Bacterial strain capable of promoting intestinal health of children and application of bacterial strain

Abstract

The invention relates to the technical field of microorganisms, and discloses a bacterial strain capable of promoting intestinal health of children and application of the bacterial strain, the bacterial strain is separated from dairy products and is named as a Bifidobacterium animalis subsp. Lactis BB1000 bacterial strain, and the bacterial strain is named as Bifidobacterium animalis subsp. Lactis BB1000 bacterial strain and is named as Bifidobacterium animalis subsp. Lactis BB1000 bacterial strain. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.30861, and the preservation date is June 5, 2024; the strain is resistant to acid and bile salt, has an antibacterial effect, has a function of enhancing intestinal epithelial barrier, and has an anti-inflammatory effect; the strain can be applied to probiotic lozenges.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a strain that promotes intestinal health in children and its applications. Background Technology

[0002] Bifidobacteria, as important probiotics in the human gut, play a vital role in maintaining intestinal health and regulating immune function. Among them, *Bifidobacterium animalis* subsp. lactis has become a research hotspot in the probiotic industry due to its excellent acid and bile salt resistance and significant health benefits.

[0003] Most probiotic strains currently on the market are imported, such as BB-12 and HN019, which have certain limitations in terms of functional characteristics, adaptability, and safety. Existing probiotic strains generally suffer from the following problems: First, they have insufficient tolerance to gastric acid and bile salts, resulting in low survival rates in the gastrointestinal environment and affecting their colonization and physiological functions in the intestine; second, they have a narrow antibacterial spectrum, with limited inhibitory effects against common pathogens such as Listeria, Salmonella, and Staphylococcus aureus; third, their mechanisms of action in improving allergy symptoms and reducing inflammation are unclear, and effective functional verification is lacking; fourth, their protective effect on the intestinal epithelial barrier function is not significant enough, making it difficult to effectively maintain intestinal integrity.

[0004] Children, as a special population, have an incompletely developed gut microbiota and an immature immune system, making them more susceptible to pathogenic infections and allergic reactions. While traditional antibiotic treatment can quickly control infections, it disrupts the balance of gut microbiota, leading to secondary infections and antibiotic resistance. Therefore, there is an urgent need to screen for indigenous probiotic strains with excellent tolerability, broad-spectrum antibacterial activity, and significant anti-allergic and anti-inflammatory effects to provide a safe and effective biological protection solution for children's gut health. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a bacterial strain that promotes children's gut health and its applications.

[0006] A strain that promotes gut health in children has been isolated from dairy products and named *Bifidobacterium animalis* subsp. *Lactis* BB1000. This strain is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30861, dated June 5, 2024.

[0007] The application of a strain that promotes children's gut health: The Bifidobacterium animalis subsp. lactis BB1000 strain was used to prepare oral remineralized probiotic lozenges.

[0008] Oral remineralizing probiotic lozenges include the following steps:

[0009] Step 1: Prepare BB1000 strain lyophilized powder with a viable count ≥102 9 CFU / g;

[0010] Step 2: Prepare a xanthan gum-locust bean gum saliva-responsive thickening compound at a mass ratio of 3:1. The viscosity is 5000-15000 mPa·s under low shear conditions and 500-1500 mPa·s under high shear conditions.

[0011] Step 3: Prepare a glycerin-propylene glycol dual moisturizing compound solution at a mass ratio of 2:1;

[0012] Step 4: CPP-ACP and HPMC E15 are encapsulated at a mass ratio of 4:1 to prepare CPP-ACP encapsulated particles;

[0013] Step 5: Prepare the rapid-disintegration outer layer formulation, containing 20% ​​sodium citrate, 20% sodium malate, and 40% xylitol;

[0014] Step 6: Prepare the adhesive core formulation, containing 15% BB1000 lyophilized powder, 25% CPP-ACP encapsulated particles, 4% responsive thickening compound, 40% L-HPC, and 8-12% dual moisturizing compound solution;

[0015] Step 7: Prepare tablets using a double-layer compression method. First, pre-compress the inner core formula into inner chips, then fill with outer particles and compress a second time to form a two-phase release structure.

[0016] Preferably, the xanthan gum has a molecular weight of 1.0-2.0 × 10⁻⁶. 6 The viscosity of a 1% aqueous solution of Da is 1200-1600 mPa·s; the molecular weight of the locust bean gum is 0.3-1.0 × 10⁻⁶. 6 The viscosity of a 1% aqueous solution of Da is 2500-3500 mPa·s.

[0017] Preferably, the glycerol has a purity ≥99.5% and a relative density of 1.249-1.251; the propylene glycol has a purity ≥99.5% and a relative density of 1.035-1.037.

[0018] Preferably, the CPP-ACP has a calcium content of 8-12%, a phosphorus content of 4-6%, and a particle size of ≤200μm; the HPMCE15 has a degree of substitution MS of 1.9-2.2 and a viscosity of 4000-5600mPa·s.

[0019] Preferably, the L-HPC has a degree of substitution of 4-7% and a viscosity of 150-400 mPa·s.

[0020] Preferably, the adhesive core formulation further contains 8% lactoperoxidase with an activity ≥150U / mg.

[0021] Preferably, in the double-layer pressing, the pre-compression pressure of the inner chip is 5-8 kN and the pre-compression time is 3-5 seconds; the pressure of the secondary pressing is 10-15 kN and the holding time is 5-8 seconds.

[0022] Preferably, the outer layer of the lozenge completely disintegrates within 5-8 minutes in artificial saliva, and the inner core continues to release for 2-4 hours through mucosal adhesion.

[0023] Preferably, the final viable count of the lozenge is ≥10. 8 CFU / tablet, total weight 400-450mg, hardness 40-80N.

[0024] The beneficial effects of this invention are as follows: the strain of this invention is acid and bile salt resistant, has antibacterial effect, enhances the intestinal epithelial barrier function, and has anti-inflammatory effect; this strain can be applied to probiotic tablets. Attached Figure Description

[0025] Figure 1 This is the oral retention time curve of the BB1000 strain of the present invention;

[0026] Figure 2 This is a radar diagram illustrating the multifunctional synergistic effect of the present invention;

[0027] Figure 3 This is a comparison of the caries prevention effect of the present invention in a 6-month human population.

[0028] Figure 4 This is an analysis of the synergistic effect of the present invention;

[0029] Figure 5 This refers to the time-series variation of the synergistic effect coefficient of the present invention. Detailed Implementation

[0030] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0031] Example 1

[0032] In this embodiment, a new strain of Bifidobacterium animalis subsp. Lactis was isolated and preserved from dairy products and named Bifidobacterium animalis subsp. Lactis BB1000 strain.

[0033] This strain is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30861 and deposit date of June 5, 2024.

[0034] Culture conditions and methods: 30-37℃, MRS medium, facultative anaerobic culture, culture time 18-24h.

[0035] The isolated and purified *Bifidobacterium animalis* subsp. *lactamase* BB1000 was activated and streaked onto MRS agar medium. It was incubated upside down at 30-37℃ for 48 hours. Colony morphology was observed, and the results are shown in the figure. The results showed that the strain grew well on MRS agar medium, with medium-sized, milky-white, convex, moist colonies with neat edges that were easy to pick. Gram staining was positive.

[0036] Bifidobacterium animalis subsp. lactis BB1000 strain was cultured at 30–37℃ for 18–24 hours to obtain a primary seed culture. This was then inoculated into MRS medium at an inoculum of 2%–8% (v / v) for a secondary activation culture of 20–24 hours to obtain a secondary seed culture. Subsequently, the secondary seed culture was inoculated into different fermenter media at the same inoculum (2%–8%, v / v) and fermented at 30–37℃, 50–100 rpm, aeration rate of 0.3–1 L / min, and pH maintained at 4.5–6.5 for 10–12 hours to obtain the final fermentation broth of Bifidobacterium animalis subsp. lactis. The fermentation broth was centrifuged at 5000–12000 rpm for 5–20 minutes, and the bacterial sludge was collected. This sludge was mixed with a freeze-drying protectant to prepare a mixture, which was then freeze-dried under vacuum to obtain BB1000 freeze-dried powder.

[0037] Freeze-drying protectant: (5-10% skim milk, 0.1%-0.5% monosodium glutamate).

[0038] Experimental Case:

[0039] 1. Tolerance to gastrointestinal fluids and bile salts.

[0040] Protocol: To test the survival rate of BB1000 strain after being stored in environments containing artificial gastric fluid, artificial intestinal fluid, and artificial bile for a certain period of time.

[0041] Artificial gastric juice containing 0.3% pepsin was adjusted to pH=3, and the survival rate of BB1000 after incubation at 37°C for 1 hour was counted.

[0042] Artificial intestinal fluid containing 0.3% pancreatic enzymes was adjusted to pH 6.8; the survival rate of BB1000 after incubation at 37°C for 1 hour was counted.

[0043] Artificial bile containing 0.3% bile salts was adjusted to pH 7, and the survival rate of BB1000 cells after incubation at 37°C for 1 hour was counted.

[0044] The survival rates of the samples after being stored at 37°C for one hour in simulated gastric juice, intestinal juice, and bile environments are shown in the table below:

[0045] Experimental environment Lactic acid bacteria survival rate (%) Artificial gastric juice (pH 3.0) 99.6 Artificial bile simulating solution (pH 6.8) 99.5 Artificial intestinal fluid (pH 7) 99.2

[0046] Results and Analysis: The data in the table show that strain BB1000 had a high survival rate after being stored at 37°C for one hour in artificial gastric juice, intestinal juice and bile, demonstrating the bacterium's good resistance to acid, bile salts and gastrointestinal environment.

[0047] 2. Ability to inhibit common pathogens

[0048] Experimental protocol: First, bacterial culture was performed. The culture medium was prepared according to the formula and autoclaved at 121°C for 20 minutes, then poured onto plates or dispensed into test tubes. Using an inoculation loop, bacterial suspension was streaked onto MRS agar plates and incubated at 37°C for 48 hours until single colonies formed. Then, a single colony was picked and inoculated into 5 mL of MRS liquid medium and fermented at 37°C for 72 hours. After incubation, the supernatant was collected by centrifugation, filtered, and stored at 4°C for later use. Simultaneously, a bacterial suspension was prepared and its concentration adjusted to 10. 6 -10 8 CFU / mL. Experimental groups included MRS, PBS, bacterial suspension, and supernatant. Inhibition zone determination was performed using the Oxford cup method: Listeria monocytogenes or Salmonella enteritidis were activated and spread onto LB agar plates, placed in Oxford cups, and BB1000 supernatant (MRS control) and bacterial suspension (PBS control) were added respectively. After diffusion overnight at 4°C, the plates were incubated at 37°C for 12 hours, and the inhibition zones were observed and their diameters measured.

[0049]

[0050] - indicates no antibacterial activity.

[0051] + indicates that the diameter of the inhibition zone is 9-14mm.

[0052] ++ indicates that the diameter of the inhibition zone is 15-20mm.

[0053] +++ indicates that the diameter of the inhibition zone is ≥20mm.

[0054] Results and Analysis: The data in the table show that strain BB1000 has a good inhibitory effect on the activity and growth of common pathogens Listeria and Salmonella, with inhibition zone diameters all above 23 mm.

[0055] 3. Zebrafish allergy model

[0056] Experimental protocol: Using AB strain zebrafish larvae (4dpf) as a model, compound 48 / 80 was used to induce mast cell degranulation, releasing trypsin-like enzymes to establish a pseudo-allergic reaction model. Zebrafish were randomly divided into normal control, model control, positive control (sodium cromoglycate), and groups with different concentrations of the test sample (BB1000 lyophilized powder, 31.2–2000 μg / mL). After 24 h of treatment, BAPNA substrate was added, and trypsin-like enzyme activity (OD value) was measured using a microplate reader as an indicator of the severity of the allergic reaction.

[0057] Results and Analysis: The level of trypsin in the model control group was significantly increased, verifying the successful establishment of the allergy model; the positive control (sodium cromoglycate) significantly reduced the level of trypsin.

[0058] 4. BB1000 enhances the intestinal epithelial barrier function and has anti-inflammatory effects.

[0059] Experimental protocol: Caco-2 cells (DMEM + 10% FBS, cultured at 37℃ and 5% CO2) were seeded in Transwell chambers and cultured for approximately 21 days to form a polarized monolayer. Four groups were established: ① Blank control (untreated); ② Model control (mixture of inflammatory factors: TNF-α 1 ng / mL + IFN-γ 10 ng / mL + IL-1β 1 ng / mL); ③ Positive control (model + reference treatment); ④ Test sample group (BB-12, with live bacteria, mildly heat-treated, and completely heat-inactivated bacteria, respectively).

[0060] Endpoint detection:

[0061] 1. Monitor changes in transepithelial resistance (TEER) in real time to evaluate barrier integrity;

[0062] 2. Collect the supernatant after 24 hours and use Luminex to detect inflammatory factors IL-6, IL-8, IL-18 and VEGF.

[0063] Results and Analysis:

[0064] 1. Live bacteria significantly improve TEER, but the effect weakens after mild heat treatment and has limited effect when completely heat-inactivated;

[0065] 2. Under the stimulation of a mixture of inflammatory factors, pretreatment with heat-inactivated BB1000 can significantly reduce the levels of IL-6, IL-18, and VEGF, suggesting that it can partially alleviate the inflammatory response.

[0066] Example 2

[0067] This embodiment proposes the application of a strain that promotes children's gut health, using Bifidobacterium animalis subsp. lactis BB1000 to prepare oral remineralized probiotic lozenges, including the following steps:

[0068] Step 1: Prepare BB1000 strain lyophilized powder with a viable count ≥102 9 CFU / g;

[0069] Step 2: Prepare a xanthan gum-locust bean gum saliva-responsive thickening compound at a mass ratio of 3:1. The viscosity is 10000 mPa·s under low shear conditions and 1000 mPa·s under high shear conditions.

[0070] Step 3: Prepare a glycerin-propylene glycol dual moisturizing compound solution at a mass ratio of 2:1;

[0071] Step 4: CPP-ACP and HPMC E15 are encapsulated at a mass ratio of 4:1 to prepare CPP-ACP encapsulated particles;

[0072] Step 5: Prepare the rapid-disintegration outer layer formulation, containing 20% ​​sodium citrate, 20% sodium malate, and 40% xylitol;

[0073] Step 6: Prepare the adhesive core formulation, containing 15% BB1000 lyophilized powder, 25% CPP-ACP encapsulated particles, 4% responsive thickening compound, 40% L-HPC, and 10% dual moisturizing compound solution;

[0074] Step 7: Prepare tablets using a double-layer compression method. First, pre-compress the inner core formula into inner chips, then fill with outer particles and compress a second time to form a two-phase release structure.

[0075] in:

[0076] The xanthan gum has a molecular weight of 1.5 × 10⁻⁶. 6 The viscosity of a 1% aqueous solution of Da is 1400 mPa·s; the molecular weight of the locust bean gum is 0.6 × 10⁻⁶. 6 The viscosity of a 1% aqueous solution of Da is 3000 mPa·s.

[0077] The glycerol has a purity of ≥99.5% and a relative density of 1.250; the propylene glycol has a purity of ≥99.5% and a relative density of 1.036.

[0078] The CPP-ACP has a calcium content of 10%, a phosphorus content of 5%, and a particle size of ≤200μm; the HPMC E15 has a degree of substitution (MS) of 2.0 and a viscosity of 4800mPa·s.

[0079] The L-HPC has a degree of substitution of 5% and a viscosity of 275 mPa·s.

[0080] The adhesive core formulation also contains 8% lactoperoxidase with an activity ≥150U / mg.

[0081] In the double-layer pressing process, the pre-compression pressure of the inner chip is 7kN and the pre-compression time is 4 seconds; the pressure of the secondary pressing is 12kN and the holding time is 6 seconds.

[0082] The outer layer of the lozenge completely disintegrates within 7 minutes in artificial saliva, and the inner core continues to release for 3 hours through mucosal adhesion.

[0083] The final viable count of the lozenge is ≥10. 8 CFU / tablet, total weight 425mg, hardness 60N.

[0084] Example 3

[0085] The difference between this embodiment and Embodiment 2 is that:

[0086] Step 2: Prepare a xanthan gum-locust bean gum saliva-responsive thickening compound at a mass ratio of 3:1. The viscosity is 5000 mPa·s under low shear conditions and 500 mPa·s under high shear conditions.

[0087] In step 6: 8% of the dual moisturizing compound solution;

[0088] The xanthan gum has a molecular weight of 1.0 × 10⁻⁶. 6 The viscosity of a 1% aqueous solution of Da is 1200 mPa·s; the molecular weight of the locust bean gum is 0.3 × 10⁻⁶. 6 The viscosity of a 1% aqueous solution of Da is 2500 mPa·s.

[0089] The glycerol has a purity of ≥99.5% and a relative density of 1.2491; the propylene glycol has a purity of ≥99.5% and a relative density of 1.035.

[0090] The CPP-ACP has a calcium content of 8%, a phosphorus content of 4%, and a particle size of ≤200μm; the HPMC E15 has a degree of substitution (MS) of 1.9 and a viscosity of 4000mPa·s.

[0091] The L-HPC has a degree of substitution of 4% and a viscosity of 150 mPa·s.

[0092] In the double-layer pressing process, the pre-compression pressure of the inner chip is 5kN and the pre-compression time is 3 seconds; the pressure of the secondary pressing is 10kN and the holding time is 5 seconds.

[0093] The outer layer of the lozenge completely disintegrates within 5 minutes in artificial saliva, and the inner core continues to release for 2 hours through mucosal adhesion.

[0094] The final viable count of the lozenge is ≥10. 8 CFU / tablet, total weight 400mg, hardness 40N.

[0095] Example 4

[0096] The difference between this embodiment and Embodiment 2 is that:

[0097] Step 2: Prepare a xanthan gum-locust bean gum saliva-responsive thickening compound at a mass ratio of 3:1. The viscosity is 15000 mPa·s under low shear conditions and 1500 mPa·s under high shear conditions.

[0098] In step 6: 12% of the dual-moisturizing compound solution;

[0099] The xanthan gum has a molecular weight of 2.0 × 10⁻⁶. 6 The viscosity of a 1% aqueous solution of Da is 1600 mPa·s; the molecular weight of the locust bean gum is 1.0 × 10⁻⁶. 6 The viscosity of a 1% aqueous solution of Da is 3500 mPa·s.

[0100] The glycerol has a purity of ≥99.5% and a relative density of 1.251; the propylene glycol has a purity of ≥99.5% and a relative density of 1.037.

[0101] The CPP-ACP has a calcium content of 12%, a phosphorus content of 6%, and a particle size of ≤200μm; the HPMC E15 has a degree of substitution (MS) of 2.2 and a viscosity of 5600mPa·s.

[0102] The L-HPC has a degree of substitution of 7% and a viscosity of 400 mPa·s.

[0103] In the double-layer pressing process, the pre-compression pressure of the inner chip is 8kN and the pre-compression time is 5 seconds; the pressure of the secondary pressing is 15kN and the holding time is 8 seconds.

[0104] The outer layer of the lozenge completely disintegrates within 8 minutes in artificial saliva, and the inner core continues to release for 4 hours through mucosal adhesion.

[0105] The final viable count of the lozenge is ≥10. 8 CFU / tablet, total weight 450mg, hardness 80N.

[0106] Example 5

[0107] This embodiment presents a method for preparing adaptive oral remineralizing probiotic lozenges containing strain BB1000, comprising the following specific steps:

[0108] Step 1: Preparation of BB1000 strain freeze-dried powder

[0109] The *Bifidobacterium animalis* subsp. *lactamase* BB1000 strain was prepared into freeze-dried powder using a standard process of strain culture, fermentation, centrifugation, and freeze-drying: after activation by culture on MRS medium, the culture was fermented in a fermenter, the bacterial sludge was collected by centrifugation, mixed with a freeze-drying protectant (5-10% skim milk, 0.1-0.5% monosodium glutamate), and then freeze-dried under vacuum to obtain a viable count ≥102. 9 BB1000 freeze-dried powder, CFU / g.

[0110] Storage conditions: The lyophilized powder is sealed in an aluminum foil bag and stored at -20°C with a relative humidity of ≤10%. It can be stored for 12 months. Before use, it needs to be equilibrated to room temperature (25°C) to avoid condensation affecting its activity.

[0111] Step 2: Formulation of responsive thickening compound

[0112] Weigh xanthan gum (food grade, molecular weight 1.0-2.0×10⁻¹⁰) precisely at a mass ratio of 3:1. 6 Da (1% aqueous solution, viscosity 1200-1600 mPa·s, 25℃) and locust bean gum (food grade, molecular weight 0.3-1.0×10⁻⁶). 6 Da (1% aqueous solution with viscosity 2500-3500 mPa·s, 25℃) is thoroughly mixed in a high-speed shear mixer (3000-5000 rpm) for 10-15 minutes to obtain a synergistically thickened compound powder.

[0113] Test method: The rheological properties of the compound powder in a 0.5% aqueous solution were determined at 25°C using a rotational viscometer (Brookfield DV-II+Pro).

[0114] This compound powder exhibits unique shear sensitivity: at low shear rates, the long-chain structure of xanthan gum and the galactomannan of locust bean gum undergo hydrogen bonding cross-linking to form a three-dimensional network structure with a viscosity of 5000-15000 mPa·s; at high shear rates, the parallel orientation of the molecular chains reduces the interaction, and the viscosity drops to 500-1500 mPa·s, exhibiting obvious pseudoplastic rheological characteristics.

[0115] Storage conditions: The compound powder should be stored in a sealed container at 25°C in a dry environment (relative humidity ≤60%), protected from light. Shelf life is 6 months. Before use, check the powder's flowability; if clumping occurs, it needs to be sieved again.

[0116] Step 3: Preparation of the dual moisturizing compound solution

[0117] Weigh glycerin (pharmaceutical grade, purity ≥99.5%, relative density 1.249-1.251, 25℃) and propylene glycol (pharmaceutical grade, purity ≥99.5%, relative density 1.035-1.037, 25℃) precisely at a mass ratio of 2:1. Mix them in a constant temperature magnetic stirrer (300-500 rpm) at 25-30℃ for 30 minutes until completely homogeneous to obtain a dual-moisturizing compound solution.

[0118] Test method: The refractive index of the compound solution was measured using a refractometer to be 1.458-1.462 (25℃). It appears as a colorless, transparent, viscous liquid. The three hydroxyl groups of glycerol provide strong hydrogen bonding, enabling prolonged adsorption of water molecules on the oral cavity surface. The dihydroxyl structure of propylene glycol maintains both hydrophilicity and permeability, allowing it to quickly penetrate the oral mucosal microstructure and carry active ingredients. The combination of the two creates a gradient moisturizing effect: propylene glycol provides initial rapid hydration (0-5 minutes), while glycerol maintains long-lasting hydration (30 minutes-2 hours).

[0119] This compound solution is used for the preparation of the core formulation in subsequent step 6.

[0120] Storage conditions: The compound solution should be stored in a sealed glass container at 15-25℃, protected from light, with a relative humidity of ≤65%. It can be stored for 3 months. Before use, check the transparency and viscosity of the liquid. If turbidity or separation occurs, it needs to be prepared again.

[0121] Step 4: Encapsulation treatment of CPP-ACP bioactive calcium complex

[0122] Casein phosphopeptide-amorphous calcium phosphate complex (CPP-ACP, calcium content 8-12%, phosphorus content 4-6%, white powder, particle size ≤200μm) was mixed with HPMC E15 (hydroxypropyl methylcellulose, degree of substitution MS 1.9-2.2, viscosity 4000-5600mPa·s) at a mass ratio of 4:1. Purified water (15-20% of the total dry weight) was added to form wet granules, which were then dried in a fluidized bed dryer at 60-80℃ until the moisture content was ≤5%. The granules were then passed through a 20-mesh sieve to obtain CPP-ACP-encapsulated particles.

[0123] Test method: Take 1g of the embedded particles and add them to 50ml of 0.1mol / L hydrochloric acid solution. Stir for 30 minutes. The amount of calcium ions released should be 60-80% of the raw material. The HPMC embedding layer can block the direct contact between CPP-ACP and organic acids, prevent premature complexation reaction, and control the release rate of calcium and phosphorus ions, thus prolonging their mineralization time on the tooth surface.

[0124] Storage conditions: Encapsulated particles are packaged in sealed aluminum foil bags and stored in a dry environment at 25°C (relative humidity ≤50%), protected from light. Shelf life is 18 months. Before use, test particle flowability and calcium ion release rate to ensure encapsulation integrity.

[0125] Step 5: Preparation of the rapid-disintegration outer layer formulation

[0126] Weigh out the following by total weight: sodium citrate (monohydrate, purity ≥99%, white crystalline powder) 20%, sodium malate (purity ≥99%, white crystalline powder) 20%, xylitol (pharmaceutical grade, purity ≥99%, crystalline powder) 40%, HPMCE15 15%, magnesium stearate (pharmaceutical grade, melting point 87-90℃) 3%, and silica (colloidal silica, specific surface area 200±25m²). 2 (g) 2%, using a wet granulation process: First, sodium citrate, sodium malate, and xylitol are mixed evenly in a V-type mixer for 5 minutes. Then, a 5% aqueous solution of HPMC (10-15% of the total dry weight) is added to form a soft mass. This mass is then granulated through a 14-mesh sieve and dried in a fluidized bed dryer at 60℃ until the moisture content is ≤3%. After granulation, the granules are mixed evenly with magnesium stearate and silica for 3 minutes to obtain rapidly disintegrating outer layer granules that completely disintegrate within 5-8 minutes in artificial saliva (pH 6.8, 37℃). Test standards: good granule flowability, angle of repose ≤45°, moisture content ≤3%, disintegration time 5-8 minutes.

[0127] Storage conditions: The outer granules are sealed in double-layered plastic bags and stored in a dry environment at 25°C (relative humidity ≤55%). They can be stored for 12 months. Before use, test the granule flowability and disintegration time to ensure compliance with standards.

[0128] Step 6: Preparation of the adhesive core formulation

[0129] Weigh out the following ingredients by total weight: 15% BB1000 lyophilized powder, 25% CPP-ACP encapsulated particles, 8% lactoperoxidase (activity ≥150U / mg, white powder), 4% responsive thickening compound (xanthan gum-locust bean gum compound powder), 40% L-HPC (low degree of substitution hydroxypropyl cellulose, degree of substitution 4-7%, viscosity 150-400mPa·s), 6% microcrystalline cellulose (pH 102, average particle size 100μm), and 2% magnesium stearate (pharmaceutical grade, melting point 87-90℃). Separately, take 8-12% of the total dry weight of the dual moisturizing compound solution (prepared in step 3) for granulation.

[0130] Key operational points: First, mix BB1000 lyophilized powder and L-HPC in a low-temperature, low-humidity environment (≤25℃, relative humidity ≤45%) using a V-type mixer for 10 minutes to prevent the strain from becoming damp and inactive. Then, add CPP-ACP encapsulated particles, lactoperoxidase, and responsive thickening compound, and dry mix for 5 minutes under the same conditions. Next, slowly add the dual-humidity compound solution at a rate of 2-3 ml / min using a peristaltic pump and mix thoroughly with a trough mixer to form a soft mass (formable when squeezed, easily crumbles under light pressure). Granulate through a 16-mesh sieve and dry in a fluidized bed dryer at 40℃ until the moisture content is 3-5% (determined by weight loss method). Finally, add microcrystalline cellulose and magnesium stearate, and mix with a V-type mixer for 5 minutes to obtain the adhesive core formulation. Test standards: Good particle flowability (angle of repose ≤40°), moisture content 3-5%, viable count ≥5×10⁻⁶. 8 CFU / g.

[0131] Storage conditions: The core granules should be used immediately or refrigerated. For short-term storage (≤7 days), store in a sealed aluminum foil bag at 2-8℃ with a relative humidity ≤40%. Viable bacteria counts should be checked daily during storage; if the count is below the standard, the granules must be prepared again.

[0132] Step 7: Preparation of biphasic tablets

[0133] The tablets are prepared using a double-layer compression technology: a rotary tablet press (ZP-19 type or equivalent) equipped with a 12mm diameter circular die is used. The first step involves pre-compressing the core formulation (200-250mg) into core chips on the tablet press at a pressure of 5-8kN for 3-5 seconds, ensuring a core density of 0.9-1.1g / cm³. 3 However, this does not destroy the activity of the strain; the second step is to fill the outer layer of rapidly disintegrating particles (150-200mg) around the inner chip and perform secondary tableting, with the pressure controlled at 10-15kN and the holding time at 5-8 seconds, to ensure that the outer layer and the inner core are firmly bonded (peel strength ≥15N) but the outer layer maintains rapid disintegration.

[0134] Through pressure gradient control and layered filler treatment, a two-phase release characteristic is achieved, characterized by rapid release from the outer layer and slow adhesion release from the inner core. Testing standards: total tablet weight 400-450mg, thickness 4.0-4.5mm, hardness 40-80N, friability ≤1%.

[0135] Step 8: Quality Control and Packaging

[0136] The prepared lozenges were subjected to quality testing:

[0137] (1) Visual inspection: The surface is smooth, without cracks, and the color is uniform;

[0138] (2) Weight variation: The coefficient of variation of single-piece weight should be ≤ ±5%;

[0139] (3) Hardness test: The hardness should be measured using a hardness tester and should be controlled within the range of 40-80N.

[0140] (4) Disintegration time test: The outer layer completely disintegrates in artificial saliva at 37°C in 5-8 minutes, and the inner core is confirmed to release continuously for 2-4 hours by adhesion test.

[0141] (5) Microbiological testing: The number of viable bacteria in BB1000 should be ≥10. 8 CFU / tablet, free from pathogenic bacteria contamination, strain identified and confirmed. After passing inspection, it is packaged in PVC / AL aluminum-plastic packaging, 10 tablets per blister pack, and stored in a dry, cool place (≤25℃, relative humidity ≤60%) away from light.

[0142] The probiotic tablets in this embodiment have the following significant technical effects:

[0143] The multifunctional synergistic caries prevention effect is outstanding: the BB1000 strain inhibits Streptococcus mutans with an inhibition ring diameter of 18.5±2.1mm and an inhibition rate of 92-96%; CPP-ACP increases the microhardness of tooth enamel by 33.3%; organic acid salts promote an increase in saliva secretion by 65-85%. Experiments show that the caries incidence rate is only 4.2%, a 78% reduction compared to traditional methods, and the caries prevention effect is 3-5 times better.

[0144] Breakthrough in individual adaptability: The responsive thickening compound achieves 5-10 times viscosity adaptive adjustment, with an overall adaptability coverage of 87% for children with different salivation abilities, and a 240% increase in oral moisture in children with severe salivation deficiency.

[0145] Long duration of action: The oral retention time is extended to 180±60 minutes, which is 9 times longer than that of traditional lozenges; the adhesion strength of the L-HPC core is increased by 4.9 times, achieving continuous release for 2-4 hours.

[0146] Optimized component compatibility: The dual-phase separation technology effectively solves the interference between functional components, increases the survival rate of live bacteria by 4.3 times, maintains the retention rate of each component by ≥92%, and reduces the premature release rate of calcium ions to only 6.8%.

[0147] Saliva dependence is significantly reduced: the saliva dependence index is reduced by 61%, and the caries prevention efficacy still reaches 52.3% in extremely low saliva environments, which is 6.2 times higher than traditional preparations.

[0148] High process controllability: The coefficient of variation of key process parameters is controlled within 5%, the product qualification rate is ≥99.2%, the batch-to-batch consistency is good, and it has good conditions for industrial production.

[0149] Experimental verification

[0150] Experiment 1: Shear Sensitivity Test of Responsive Thickening Complex

[0151] 1. Experimental Objective

[0152] The viscosity-modulating ability of the xanthan gum-locust bean gum 3:1 compound under different shear rates was verified, demonstrating its adaptability to different salivary environments.

[0153] 2. Preparation of experimental samples

[0154] Experimental group: Xanthan gum-locust bean gum compound (mass ratio 3:1);

[0155] Control group 1: pure xanthan gum;

[0156] Control group 2: Pure locust bean gum;

[0157] Control group 3: All samples of commercially available thickeners were prepared into 0.5% aqueous solutions and equilibrated at 25°C for 24 hours.

[0158] 3. Experimental conditions

[0159] Test equipment: Brookfield DV-II+Pro rotational viscometer;

[0160] Test temperature: 25℃ (consistent with this embodiment);

[0161] Shear rate range: 0.1-1000s -1 - Number of parallel samples: n = 6.

[0162] 4. Experimental Procedure

[0163] (1) Each sample solution is placed into a viscometer test cup with a sample volume of 1.2 mL;

[0164] (2) Set the temperature to 25℃ and allow it to equilibrate for 5 minutes;

[0165] (3) According to shear rates of 0.1, 1, 10, 100, and 1000 s -1 Test in the following order;

[0166] (4) Record the viscosity value after stabilizing for 60 seconds at each shear rate;

[0167] (5) Calculate the shear thinning index n (n = d(logη) / d(logγ));

[0168] (6) Repeat the test 6 times and calculate the mean and standard deviation.

[0169] 5. Experimental Results

[0170] Viscosity test results at different shear rates:

[0171]

[0172] Key parameter comparison:

[0173]

[0174] 6. Analysis and Summary

[0175] The experimental group (xanthan gum-locust bean gum 3:1 compound) showed excellent shear sensitivity, with a viscosity adjustment factor of 33.8 times (15,200→450 mPa·s), which was much higher than the 2.5-2.9 times of the control groups.

[0176] The shear thinning index n = 0.18 indicates that it has strong pseudoplastic fluid characteristics and can automatically adjust its viscosity according to the flow state of saliva in the oral cavity: it forms a high-viscosity network structure to prolong the residence time under low shear conditions, and reduces the viscosity to ensure normal release under high shear conditions, effectively solving the problem of individual differences in adaptation for children with different saliva secretion abilities.

[0177] Experiment 2: Long-term release test of L-HPC mucosal adhesion core

[0178] 1. Experimental Objective

[0179] To verify the long-term release characteristics of the L-HPC mucosal adhesion core technology, the retention time and activity maintenance ability of the BB1000 strain in the oral environment were determined.

[0180] 2. Preparation of experimental samples

[0181] Experimental group: biphasic tablets containing L-HPC adhesive core;

[0182] Control group 1: Traditional HPMC lozenges;

[0183] Control group 2: Regular immediate-release lozenges;

[0184] Control group 3: All samples of commercially available probiotic tablets contained the same concentration of fluorescently labeled BB1000 strain (≥10). 8 CFU / tablet, meeting the quality standards of step 8 in this embodiment).

[0185] 3. Experimental conditions

[0186] Test environment: Artificial oral cavity simulator (37℃, pH 6.8, meeting artificial saliva conditions);

[0187] Saliva flow rate: 0.5 mL / min (simulating normal saliva secretion);

[0188] Testing equipment: Fluorescence microscope (Olympus BX53) + viable bacteria counter;

[0189] Volunteers: 20 healthy adults (aged 25-35).

[0190] 4. Experimental Procedure

[0191] (1) In vitro release test: The sample was placed in the artificial oral cavity device and the number of BB1000 viable bacteria was measured at regular intervals;

[0192] (2) Adhesion strength test: The adhesion force between the sample and the artificial membrane was measured using a texture analyzer;

[0193] (3) In vivo retention test: After volunteers ingested the sample, oral swabs were collected at regular intervals for bacterial strain detection;

[0194] (4) Fluorescence tracking: The distribution and retention of labeled strains in the oral cavity were observed using a fluorescence microscope;

[0195] (5) Activity evaluation: The plate count method was used to detect the changes in the number of viable bacteria at different time points.

[0196] 5. Experimental Results

[0197] Time point viable bacteria count in the experimental group Control group 1 viable bacteria count Control group 2 viable bacteria count Control group 3 viable bacteria count 0.5h <![CDATA[8.2±0.5×10 7 ]]> <![CDATA[6.1±0.8×10 7 ]]> <![CDATA[3.2±0.6×10 6 ]]> <![CDATA[4.5±0.7×10 6 <!-- 10 -->]]> 1h <![CDATA[7.8±0.6×10 7 ]]> <![CDATA[4.2±0.5×10 6 ]]> <![CDATA[8.5±1.2×10 4 ]]> <![CDATA[1.8±0.3×10 5 ]]> 2h <![CDATA[6.5±0.4×10 7 ]]> <![CDATA[1.5±0.3×10 5 ]]> <![CDATA[<10 3 ]]> <![CDATA[2.1±0.4×10 4 ]]> 4h <![CDATA[4.2±0.7×10 6 ]]> <![CDATA[3.2±0.8×10 4 ]]> <![CDATA[<10 3 ]]> <![CDATA[<10 3 ]]> 6h <![CDATA[1.8±0.5×10 5 ]]> <![CDATA[<10 3 ]]> <![CDATA[<10 3 ]]> <![CDATA[<10 3 ]]>

[0198] Sample group Peel strength (N) Effective duration of action (h) 4-hour viability rate (%) Comparison with implementation methods experimental group 18.2±2.8 4.2±0.6 5.1±0.8 ≥15N (Compliant) Control group 1 3.2±0.8 1.8±0.3 0.08±0.02 <15N Control group 2 0.5±0.2 0.3±0.1 <0.001 <15N Control group 3 1.1±0.4 0.8±0.2 0.02±0.01 <15N

[0199] Figure 1 The oral retention time curve of strain BB1000 is shown.

[0200] 6. Analysis and Summary

[0201] The experimental group used L-HPC mucosal adhesion core technology, with a peel strength of 18.2±2.8N, exceeding the ≥15N standard set in the implementation method, which is 5.7 times that of traditional HPMC. The effective action time was extended to 4.2±0.6 hours, and the viable bacteria retention rate after 4 hours was 5.1%, which was significantly better than all control groups.

[0202] Fluorescence tracking showed that the BB1000 strain can form a stable adhesion layer on the oral mucosa and achieve long-lasting sustained release through the strong mucosal affinity of L-HPC. This overcomes the technical difficulties of traditional lozenges, such as short duration of action and susceptibility to swallowing, and provides a solution for the long-lasting use of oral probiotic preparations.

[0203] Experiment 3: Evaluation of the Synergistic Anti-Caries Effect of Multifunctional Devices

[0204] 1. Experimental Objective

[0205] To verify the synergistic anti-caries effect of the BB1000 strain's antibacterial activity, CPP-ACP remineralization, and saliva stimulation by organic acid salts, and to evaluate the technical advantages of multifunctional integration compared to single-function.

[0206] 2. Preparation of experimental samples

[0207] Experimental group: Tri-functional lozenges containing BB1000 + CPP-ACP + organic acid salts;

[0208] Control group 1: Single-function lozenges containing only BB1000;

[0209] Control group 2: Single-function lozenges containing only CPP-ACP;

[0210] Control group 3: Single-function lozenges containing only organic acid salts;

[0211] Control group 4: Fluoride toothpaste (commercially available positive control) - Control group 5: Placebo (negative control).

[0212] 3. Experimental conditions

[0213] Experimental period: 6-month follow-up

[0214] Subjects: 180 children aged 6-12 years were randomly divided into 6 groups (30 cases in each group);

[0215] Detection methods: agar diffusion method (antibacterial), microhardness tester (remineralization), saliva flow meter (secretory stimulation);

[0216] Evaluation indicators: caries incidence, changes in enamel hardness, and changes in saliva secretion.

[0217] 4. Experimental Procedure

[0218] (1) Antibacterial effect test: The inhibitory ability of each sample against Streptococcus mutans was detected by the agar diffusion method;

[0219] (2) Remineralization effect test: The mineralization effect of CPP-ACP was evaluated using an artificial demineralization-remineralization cycle model;

[0220] (3) Saliva stimulation test: to determine the promoting effect of organic acid salts on saliva secretion;

[0221] (4) Human caries prevention trial: During the 6-month use period, the occurrence of caries in the subjects was checked regularly;

[0222] (5) Synergistic effect analysis: Statistical analysis is used to evaluate the synergistic effect of multifunctional synergy compared with single function.

[0223] 5. Experimental Results

[0224] In vitro functional test results

[0225]

[0226] Results of a 6-month human caries prevention trial

[0227]

[0228] Figure 2 A radar chart showing the multi-functional synergistic effect was displayed;

[0229] Figure 3 It shows a comparison of the caries prevention effects in a 6-month population.

[0230] Figure 4 The analysis of synergistic effects was presented;

[0231] Figure 5 It shows the time-series changes in the synergy coefficient.

[0232] 6. Analysis and Summary

[0233] The experimental group achieved multifunctional synergy of BB1000 antibacterial activity, CPP-ACP remineralization, and saliva stimulation by organic acid salts, with a comprehensive efficacy index of 8.9±1.2, which was significantly higher than the 2.1-3.8 of each single-function control group.

[0234] A 6-month human trial showed that the incidence of tooth decay in the experimental group was only 4.2%, with a relative risk reduction of 78.1%, which is basically consistent with the 78% reduction rate in the technical effect section. The caries prevention effect was 3.2 times better than the best single-function product and 67% better than commercially available fluoride toothpaste (12.8%).

[0235] The synergistic effect coefficient gradually increased from 1.2 to 3.9 over 6 months, indicating a significant time-cumulative synergistic effect among the multifunctional components. This result confirms the significant advantages of multifunctional integrated technology compared to traditional single-function solutions, providing an innovative multidimensional protective strategy for childhood dental caries prevention.

[0236] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A bacterial strain that promotes gut health in children, characterized in that, This strain was isolated from dairy products and named Bifidobacterium animalis subsp. Lactis BB1000. It is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30861 and deposit date of June 5, 2024.

2. The application of a bacterial strain that promotes intestinal health in children, characterized in that, The Bifidobacterium animalis subsp. lactis BB1000 strain was used to prepare oral remineralization probiotic lozenges.

3. The application according to claim 1, characterized in that, Oral remineralizing probiotic lozenges include the following steps: Step 1: Prepare BB1000 strain lyophilized powder with a viable count ≥102 9 CFU / g; Step 2: Prepare a xanthan gum-locust bean gum saliva-responsive thickening compound at a mass ratio of 3:

1. The viscosity is 5000-15000 mPa·s under low shear conditions and 500-1500 mPa·s under high shear conditions. Step 3: Prepare a glycerin-propylene glycol dual moisturizing compound solution at a mass ratio of 2:1; Step 4: CPP-ACP and HPMC E15 are encapsulated at a mass ratio of 4:1 to prepare CPP-ACP encapsulated particles; Step 5: Prepare the rapid-disintegration outer layer formulation, containing 20% ​​sodium citrate, 20% sodium malate, and 40% xylitol; Step 6: Prepare the adhesive core formulation, containing 15% BB1000 lyophilized powder, 25% CPP-ACP encapsulated particles, 4% responsive thickening compound, 40% L-HPC, and 8-12% dual moisturizing compound solution; Step 7: Prepare tablets using a double-layer compression method. First, pre-compress the inner core formula into inner chips, then fill with outer particles and compress a second time to form a two-phase release structure.

4. The application according to claim 1, characterized in that, The xanthan gum has a molecular weight of 1.0-2.0 × 10⁻⁶. 6 The viscosity of a 1% aqueous solution of Da is 1200-1600 mPa·s; the molecular weight of the locust bean gum is 0.3-1.0 × 10⁻⁶. 6 The viscosity of a 1% aqueous solution of Da is 2500-3500 mPa·s.

5. The application according to claim 1, characterized in that, The glycerol has a purity of ≥99.5% and a relative density of 1.249-1.251; the propylene glycol has a purity of ≥99.5% and a relative density of 1.035-1.

037.

6. The application according to claim 1, characterized in that, The CPP-ACP has a calcium content of 8-12%, a phosphorus content of 4-6%, and a particle size of ≤200μm; the HPMC E15 has a degree of substitution (MS) of 1.9-2.2 and a viscosity of 4000-5600mPa·s.

7. The application according to claim 1, characterized in that, The L-HPC has a degree of substitution of 4-7% and a viscosity of 150-400 mPa·s.

8. The application according to claim 1, characterized in that, The adhesive core formulation also contains 8% lactoperoxidase with an activity ≥150U / mg.

9. The application according to claim 1, characterized in that, In the double-layer pressing process, the pre-compression pressure of the inner chip is 5-8 kN, and the pre-compression time is 3-5 seconds; the pressure of the secondary pressing is 10-15 kN, and the holding time is 5-8 seconds.

10. The application according to claim 1, characterized in that, The outer layer of the lozenge completely disintegrates within 5-8 minutes in artificial saliva, while the inner core continues to release for 2-4 hours through mucosal adhesion.

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