5–2.5% casein hydrolysate, 0.1–0.2% Tween-80, and the remainder being water.

[0020] High-density culture with feed was carried out in a 10L automated fermenter, with the glucose concentration controlled at 4-6 g / L;

[0021] Fermentation conditions: pH 6.2–6.5, temperature 37±1℃, stirring speed 150–200 r / min, fermentation time 18–22 hours.

[0022] A ninth aspect of the present invention is to provide a method for preparing the microbial preparation, comprising the steps of mixing Lactobacillus rhamnosus XB01 with a cryoprotectant and freeze-drying the mixture, wherein the cryoprotectant comprises trehalose and skim milk.

[0023] Preferably, the freeze-drying preservation method of Lactobacillus rhamnosus XB01 with a cryoprotectant specifically includes:

[0024] The fermented cells are mixed with a cryoprotectant, which contains skim milk and trehalose.

[0025] Pre-freeze at -35℃ to -45℃, then vacuum dry until moisture content is ≤3%.

[0026] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0027] (1) Novel antibacterial spectrum: Lactobacillus rhamnosus XB01 breaks through the traditional antibacterial range of Lactobacillus rhamnosus. For the first time, it systematically confirms and protects its significant inhibitory effect on Vibrio parahaemolyticus, Gardnerella vaginalis and pharyngitis-related pathogens, providing unique technical support for the development of new products.

[0028] (2) Application innovation: The innovative application of Lactobacillus rhamnosus XB01 to pharyngitis intervention and the design of targeted local drug delivery formulations provide a new, non-antibiotic solution for directly maintaining upper respiratory tract health through microbial regulation, with a clear market application prospect.

[0029] (3) Significant synergistic effect: Experiments have confirmed that a specific combination of XB01 and XB02 has a clear synergistic enhancement effect on sleep promotion, and the effect is not simply additive. This provides core technology and data support for developing probiotic products that improve sleep with higher efficiency and lower dosage. Attached Figure Description

[0030] Figure 1 This is a Gram staining image of Lactobacillus rhamnosus XB01 of the present invention. Detailed Implementation

[0031] The core of this invention is to provide a novel *Lactobacillus rhamnosus* strain XB01 and its novel application in inhibiting specific pathogens and preventing or improving pharyngeal inflammation. Furthermore, this invention also relates to microbial preparations containing this strain, particularly its application in combination with *Bifidobacterium animalis* subsp. *lactobacter* XB02 in synergistically promoting sleep. The main technical solutions claimed by this invention are as follows:

[0032] First, we provide a strain of *Lactobacillus rhamnosus* XB01 with Tibetan identification number CGMCC No. 34941. This strain has been shown for the first time to be used in the preparation of products that inhibit *Vibrio parahaemolyticus*, *Helicobacter pylori*, *Gardnerella vaginalis*, *Streptococcus pneumoniae*, and *Staphylococcus aureus*, as well as for the preparation of products for the prevention or improvement of pharyngitis.

[0033] Secondly, a microbial preparation containing XB01 is provided, which is designed for local application to the pharynx, such as a spray, lozenge, mouthwash, or gel, so that the bacteria or its active ingredients can act directly on the pharyngeal mucosa to competitively inhibit pathogen colonization and relieve inflammation.

[0034] Then, a microbial composition comprising *Lactobacillus rhamnosus* XB01 and *Bifidobacterium animalis* subsp. *lactobacter* XB02 (CGMCC No. 34942) is provided. This particular combination exhibits a synergistic effect in improving sleep quality, significantly superior to the use of a single strain.

[0035] Finally, in terms of product form, an application product can be provided, which may be in various forms containing an effective amount of live bacteria, inactivated bacteria, bacterial lysates, or metabolites, such as functional foods, pharmaceuticals, health products, or feed additives.

[0036] I. Strain Screening and Identification

[0037] The strain XB01 of this invention was isolated from intestinal samples of healthy infants and obtained through screening under specific culture media and conditions:

[0038] Initial screening medium: MRS agar medium supplemented with 0.3% ox bile salt and 0.05% pepsin, pH 2.5–3.0, to simulate the gastrointestinal environment;

[0039] Screening conditions: Anaerobic culture at 37℃ for 48 hours, selecting colonies resistant to acid bile salts;

[0040] Identification method: The bacteria were identified as *Lactobacillus rhamnosus* by colony morphology, Gram staining, 16S rRNA gene sequence analysis and whole genome sequencing.

[0041] Based on 16S rRNA gene sequence analysis (SEQ ID NO:1) and average nucleotide identity (ANI) analysis, XB01 showed <95% similarity to known Lactobacillus rhamnosus strains, making it a newly discovered strain.

[0042] II. Characteristics of the strain

[0043] This Lactobacillus rhamnosus XB01 has the following biological characteristics:

[0044] Morphological characteristics: Gram-positive bacilli, colonies are milky white, round, and have neat edges.

[0045] Physiological characteristics: Facultative anaerobic, does not form spores, optimal growth temperature is 37℃, and optimal pH is 6.2~6.5.

[0046] Tolerance: Survival rate ≥85% in pH 2.5–3.0 environment, and survival rate ≥80% in 0.3% bile salt condition.

[0047] Antibacterial activity: It has a significant inhibitory effect on Vibrio parahaemolyticus, Helicobacter pylori and Gardnerella vaginalis.

[0048] Safety: It does not carry known virulence genes or drug resistance genes and is sensitive to common antibiotics.

[0049] III. Large-scale fermentation process

[0050] To achieve high-density culture of the strain, this invention optimizes the fermentation medium and process conditions:

[0051] (1) Culture medium composition:

[0052] Carbon source: 12-15% skim milk powder, 8-12% glucose.

[0053] Nitrogen source: 0.5-1.5% yeast extract, 1.5-2.5% casein hydrolysate.

[0054] Growth factors: 0.1-0.2% Tween-80, 0.05-0.1% MgSO4·7H2O, 0.05-0.1% MnSO4·H2O.

[0055] The rest is water.

[0056] (2) Fermentation conditions:

[0057] Inoculation dosage: 2-4%.

[0058] Temperature: 37±1℃.

[0059] pH control: 6.2–6.5, maintained by automatic addition of NaOH solution.

[0060] Stirring speed: 150~200r / min.

[0061] Feeding strategy: When the glucose concentration is below 4 g / L, add glucose to maintain the concentration at 4-6 g / L.

[0062] Fermentation time: 18-22 hours.

[0063] This process can achieve a viable bacterial count of 4.2 × 10⁻⁶. 10 The CFU / mL concentration is more than 10 times higher than that of conventional batch culture.

[0064] IV. Freeze-drying preservation technology

[0065] To improve strain stability and extend shelf life, this invention optimizes the freeze-drying protectant and process:

[0066] Protectant composition: 12% skim milk, 5% trehalose, 3% glycerol, 2% sucrose, and the remainder is water.

[0067] Pre-freezing conditions: Pre-freeze at -40℃ for 2 to 4 hours.

[0068] Vacuum drying: cold trap temperature -55℃, vacuum degree <10Pa, drying time 24-36 hours.

[0069] Moisture control: Final moisture content ≤3%.

[0070] This method ensures that the survival rate of the strain is ≥80% after 12 months of storage at 4°C.

[0071] The present invention will now be described in detail and specifically through specific embodiments to enable a better understanding of the invention. However, the following embodiments do not limit the scope of the invention.

[0072] Example 1: Screening and Identification of Lactobacillus rhamnosus XB01

[0073] (1) Sample source and pretreatment

[0074] Collect 10 stool samples from healthy infants (6 months old). Add 1g of each sample to 9mL of sterile saline, mix thoroughly, and then serially dilute to 10⁻⁶. -3 .

[0075] (2) Initial screening of acid- and bile-tolerant strains

[0076] 100 μL of the diluted solution was spread onto MRS agar plates containing 0.3% ox bile salts and 0.05% pepsin (pH 3.0) and anaerobically cultured at 37°C for 48 hours. Single colonies with good growth were selected for purification, yielding a total of 85 acid-tolerant bile salt-resistant lactic acid bacteria.

[0077] (3) Screening for antibacterial activity

[0078] The antibacterial activity of the initially screened strains against Vibrio parahaemolyticus, Helicobacter pylori, and Gardnerella vaginalis was evaluated using the agar diffusion method. The results showed that 12 strains had inhibitory effects against at least one pathogen, of which 3 strains (including XB01) had significant inhibitory effects against all three pathogens.

[0079] (4) Strain identification

[0080] Systematic evaluation of XB01:

[0081] Morphological characteristics: Gram-positive short rod-shaped bacteria; colonies are milky white, round, and smooth. Figure 1 As shown.

[0082] Physiological and biochemical characteristics: It can ferment glucose, lactose, and maltose, but not arabinose or xylose.

[0083] 16S rRNA gene sequence analysis: 99.5% homology with the standard strain of Lactobacillus rhamnosus.

[0084] Whole genome sequencing: The genome size is 2.85 Mb, and the GC content is 47.2%. It was finally identified as Lactobacillus rhamnosus and named Lactobacillus rhamnosus XB01. It was deposited at the China General Microbiological Culture Collection Center on June 18, 2025, with the accession number CGMCC No. 34941.

[0085] Example 2: In vitro antibacterial evaluation of Lactobacillus rhamnosus XB01

[0086] To evaluate the in vitro antibacterial activity of *Lactobacillus rhamnosus* XB01, the strain screened in Example 1 was streaked and purified. *Lactobacillus rhamnosus* HN001 was used as a control strain. Both strains were inoculated into 50 ml of MRS liquid medium and cultured at 37°C for 16 h. The fermentation broth was then centrifuged at 10000 rpm for 10 min, and the supernatant and bacterial cells were collected separately. The bacterial cells were washed twice with 0.9% sterile saline, and then OD was prepared using 0.9% sterile saline. 600 The bacterial suspension was prepared at a concentration of 0.5–0.8 g and stored at 4°C for future use. *Aeromonas coli* BNCC 377479, *Streptococcus agalactiae* BNCC 357545, and *Escherichia coli* ATCC 35469 were selected as test strains for in vitro antibacterial experiments. The inhibition zone test was performed on the fermentation supernatant and bacterial suspension of each strain using the agar perforation method.

[0087] Lactic acid was used to adjust the pH of the MRS culture medium to be the same as that of the supernatant as a blank control. A 0.05 mg / mL levofloxacin solution served as a positive control for inhibiting *Aeromonas coli* BNCC 377479, *Streptococcus agalactiae* BNCC 357545, and *Escherichia coli* ATCC 35469. The size of the inhibition zone was measured; a zone larger than the MRS control was considered to have antibacterial activity. Strains with weak inhibitory effects were removed, and the results for strains with significant inhibitory effects were evaluated. The results are shown in Table 1.

[0088] Table 1 Evaluation of antibacterial effect

[0089] experimental group Molybditis kohlii agalactococcus Escherichia coli Fergusonii Positive control (levofloxacin) +++ +++ +++ Blank control (MRS) - - - XB01 strain (bacterial suspension) ++a ++a ++a XB01 strain (supernatant) ++a ++a +a HN001 strain (bacterial suspension) + + + HN001 strain (supernatant) + - -

[0090] Note: "-" indicates no obvious inhibition zone, "+" indicates inhibition zone diameter is between 3mm and 5mm, "++" indicates inhibition zone diameter is between 5mm and 10mm, and "+++" indicates inhibition zone diameter is >10mm. a is significantly different from HN001.

[0091] Experimental results showed that the bacterial suspension and supernatant of strain XB 01 had significant inhibitory effects on *Aerobicus kohlii* BNCC 377479, *Streptococcus agalactiae* BNCC 357545, and *Escherichia coli* ATCC 35469, and the antibacterial ability was significantly better than that of *Lactobacillus plantarum* HN 001.

[0092] Example 3: Effect of Lactobacillus rhamnosus XB01 on Pharyngitis

[0093] The study selected individuals aged 35–65 years diagnosed with pharyngitis, who met the following criteria: accompanied by dry throat / foreign body sensation, duration of illness ≥72 hours, and no use of antibiotics, immunomodulators, or other pharyngitis treatments in the past week. Individuals with allergies, severe liver or kidney dysfunction, immunodeficiency, or who are pregnant / lactating were excluded. A total of 72 individuals were randomly divided into two groups of 36 each. The experimental group received daily oral administration of Lactobacillus rhamnosus XB01 (2 × 10⁻⁶). 10 CFU / sachet) probiotic preparation, once a day, one sachet each time. The control group took a placebo (containing no live bacteria, maltodextrin and sweetener) with the same appearance and taste as the experimental group, and took it in the same way as the experimental group. During the test, both groups were required to avoid spicy and cold foods, quit smoking and drinking alcohol, and drink ≥1500ml of water per day for 7 consecutive days.

[0094] Pharyngitis symptom scores were collected before intervention (day 0) and on day 7 (using the Visual Analogue Scale (VAS), 0–10 points, with higher scores indicating more severe symptoms; a sore throat score ≥3 was considered pharyngitis symptom). Scores for sore throat (intensity of pain during swallowing), dry throat (frequency of throat dryness), and foreign body sensation (duration of foreign body sensation in the throat) were also recorded. The results are shown in Table 2.

[0095] Table 2 Changes in Pharyngitis Symptom Scores

[0096]

[0097]

[0098] Note: * indicates that compared with the control group at the same time point, P<0.05; compared with the group before intervention, P<0.05.

[0099] The experimental results showed that after 7 days of intervention with Lactobacillus rhamnosus XB01, the experimental group had significantly lower levels of symptoms such as sore throat, dry throat, and foreign body sensation compared to the control group, and all symptoms improved. This indicates that the Lactobacillus rhamnosus XB01 probiotic preparation can quickly and continuously relieve pharyngitis symptoms.

[0100] Example 4: Evaluation of the anti-fatigue effect of Lactobacillus rhamnosus XB01

[0101] (1) Experimental materials

[0102] Experimental animals: 120 SPF-grade ICR male mice, weighing 18-22g, were randomly divided into 4 groups. Preparation of bacterial agent: XB01 was fermented and cultured, and the bacterial cells were collected by centrifugation and resuspended in physiological saline to the required concentration.

[0103] (2) Experimental grouping and treatment

[0104] Control group: normal saline administered by gavage

[0105] Low-dose group: XB011×10 8CFU / day

[0106] High-dose group: XB011×10 9 CFU / day

[0107] Positive control group: Commercial Lactobacillus rhamnosus GG 1×10 9 CFU / day

[0108] The patients were given gavage for 28 consecutive days, and various indicators were measured one hour after the last gavage.

[0109] (3) Weighted swimming experiment

[0110] Mice were placed in a swimming tank with a tail base loaded with a lead wire equal to 5% of their body weight. The water temperature was 25±1℃ and the depth was 30cm. The time to exhaustion (from the moment the mouse entered the water until its head sank to the surface for 10 seconds) was recorded. The results are shown in Table 3.

[0111] Table 3 Comparison of weight-bearing swimming time among different groups of mice

[0112]

[0113] Note: Compared with the control group, *P<0.05, **P<0.01

[0114] (4) Biochemical index determination

[0115] Immediately after the swimming experiment, blood samples were taken to measure blood lactate (BLA) and blood urea nitrogen (BUN) levels. After euthanasia, liver samples were taken to measure liver glycogen content. The results are shown in Table 4.

[0116] Table 4 Comparison of biochemical indicators among mice in each group

[0117]

[0118] Note: Compared with the control group, *P<0.05, **P<0.01

[0119] Experimental results showed that XB01 could significantly prolong the swimming time of mice under load, reduce blood lactate and urea nitrogen levels after exercise, and increase liver glycogen reserves. Moreover, its effect was better than that of the commercial strain GG, proving that it has a significant anti-fatigue effect.

[0120] Example 5: Evaluation of the sleep-promoting effect of Lactobacillus rhamnosus XB01 combined with Bifidobacterium animalis subsp. lactis XB02

[0121] (1) Experimental materials

[0122] Laboratory animals: 90 male SPF-grade C57BL / 6J mice, weighing 20–25g.

[0123] Preparation of bacterial agents: XB01 and XB02 were fermented and cultured separately, the bacterial cells were collected by centrifugation and resuspended in physiological saline.

[0124] (2) Experimental grouping and treatment

[0125] Control group: normal saline administered by gavage

[0126] XB01 single-cell group: 1×10 9 CFU / day

[0127] XB02 single-cell group: 1×10 9 CFU / day

[0128] Combined group: XB015×10 8 CFU+XB025×10 8 CFU / day

[0129] Positive control group: GABA 5mg / kg

[0130] A pentobarbital sodium-induced sleep experiment was conducted by continuous gavage for 30 days.

[0131] (3) Sodium pentobarbital-induced sleep experiment

[0132] Mice were injected intraperitoneally with a subthreshold dose of sodium pentobarbital (35 mg / kg) to observe sleep latency and sleep duration. The results are shown in Table 5.

[0133] Table 5 Comparison of sleep indicators among different groups of mice

[0134]

[0135] Note: Compared with the control group, *P<0.05, **P<0.01; compared with the single-strain group, △P<0.05

[0136] (4) GABA content determination

[0137] After euthanizing the mice, the cerebral cortex was harvested, and the GABA content was determined by HPLC. The results showed that the GABA content in the brain of the combined group was 35.6±4.2 μg / g, which was significantly higher than that in the control group (24.3±3.1 μg / g) and the single-bacterial group (XB01 group: 28.7±3.5 μg / g; XB02 group: 30.2±3.8 μg / g).

[0138] Experimental results show that the combined use of XB01 and XB02 has a significant synergistic sleep-promoting effect, which is better than the use of single bacteria. The mechanism may be related to the increase of GABA content in the brain.

[0139] Example 6: Large-scale fermentation of Lactobacillus rhamnosus XB01

[0140] (1) Culture medium preparation

[0141] The basal culture medium consisted of 12% skim milk powder, 10% glucose, 1% yeast extract, 2% casein hydrolysate, 0.15% Tween-80, and the remainder was water; pH 6.5, sterilized at 121°C for 15 minutes.

[0142] (2) Fermentation process

[0143] Seed culture preparation: The frozen bacterial strain was inoculated into MRS liquid medium and incubated at 37°C for 18 hours.

[0144] Fermentation tank cultivation: Inoculate 4% into a 10L automatic fermenter, and control the following conditions: temperature: 37±0.5℃;

[0145] pH: 6.2–6.5 (controlled by automatic addition of 5M NaOH); stirring speed: 180 r / min.

[0146] Feeding strategy: When the glucose concentration is below 4 g / L, add 50% glucose solution to maintain the concentration at 4-6 g / L.

[0147] (3) Results

[0148] During the fermentation process, samples were taken every 2 hours to determine the viable cell count and pH. The results are shown in Table 6.

[0149] Table 6. Cell growth during fermentation.

[0150]

[0151] Note: * indicates that glucose supplementation should begin at this point.

[0152] The results showed that after 20 hours of fermentation, the viable cell count reached 4.2 × 10⁻⁶. 10 CFU / mL, compared to conventional batch culture (3.8 × 10⁻⁶). 9 The CFU / mL ratio increased by more than 10 times.

[0153] Comparison of antibacterial spectra of different Lactobacillus rhamnosus strains in Comparative Example 1

[0154] Three commercially available strains of Lactobacillus rhamnosus IDCC 3201, Lactobacillus rhamnosus GR-1, and Lactobacillus rhamnosus TR08 were selected and compared with the present invention XB01. The antibacterial effects against Gardnerella vaginalis and Streptococcus pneumoniae were compared using the same method as in Example 2. The results are shown in Table 7.

[0155] Table 7 Comparison of antibacterial activities of different Lactobacillus rhamnosus strains

[0156]

[0157] The results showed that the inhibitory effects of strains IDCC 3201, GR-1 and TR08 on these two bacteria were generally weak or unstable, while XB01 showed a significant and stable strong inhibitory effect, highlighting its strain-specific advantage.

[0158] Comparative Example 2: Comparison of Anti-fatigue Effects of Different Strains

[0159] To demonstrate the unique advantages of XB01, the anti-fatigue effects of various commercially available probiotic strains were compared. The experimental method was the same as in Example 4, and the results are shown in Table 8.

[0160] Table 8 Comparison of anti-fatigue effects of different strains

[0161]

[0162] The results showed that XB01 was significantly more effective than other control strains in combating fatigue.

[0163] Comparison of the sleep-promoting effects of different strain combinations in Comparative Example 3

[0164] XB01 was combined with XB02, another strain of Bifidobacterium lactis (Bifidobacterium animalis subsp. lactis BLa80), and a strain of Lactobacillus acidophilus (Lactobacillus acidophilus LIHUO 1978), respectively, and the sleep-promoting effects of different strain combinations were compared. The experimental method was the same as in Example 5. The results are shown in Table 9.

[0165] Table 9 Comparison of sleep-promoting effects of different strain combinations

[0166]

[0167] The results showed that the dormancy-promoting effect of XB01 when combined with other bacterial strains did not reach the synergistic level when combined with XB02, indicating that the synergistic effect of XB01 and XB02 is strain-specific and cannot be achieved by combining with any Bifidobacterium or Lactobacillus.

[0168] The above examples and comparative examples fully demonstrate the outstanding innovation, technological progress, and practical value of the Lactobacillus rhamnosus XB01 and its applications provided by the present invention in expanding the antibacterial spectrum, innovatively applying it to pharyngitis intervention, and producing a synergistic sleep-promoting effect when used in combination with a specific partner strain XB02.

[0169] Furthermore, by optimizing the fermentation process, this invention achieves a viable cell count of 4.2 × 10⁻⁶. 10 The CFU / mL concentration is significantly higher than that of conventional processes. Furthermore, by optimizing the freeze-drying process, the stability of the strain in storage and the gastrointestinal environment has been significantly improved, making it widely applicable in probiotic preparations, functional foods, pharmaceuticals, and health products.

[0170] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus XB01, characterized in that, The Lactobacillus rhamnosus XB01 was deposited at the China General Microbiological Culture Collection Center on June 18, 2025, with accession number CGMCC No. 34941.

2. The use of *Lactobacillus rhamnosus* XB01 as described in claim 1 in the preparation of a product for inhibiting pathogenic bacteria, characterized in that... The pathogenic bacteria are selected from at least one of Vibrio parahaemolyticus, Helicobacter pylori, Gardnerella vaginalis, Streptococcus pneumoniae, and Staphylococcus aureus.

3. The use of the Lactobacillus rhamnosus XB01 of claim 1 in the preparation of a product for the prevention or improvement of pharyngitis.

4. A microbial preparation, characterized in that, It contains an effective amount of Lactobacillus rhamnosus XB01 as described in claim 1 and a pharmaceutically or food-grade acceptable carrier.

5. The microbial preparation according to claim 4, characterized in that, The formulation is a dosage form suitable for local application to the pharynx, including sprays, lozenges, mouthwashes, or gels.

6. A microbial composition, characterized in that, It comprises *Lactobacillus rhamnosus* XB01 and *Bifidobacterium animalis* subsp. *lactobacter* XB02 as described in claim 1, wherein the preservation number of *Bifidobacterium animalis* subsp. *lactobacter* XB02 is CGMCC No. 34942.

7. The microbial composition according to claim 6, characterized in that, The ratio of viable bacteria of Lactobacillus rhamnosus XB01 to Bifidobacterium animalis subsp. lactis XB02 is 1:10 to 10:

1.

8. The use of the microbial composition of claim 6 or 7 in the preparation of a product for improving sleep quality or prolonging sleep time.

9. A topical product for intervening in pharyngitis, characterized in that, The product contains inactivated cells, cell lysates, or metabolites of *Lactobacillus rhamnosus* XB01 as described in claim 1.

10. A method for preparing the microbial preparation of claim 4, characterized in that, The method includes the steps of mixing Lactobacillus rhamnosus XB01 with a cryoprotectant and freeze-drying it, the cryoprotectant comprising trehalose and skim milk.

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