Bifidobacterium animalis subsp. Lactis XB02 capable of inhibiting pathogenic bacteria and improving inflammation and application of bifidobacterium animalis subsp. Lactis XB02

Abstract

The invention belongs to the technical field of microorganisms, and discloses a bifidobacterium animalis subsp. Lactis XB02 strain capable of inhibiting pathogenic bacteria and improving inflammation, the preservation number of the bifidobacterium animalis subsp. Lactis XB02 strain is CGMCC (China General Microbiological Culture Collection Center) No.34942, and the preservation date is June 18, 2025. Meanwhile, the probiotic composition adopting the bacterial strain XB02 also comprises a lactobacillus rhamnosus XB01 bacterial strain. The XB02 strain disclosed by the invention has excellent bacteriostatic activity, especially for vibrio parahaemolyticus and gardneria; the atopic dermatitis symptom can be obviously improved; the intestinal mucosa permeability is reduced, and the intestinal barrier function is enhanced; and when the strain is combined with the XB01 strain for use, a synergistic sleep promoting effect is shown. The invention further provides a probiotic composition containing the strain, a preparation and application of the probiotic composition and the preparation in food, medicine or health care products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to an animal Bifidobacterium lactis XB02 strain with the functions of inhibiting pathogenic bacteria and improving inflammation, and application thereof, in particular to the application of the strain in inhibiting specific pathogenic bacteria, improving atopic dermatitis, improving intestinal mucosal permeability, and enhancing the sleep-promoting effect in combination with Lactococcus lactis XB01. BACKGROUND

[0002] Bifidobacterium and Lactobacillus are important probiotics in the human intestinal tract, which have physiological functions such as maintaining intestinal flora balance, inhibiting growth of pathogenic bacteria, and enhancing immune regulation. In recent years, research has found that probiotics also have potential application value in skin health and sleep regulation.

[0003] In the prior art, there are some research reports on Bifidobacterium and Lactobacillus. For example, a patent has disclosed the application of animal Bifidobacterium lactis in inflammatory bowel disease (CN119633024A), but it does not disclose the XB02 strain of the present application, nor does it disclose the application of the strain in inhibiting Vibrio parahaemolyticus and Gardnerella, improving atopic dermatitis, and enhancing the sleep-promoting effect in combination with specific Lactococcus lactis.

[0004] In terms of bacteriostatic function, the prior art is mostly focused on the inhibition of common pathogenic bacteria such as Escherichia coli and Staphylococcus aureus, and there are few reports on the bacteriostatic activity of specific pathogenic bacteria such as Vibrio parahaemolyticus and Gardnerella. In terms of improving atopic dermatitis, existing research mostly uses a combination of multiple strains, and the effectiveness of a single strain is insufficient. In terms of improving sleep, although some studies have shown that certain probiotics (such as Lactobacillus plantarum P72 and Lacticaseibacillus paracasei Lpc-37) have the potential to improve sleep, the synergistic effect of different strains has not been thoroughly studied.

[0005] In addition, probiotics are easily inactivated during processing, storage, and gastrointestinal transportation, which limits their application effect. Existing embedding techniques such as single emulsion or polysaccharide microcapsules have limited protection effect and insufficient controlled release performance. Therefore, it is crucial to develop new probiotic embedding techniques to improve their stability. SUMMARY

[0006] The primary purpose of the present application is to provide a new animal Bifidobacterium lactis XB02 strain with the functions of inhibiting pathogenic bacteria and improving inflammation, with the preservation number of CGMCC No.34942.

[0007] A third object of the present application is to provide a probiotic composition comprising the XB02 strain and / or the XB01 strain of Lactobacillus rhamnosus XB01, which has a preservation number of CGMCC No. 34941.

[0008] A third object of the present application is to provide the use of the XB02 strain and / or the probiotic composition in the following aspects:

[0009] Inhibiting pathogenic bacteria such as Vibrio parahaemolyticus and Gardnerella;

[0010] Improving and / or treating atopic dermatitis;

[0011] Improving intestinal mucosal permeability and enhancing intestinal barrier function;

[0012] In combination with the XB01 strain, synergistically promoting sleep and improving sleep quality.

[0013] A fourth object of the present application is to provide a method for improving the stability of the strain, which is encapsulated by a W1 / O / W2 double emulsion embedding system stabilized by potato protein.

[0014] To achieve the above object, the present application adopts the following technical solutions:

[0015] The first aspect of the present application is to provide a strain of Lactobacillus animalis subsp. lactis XB02, which has been preserved in the China General Microbiological Culture Collection Center on June 18, 2025, with a preservation number of CGMCC No. 34942.

[0016] The second aspect of the present application is to provide a probiotic composition comprising live bacteria, inactivated bacteria, bacterial fragments or metabolites of the Lactobacillus animalis subsp. lactis XB02 strain of claim 1.

[0017] Preferably, the probiotic composition further comprises live bacteria, inactivated bacteria, bacterial fragments or metabolites of the Lactobacillus chamois XB01 strain;

[0018] The Lactobacillus chamois XB01 strain has been preserved in the China General Microbiological Culture Collection Center on June 18, 2025, with a preservation number of CGMCC No. 34941

[0019] Preferably, the ratio of the number of live bacteria of the Lactobacillus animalis subsp. lactis XB02 strain to the Lactobacillus chamois XB01 strain is 1:5 to 5:1.

[0020] Preferably, the composition further comprises a pharmaceutically or dietetically acceptable carrier.

[0021] More preferably, the carrier comprises a lyoprotectant, diluent or excipient.

[0022] A third aspect of the present application provides use of the Bifidobacterium animalis lactis XB02 strain or any of the probiotic compositions described in the preparation of a medicament or food for inhibiting pathogenic microorganisms.

[0023] Preferably, the pathogenic microorganisms comprise Vibrio parahaemolyticus and Gardnerella.

[0024] A fourth aspect of the present application provides use of the Bifidobacterium animalis lactis XB02 strain or any of the probiotic compositions described in the preparation of a medicament or food for improving and / or treating atopic dermatitis.

[0025] A fifth aspect of the present application provides use of the Bifidobacterium animalis lactis XB02 strain or any of the probiotic compositions described in the preparation of a medicament or food for improving intestinal mucosal permeability and enhancing intestinal barrier function.

[0026] A sixth aspect of the present application provides use of any of the probiotic compositions described in the preparation of a medicament or food for promoting sleep and improving sleep quality.

[0027] A seventh aspect of the present application provides a method for improving the stability of probiotics, wherein the Bifidobacterium animalis lactis XB02 strain and / or the Lactobacillus rhamnosus XB01 strain is encapsulated using a W1 / O / W2 double emulsion embedding system.

[0028] Preferably, the double emulsion embedding system uses potato protein as a stabilizer.

[0029] The present application has the following technical effects compared with the prior art by using the above technical solutions:

[0030] (1) Novelty of the strain and uniqueness of the function: the XB02 strain provided by the present application is a new Bifidobacterium animalis lactis with the preservation number of CGMCC No.34942. The strain exhibits significant bacteriostatic activity against Vibrio parahaemolyticus and Gardnerella, filling the gap in the prior art for these two specific targets.

[0031] (2) Improvement of atopic dermatitis: the XB02 strain alone can effectively relieve the symptoms of atopic dermatitis, providing reliable evidence for the application of single strains and avoiding the uncontrollability of multi-strain combinations.

[0032] (3) Improving intestinal barrier: XB02 strain can significantly reduce serum endotoxin, up-regulate tight junction proteins, improve intestinal mucosal permeability, and repair intestinal barrier function through multi-targets, with clear effect.

[0033] (4) Synergistic sleep promotion: XB02 combined with XB01 strain shows significant synergistic effect in promoting sleep, which is better than single strain, providing a new idea for developing probiotic products to improve sleep.

[0034] (5) High stability: The use of potato protein double emulsion embedding technology greatly improves the stability of the strain in storage and gastrointestinal environment, ensuring the application effect of probiotics. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Gram staining of the animal Bifidobacterium lactis XB02 strain of the present application. DETAILED DESCRIPTION

[0036] The core scheme of the present application is to provide an animal Bifidobacterium lactis XB02 strain with the ability to inhibit pathogenic bacteria and improve inflammation, and its application. In particular, the application of the strain in inhibiting specific pathogenic bacteria, improving atopic dermatitis, improving intestinal mucosal permeability, and using it in combination with Rhamnosus casei XB01 to enhance the sleep-promoting effect, as well as the probiotic composition, preparation containing the strain and its application in food, medicine or health products are involved. The main technical scheme claimed in the present application is as follows:

[0037] I. Strain screening and identification

[0038] The animal Bifidobacterium lactis XB02 strain described in the present application is isolated and screened from the feces of healthy infants. Through 16S rDNA sequence analysis and whole genome sequencing, it is identified as animal Bifidobacterium lactis. The strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number CGMCC No. 34942 and the preservation date June 18, 2025.

[0039] The Rhamnosus casei XB01 strain used in the present application is also isolated from healthy infant intestinal samples, and is obtained by specific culture medium and conditions, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number CGMCC No. 34941 and the preservation date June 18, 2025.

[0040] II. Bacteriostatic effect

[0041] It was found by the antibacterial circle experiment that the fermentation supernatant of the XB02 strain had a significant inhibitory effect on Vibrio parahaemolyticus and Gardnerella, and the diameters of the antibacterial circles were 15.8±0.5 mm and 14.2±0.4 mm, respectively, which was better than the effect of Comparative Example 1 (known antibacterial strain Lactobacillus gasseri MS-72).

[0042] III. Improving atopic dermatitis

[0043] In the atopic dermatitis mouse model, XB02 strain (1×109CFU / day, for 4 weeks) can significantly reduce the dermatitis score, reduce the scratching frequency, and reduce the levels of IgE and inflammatory factors (IL-4, IL-13) in serum.

[0044] IV. Improving intestinal mucosal permeability

[0045] In the DSS-induced colitis mouse model, XB02 strain intervention can significantly reduce the levels of endotoxin (LPS) and D-lactic acid in serum, and up-regulate the expression of tight junction protein (Occludin, ZO-1) in colon tissue, indicating that it can effectively improve the intestinal mucosal permeability and repair the intestinal barrier.

[0046] V. Combined sleep-promoting effect

[0047] When XB02 strain and XB01 strain are used in combination at a ratio of 1:1 in the sleep-deprived mouse model, a synergistic sleep-promoting effect is exhibited, the sleep latency is significantly shortened, and the sleep duration is prolonged, and the effect is better than that of a single strain.

[0048] VI. Preparation and stability

[0049] In order to improve the stability of the strain, the XB02 and XB01 strains are encapsulated by using a W1 / O / W2 double emulsion embedding system stabilized by potato protein. After simulation of gastrointestinal digestion, the survival rate of the bacterial cells is as high as 86%, and after storage at 4℃ for 35 days, the viable bacterial count is still maintained at 8.06log10 CFU / mL, which is significantly higher than that of the free bacteria (4.35log10 CFU / mL) without embedding.

[0050] The present application will be described in detail and specifically through specific examples below, so that it can be better understood. However, the following examples do not limit the scope of the present application.

[0051] Example 1 Screening and identification of animal Bifidobacterium lactis XB02 strain

[0052] Isolation and purification: sample was taken from the intestinal fecal sample of healthy infants, and MRS medium (containing cysteine hydrochloride) was used for anaerobic culture (37℃, 72h). Single colonies were picked and repeatedly streaked and purified.

[0053] Morphological observation: The colony was ivory white, round, smooth surface, and regular edge. Gram staining was positive, and the cell was polymorphic rod as shown in the figure. Figure 1

[0054] Molecular biology identification: The genomic DNA of the strain was extracted, and the 16S rDNA sequence was amplified and sequenced. The sequencing results were compared with the NCBI database, and the homology was more than 99%, which was identified as Bifidobacterium animalis subsp. lactis. The whole genome of the strain was sequenced, and the complete genomic information was obtained.

[0055] Preservation: The strain was named XB02 and preserved in the China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 34942.

[0056] Example 2 Bacteriostatic experiment

[0057] Strain preparation: XB02 strain, Comparative Example 1 (Lactobacillus gasseri MS-72) and Comparative Example 2 (known bacteriostatic Lactobacillus plantarum EH1) were inoculated in MRS liquid medium, respectively, and cultured at 37°C for 48h under anaerobic condition. The supernatant was collected by centrifugation, filtered with 0.22μm filter membrane to remove bacteria, and obtained cell-free fermentation supernatant (CFS).

[0058] Indicator bacteria: Vibrio parahaemolyticus ATCC 17802, Gardnerella vaginalis ATCC 14018, Helicobacter pylori ATCC43504.

[0059] Bacteriostatic circle method: The indicator bacteria suspension was coated on the corresponding solid medium plate. Oxford cups were placed in the center of the plate, and 100μL of the above CFS was added. After 24-48h culture at 37°C, the diameter of the bacteriostatic circle was measured.

[0060] Results: As shown in the following table, XB02 strain had obvious inhibitory effect on Vibrio parahaemolyticus, Gardnerella vaginalis and Helicobacter pylori, and the effect was better than that of Comparative Example 1 and Comparative Example 2.

[0061] Table 1 Diameter of bacteriostatic circle (mm, mean ± standard deviation, n = 3)

[0062]

[0063] Example 3 Experiment for improving atopic dermatitis ​

[0064] Animal model: BALB / c mice were selected and randomly divided into 5 groups: normal control group, model control group, XB02 low-dose group (1 x 10 8 CFU / day), XB02 high-dose group (1 x 10 9 CFU / day), positive control group (commercial Bifidobacterium longum 1714).

[0065] Modeling and intervention: the atopic dermatitis model was established by repeatedly stimulating the back skin of mice with dust mite allergens. At the same time of modeling, each group was given corresponding bacterial solution or normal saline by gavage for 4 weeks.

[0066] Evaluation index: dermatitis score and scratching frequency were recorded every week. At the end of the experiment, serum IgE, IL-4, and IL-13 levels were detected.

[0067] Results: As shown in Table 2, compared with the model control group, the XB02 high-dose group could significantly reduce the dermatitis score, scratching frequency, and serum IgE, IL-4, and IL-13 levels, and the effect was comparable to that of the positive control group, and even better in some indicators.

[0068] Table 2 Effect of XB02 on atopic dermatitis mice (mean ± standard deviation, n = 8)

[0069]

[0070]

[0071] Note: p < 0.05, *p < 0.01 compared with the model control group.

[0072] Example 4 Experiment of improving intestinal mucosal permeability

[0073] Animal model: C57BL / 6 mice were selected and randomly divided into 4 groups: normal control group, DSS model group, XB02 intervention group (1 x 10 9 CFU / day), Comparative Example 3 (a mixture of Limosilactobacillus fermentum TD-3 and Lactococcus lactis MQ1-1 known to improve intestinal barrier).

[0074] Modeling and intervention: 3% DSS drinking water was used to feed for 7 days to induce acute colitis. XB02 intervention group and Comparative Example 3 started to give bacterial solution by gavage at the same time as DSS was given, for 7 days.

[0075] Evaluation index: At the end of the experiment, blood was collected to detect the levels of serum endotoxin (LPS) and D-lactic acid. The colon tissue was taken to detect the expression of tight junction proteins Occludin and ZO-1 by Western Blot.

[0076] Results: As shown in Table 3, the serum LPS and D-lactic acid levels of the DSS model group were significantly increased, while XB02 intervention could effectively reverse this trend and significantly up-regulate the protein expression of Occludin and ZO-1 in colon tissue, with better effect than Comparative Example 3.

[0077] Table 3 Effect of XB02 on intestinal mucosal permeability of DSS colitis mice (mean ± standard deviation, n = 6)

[0078]

[0079] Note: Compared with the DSS model group, p < 0.05, *p < 0.01. Protein expression is expressed as fold relative to the normal control group.

[0080] Example 5 Combined sleep-promoting experiment

[0081] Animal model and grouping: The threshold sub-dose hypnotic model and sleep deprivation model of sodium pentobarbital were used. ICR mice were randomly divided into 6 groups: normal control group, sleep deprivation model group, XB01 single strain group (1 × 10 9 CFU / day), XB02 single strain group (1 × 10 9 CFU / day), XB01 + XB02 combined group (0.5 × 10 9 CFU / day each), positive control group (Lactobacillus plantarum P72, 1 × 10 9 CFU / day).

[0082] Intervention and testing: Intragastric intervention for 3 weeks. In the sodium pentobarbital experiment, the sleep latency and sleep time of mice were recorded. After sleep deprivation, behavioral tests were performed.

[0083] Results: As shown in Table 4, in the sleep deprivation model, the XB01 + XB02 combined group significantly outperformed each single strain group and the positive control group in terms of shortening sleep latency and prolonging sleep time, indicating that the two strains have a synergistic sleep-promoting effect.

[0084] Table 4 Effect of strain combination on sleep-promoting effect in sleep-deprived mice (mean ± standard deviation, n = 10)

[0085]

[0086] Note: Compared with the sleep deprivation model group, p < 0.05, *p < 0.01.

[0087] Embedding and stability test of Example 6 strains

[0088] Preparation of double emulsion embedding system: XB02 and XB01 strains were suspended in physiological saline (W1 phase). PGPR was dissolved in soybean oil (O phase). W1 phase was slowly added to O phase, and high-speed homogenization was performed to form W1 / O primary emulsion. The primary emulsion was added to an aqueous solution containing 1.5% (w / w) potato protein (PP) (W2 phase), and shearing was performed to form a W1 / O / W2 double emulsion.

[0089] Embedding efficiency and stability test: The encapsulation efficiency of the double emulsion was determined. The embedded bacterial powder and the unembedded freeze-dried bacterial powder were stored at 4°C and 25°C, respectively, and the viable cell count was detected regularly. In vitro simulated gastrointestinal digestion experiments were performed, and the viable cell count after digestion was detected.

[0090] Results: The encapsulation efficiency of XB02 / XB01 in potato protein double emulsion reached 93.5%. After 35 days of storage at 4°C, the viable cell count of the embedded group remained above 8.0 log10 CFU / mL, while that of the unembedded group decreased to below 4.5 log10 CFU / mL. After simulated gastrointestinal digestion, the survival rate of the embedded strain group was as high as 85%, which was significantly higher than that of the unembedded group (about 30%).

[0091] Comparative Example 1 Bacteriostatic experiment

[0092] Test method: Using the bacteriostatic circle method, the cell-free fermentation supernatant of XB02, Comparative Example 1 (Lactobacillus gasseri MS-72) and Comparative Example 2 (Lactiplantibacillus plantarum EH1) was added to a flat plate Oxford cup coated with pathogenic bacteria, and the diameter of the bacteriostatic circle was measured after incubation.

[0093] Results: As shown in Table 5, the inhibition of Vibrio parahaemolyticus and Gardnerella by XB02 strain was significantly stronger than that of the two control strains, which proved its unique and excellent potential in inhibiting these two specific pathogenic bacteria.

[0094] Table 5: Comparison of bacteriostatic effect of XB02 strain and MS-72 strain

[0095]

[0096] Comparative Example 2 Improvement of atopic dermatitis (AD)

[0097] Test method: Establish AD model induced by dust mite allergen in mice. Set XB02 low and high dose groups, and set positive control group (commercial Bifidobacterium longum 1714). After intervention, evaluate dermatitis score, scratching frequency and serum immune indicators (IgE, IL-4, IL-13).

[0098] Results: As shown in Table 6, XB02 strain can significantly alleviate AD symptoms and reduce allergic immune response. The high dose of single strain is equivalent to the effect of commercial strain 1714, and is slightly better in some indicators.

[0099] Table 6 Comparison of XB02 strain and commercial strain 1714 strain on antibacterial effect

[0100]

[0101]

[0102] Note: Compared with the model control group, **p<0.01

[0103] Example 3 Improvement of intestinal mucosal permeability

[0104] Test method: Use DSS-induced mouse colitis model. Set XB02 intervention group and example 3 group (mixture of TD-3 and MQ1-1). Detect serum endotoxin (LPS) and D-lactic acid levels, and analyze the expression of tight junction protein (Occludin, ZO-1) in colon tissue by Western Blot.

[0105] Results: As shown in Table 7, XB02 is better than the strain mixture of example 3 in reducing intestinal permeability indicators (LPS, D-lactic acid) and up-regulating tight junction protein expression, indicating that it has better effect in repairing damaged intestinal barrier.

[0106] Table 7 Comparison of XB02 strain and mixture of TD and MQ1-1 on intestinal barrier improvement

[0107]

[0108] Example 4 Sleep-promoting experiment

[0109] Test method: Use sleep-deprived mouse model. Set XB01 single strain group, XB02 single strain group, XB01+XB02 combined group, and positive control group (commercial Lactobacillus plantarum P72, i.e. example 4). Record sleep latency and sleep duration by pentobarbital sodium experiment.

[0110] Results: As shown in Table 8, the combination of XB01 and XB02 significantly outperforms either single strain and the known sleep-promoting strain P72 in shortening sleep onset latency and prolonging sleep duration, fully demonstrating the synergistic effect between the two, which is one of the core innovations of the present application.

[0111] Table 8 Comparison of sleep-promoting effects of XB01, XB02 and their combination with commercial Lactobacillus plantarum P72 strain

[0112]

[0113]

[0114] Strain stability experiment of Comparative Example 5

[0115] Test method: XB02 / XB01 bacteria powder embedded in double emulsion and Comparative Example 5 (unembedded freeze-dried bacteria powder) were stored at 4℃ and 25℃, respectively, and the viable cell count was detected regularly; and in vitro simulated gastrointestinal digestion experiment was conducted to detect the viable cell count after digestion.

[0116] Storage stability: After 35 days of storage at 4℃, the viable cell count of the embedded group remained at 8.06 log10 CFU / mL, while that of the unembedded group decreased to 4.35 log10 CFU / mL.

[0117] Digestive tolerance: After simulated gastrointestinal digestion, the survival rate of the embedded strain was as high as 86%, which was significantly higher than that of the unembedded group (about 30%).

[0118] The results show that the double emulsion embedding technology used in the present application can significantly improve the storage stability and gastrointestinal tolerance of XB02 and XB01 strains, effectively ensuring the number of viable bacteria before the probiotic product reaches the intestine.

[0119] The above describes the specific embodiments of the present application in detail, but it is only as an example, the present application is not limited to the above described specific embodiments. For those skilled in the art, any equivalent modification and replacement of the present application are also within the scope of the present application. Therefore, any equivalent transformation and modification without departing from the spirit and scope of the present application should be covered within the scope of the present application.

Claims

1. An animal Bifidobacterium lactis XB02 strain having the characteristics of inhibiting pathogenic bacteria and improving inflammation, characterized in that, The animal Bifidobacterium lactis XB02 strain is preserved in the China General Microbiological Culture Collection Center on June 18, 2025, and the preservation number is CGMCC No. 34942.

2. A probiotic composition, characterized in that, The live bacteria, inactivated bacteria, bacterial fragments or metabolites of the animal Bifidobacterium lactis XB02 strain of claim 1.

3. The probiotic composition as claimed in claim 2, wherein, The probiotic composition further comprises live bacteria, inactivated bacteria, bacterial fragments or metabolites of the Bifidobacterium lactis XB01 strain; The Bifidobacterium lactis XB01 strain is preserved in the China General Microbiological Culture Collection Center on June 18, 2025, and the preservation number is CGMCC No. 34941.

4. The probiotic composition according to claim 3, characterized in that, The ratio of the viable bacterial number of the animal Bifidobacterium lactis XB02 strain to the Bifidobacterium lactis XB01 strain is 1:5 to 5:

1.

5. The probiotic composition according to claim 3, characterized in that, The composition further comprises a pharmaceutically or food-acceptable carrier; The carrier comprises a freeze-drying protective agent, a diluent or an excipient.

6. Use of the animal Bifidobacterium lactis XB02 strain of claim 1 or the probiotic composition of any one of claims 3-5 in the preparation of a medicament or food for inhibiting pathogenic microorganisms. The pathogenic microorganisms include Vibrio parahaemolyticus and Gardnerella.

7. Use of the animal Bifidobacterium lactis XB02 strain of claim 1 or the probiotic composition of any one of claims 3-5 in the preparation of a medicament or food for improving and / or treating atopic dermatitis.

8. Use of the animal Bifidobacterium lactis XB02 strain of claim 1 or the probiotic composition of any one of claims 3-5 in the preparation of a medicament or food for improving intestinal mucosal permeability and enhancing intestinal barrier function.

9. Use of the probiotic composition of any one of claims 3-5 in the preparation of a medicament or food for promoting sleep and improving sleep quality.

10. A method of improving stability of probiotic bacteria, characterized by, The animal Bifidobacterium lactis XB02 strain of claim 1 and / or the Bifidobacterium lactis XB01 strain of claim 3 are encapsulated using a W1 / O / W2 double emulsion embedding system.

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