Lactobacillus reuteri RC-19 helpful for moisturizing human body

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses lactobacillus reuteri RC-19 beneficial to moisturizing of a human body. The strain is preserved in the China Center for Type Culture Collection (CCTCC) on June 6, 2025, and the preservation number of the strain is CCTCC No. M20251292. The bacterial strain can promote keratinocytes to secrete hyaluronic acid (HA) and enhance a skin barrier function (TEER is increased, and Occludin / Claudin-1 is up-regulated) mechanism, so that the moisture degree of the skin is remarkably improved. The human body trial verifies that the skin moisture content can be increased by 57.1% after the emulsion (the formula comprises RC-19 fermentation filtrate, sodium hyaluronate and the like) containing the emulsion is continuously used for 4 weeks, and the safety is high. The invention provides an endogenous and long-acting probiotic solution for improving the dry skin.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a type of Lactobacillus reuteri RC-19 that helps with human hydration. Background Technology

[0002] The body's hydration primarily depends on the skin and mucous membranes' ability to retain moisture, which is regulated by factors such as the stratum corneum barrier function, the content of natural moisturizing factors (such as hyaluronic acid, ceramides, and free amino acids), and the expression level of aquaporins (AQP). With age, environmental stimuli (such as dryness and ultraviolet radiation), or lifestyle changes (such as excessive cleansing), the human body often experiences problems such as dry skin, peeling, and mucous membrane sensitivity. Traditional methods of improvement often rely on exogenous hydration (such as applying moisturizing cream) or supplementing with chemically synthesized moisturizers (such as glycerin and sodium hyaluronate), but these methods have limitations such as short-term effects and the potential to cause allergies or dependence.

[0003] In recent years, the potential role of probiotics in skin and mucous membrane health has gradually attracted attention. Some studies have shown that certain lactic acid bacteria can exert moisturizing effects by regulating the skin microecology and promoting barrier repair. However, existing strains mostly target anti-inflammation or anti-oxidation, and the development of functional strains that "actively enhance endogenous hydration" remains a gap. Lactobacillus reuteri is a group of symbiotic probiotics widely found in the human gut and urogenital tract. It is known to have functions such as regulating immunity and inhibiting pathogens, but the specific functions of its subspecies or strains in maintaining skin / mucous membrane hydration have not yet been systematically explored.

[0004] Therefore, developing a novel strain of Lactobacillus reuteri that can improve human hydration is of significant scientific importance and practical value. Summary of the Invention

[0005] This invention first provides a strain of *Lactobacillus reuteri* (… Limosilacto bacillus reuteri RC-19, with accession number China Center for Type Culture Collection (CCTCC) NO: M20251292 (deposited on June 6, 2025), is classified as *Lactobacillus reuteri* RC-19. Limosilacto bacillus reuteri RC-19).

[0006] The present invention also provides the application of the above-mentioned *Lactobacillus reuteri* RC-19 in the preparation of topical formulations that improve the hydration of human skin.

[0007] In some embodiments, the topical formulation works through at least one of the following mechanisms: (1) Promotes the secretion of hyaluronic acid (HA) by keratinocytes in the skin; (2) Enhance the skin barrier function.

[0008] In some embodiments, the topical preparation is a lotion, cream, gel, or mask.

[0009] In some embodiments, the emulsion comprises the following ingredients by weight percentage: 0.1-5% fermentation filtrate of *Lactobacillus reuteri* RC-19; Sodium hyaluronate 0.01-0.5%; Squalane 0.5-3%; Glycerin 2-8%; Emulsifier 0.5-2%; Preservative 0.1-0.5%; The remainder consists of deionized water and pH adjuster.

[0010] The present invention also provides a composition comprising the above-mentioned *Lactobacillus reuteri* (…). Limosilacto bacillus reuteri RC-19 and pharmaceutically acceptable carriers.

[0011] The present invention also provides the use of the above-described composition in the preparation of topical formulations that improve the hydration of human skin.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Innovation: It is the first time that Lactobacillus reuteri RC-19 has been found to have the function of specifically improving human body hydration, which is different from the anti-inflammatory or intestinal regulation effects of traditional probiotics; (2) High efficiency: It enhances hydration through a multi-target mechanism (promoting HA / ceramide synthesis and strengthening the barrier), and its effect is better than that of a single moisturizing ingredient (such as exogenous hyaluronic acid). (3) Wide range of applications: It can be developed into cosmetics and other types of products to cover the needs of different groups of people (such as dry skin, sensitive skin, and dry skin in middle-aged and elderly people).

[0013] Preservation Instructions Lactobacillus reuteri RC-19 ( Limosilacto bacillus reuteri RC-19 This bacterium, *Lactobacillus reuteri* RC-19, was isolated from healthy breast milk samples and deposited at the China Center for Type Culture Collection (CCTCC) on June 6, 2025. Its classification name is *Lactobacillus reuteri* RC-19. Limosilacto bacillus reuteri RC-19 The accession number is CCTCCNO:M20251292, and the address is Wuhan University, Wuhan, Hubei, China. Attached Figure Description

[0014] Figure 1 This is a graph showing the results of HA secretion. * indicates a significant difference compared to the blank control group. P <0.05, *** indicates a significant difference compared to the blank control group. P<0.001.

[0015] Figure 2 This graph shows the relative expression levels of HAS2 mRNA. * indicates a significant difference compared to the blank control group. P <0.05, *** indicates a significant difference compared to the blank control group. P <0.001.

[0016] Figure 3 This is a graph showing the relative fluorescence intensity of Occludin. * indicates a significant difference compared to the model control group. P <0.05. ** indicates a significant difference compared to the model control group. P <0.01.

[0017] Figure 4 This is a graph showing the relative fluorescence intensity of Claudin-1. * indicates a significant difference compared to the model control group. P <0.05. ** indicates a significant difference compared to the model control group. P <0.01. Detailed Implementation

[0018] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0019] Example 1. Isolation and identification of Lactobacillus reuteri RC-19 The strain was isolated from healthy breast milk samples and purified through the following steps: (1) Sample processing: Take 100 μL of healthy breast milk sample and serially dilute to 10 -6 (1) Spread on MRS agar plates (containing 0.5% calcium carbonate) and anaerobic culture at 37℃ for 48h; (2) Initial screening: single colonies with obvious calcium dissolution zones were picked and purified by streaking until the morphology was consistent; (3) Physiological and biochemical identification: Gram staining (positive cocci), catalase test (negative), and sugar fermentation test (fermentation of glucose to produce acid and gas, but not fermentation of lactose) were used to preliminarily determine that it belonged to the genus Lactobacillus; (4) Molecular identification: genomic DNA was extracted, 16S rRNA gene was amplified (primer 27F / 1492R), and after sequencing, it was compared with the NCBI database to confirm that it belonged to Lactobacillus reuteri; further whole genome sequencing (Illumina NovaSeq platform) was used to compare and found that it carried a unique mucus binding protein encoding gene (mub) and hyaluronic acid synthase activation gene cluster (hasA-hasB), which was named RC-19.

[0020] Preservation information: Deposited at the China Center for Type Culture Collection (CCTCC) on June 6, 2025, and classified as: *Lactobacillus reuteri* RC-19 ( Limosilacto bacillus reuteriRC-19), accession number: CCTCC NO:M20251292, address: Wuhan University, Wuhan, Hubei, China.

[0021] Example 2: In vitro validation of Lactobacillus reuteri RC-19 promoting hyaluronic acid (HA) synthesis in human immortalized keratinocytes (HaCaT). Experimental materials Cell line: Human immortalized keratinocytes (HaCaT, purchased from China Center for Type Culture Collection). Culture medium: DMEM high glucose medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, Gibco); Test strain: *Lactobacillus reuteri* RC-19 (preservation number: CCTCC NO: M20251292, passage number ≤10, resuspended in sterile physiological saline to OD). 600 =0.5, approximately 1×10 8 (CFU / mL) Main reagents: Hyaluronic acid (HA) ELISA kit (Corgenix, catalog number: HYAL-100); qPCR kit (Takara, RR820A); RNA extraction kit (Omega, R6867-01). Instruments: CO2 incubator (Thermo, 3111), microplate reader (Biotek, Synergy H1), real-time quantitative PCR instrument (ABI, 7500).

[0022] Experimental methods Cell culture and grouping: HaCaT cells were seeded in 6-well plates (2 × 10⁻⁶ cells per well). 5 Cells / well), culture medium volume 2 mL / well, incubated at 37℃, 5% CO2 for 24 h until 80% confluence. The original culture medium was discarded, and the cells were washed twice with PBS and randomly divided into 3 groups (n=6):

[0023] Blank control group (Control): Add 2 mL of serum-free DMEM medium; RC-19 supernatant group: Add 2 mL of RC-19 bacterial culture (OD) 600 =0.5) and serum-free DMEM were co-incubated at a 1:1 ratio for 24 hours. The supernatant was then filtered through a 0.22μm filter membrane for sterilization. Heat-killed RC-19 group: RC-19 bacterial solution was boiled at 100℃ for 10 min to inactivate the bacteria, and then incubated with serum-free DMEM at a 1:1 ratio for 24 h. The supernatant was then used to exclude the effects of bacterial metabolites.

[0024] HA secretion assay: After culturing for another 48 hours, cell supernatants were collected from each group and centrifuged at 1000×g for 5 min to remove cell debris. The HA concentration (ng / mL) in the supernatant was measured according to the HA ELISA kit instructions, and the mean ± standard deviation (Mean ± SD) for each group was calculated.

[0025] HAS2 gene expression detection: Cells from each group were collected, total RNA was extracted using the TRIzol method, and reverse transcribed into cDNA (1 μg RNA / 20 μL reaction system). GAPDH was used as an internal control, and the primer sequences are as follows:

[0026] HAS2 Upstream: 5'-TGAGAGGTTTCTATGTGTCCT-3'; Downstream: 5'-CGTACAGTCCAAATGAGAAGT-3'; GAPDH Upstream: 5'-GTCTTCACCACCATGGAGAA-3'; Downstream: 5'-AGGAGGCATTGCTGATGAT-3'.

[0027] qPCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, 40 cycles. Using 2... ⁻ΔΔCt The relative expression level was calculated using this method. Experimental results are as follows: Figures 1-2 As shown.

[0028] HA secretion levels, such as Figure 1 As shown: The HA concentration in the RC-19 supernatant group was significantly higher than that in the blank control group ( p <0.001). This indicates that the metabolites of RC-19 are key to promoting HA synthesis. HAS2 gene expression is as follows: Figure 2 As shown, the relative expression level of HAS2 mRNA in the RC-19 supernatant group was significantly higher than that in the blank control group (p<0.001). This verifies that the *Lactobacillus reuteri* RC-19 of the present invention can promote HA synthesis by activating HAS2 transcription, demonstrating its certain moisturizing ability.

[0029] Example 3: Validation of Lactobacillus reuteri RC-19 enhancing the barrier function of an in vitro reconstructed human epidermal (RHE) model. Experimental materials In vitro reconstructed human epidermal model (RHE, MatTek, EPI-200). Stimulant: Tumor necrosis factor-α (TNF-α, 10 ng / mL, R&D Systems). Test strain: Lactobacillus reuteri RC-19 (same as in Example 2); Detection indicators: transepithelial electrical resistance (TEER, Millicell-ERS system); tight junction proteins Occludin and Claudin-1 (immunofluorescence staining).

[0030] Experimental methods Model construction and grouping: RHE models were cultured until the barrier matured (TEER stabilized at 400-500 Ω·cm²), and then randomly divided into 4 groups (n=4): Normal group: No processing; Model group: serum-free medium containing 10 ng / mL TNF-α was added and cultured for 24 h to induce barrier damage, and cultured under the same conditions as the normal group; RC-19 pre-treatment group: After the barrier matures, RC-19 fermentation filtrate (1×10⁻⁶) is added first. 8 Co-cultured with CFU / mL for 24 h, then TNF-α was added to induce damage; Positive control: After barrier damage, culture medium containing 10 μM dexamethasone was added and cultured for 24 h.

[0031] TEER value detection: After each group was treated, the TEER value (Ω·cm²) was measured using the Millicell-ERS system, and the recovery rate was calculated (Recovery rate = (TEER of model group - TEER of treatment group) / (TEER of normal group - TEER of model group) × 100%).

[0032] Tight junction protein expression detection: Each model was fixed in 4% paraformaldehyde, permeabilized (0.1% Triton X-100), and then blocked (5% BSA). Anti-Occludin (1:200, Abcam) and anti-Claudin-1 (1:200, Cell Signaling) antibodies were added, and the models were incubated overnight at 4°C. After labeling with fluorescent secondary antibody (1:500, Alexa Fluor 488), protein expression was observed under a laser confocal microscope (mean fluorescence intensity, MFI).

[0033] The relative fluorescence intensity is calculated using the following formula: Relative fluorescence intensity (%) = (average fluorescence intensity of sample group / average fluorescence intensity of model group) x 100%.

[0034] Experimental results TEER value and recovery rate: TEER in the normal group: (420±30) Ω·cm²; Model group TEER: (150±20) Ω·cm² (severe barrier damage); The TEER of the RC-19 pretreated group was (380±35) Ω·cm², and the recovery rate was (380-150) / (420-150)×100%=85.2%±5.3%, which was significantly higher than that of the model group (p<0.01 vs model group). Positive control group TEER: (320±25) Ω·cm², recovery rate 61.9%±4.1% (RC-19 showed better results than the positive control).

[0035] Results of tight junction protein expression are as follows Figures 3-4 As shown, the relative fluorescence intensity of Occludin and Claudin-1 in the RC-19 pretreated group was significantly higher than that in the model group. p <0.01) and positive control group ( p <0.01), close to the level of the normal group.

[0036] Example 4: Preparation and quality evaluation of a moisturizing emulsion containing *Lactobacillus reuteri* RC-19 The formulation design (per 100g emulsion) is shown in Table 1: Table 1. Formulation Design

[0037] Preparation process: Preparation of fermentation filtrate: RC-19 strain was inoculated into MRS liquid medium (containing 0.5% calcium carbonate), anaerobic cultured at 37℃ for 48h, centrifuged at 4000×g for 10min to collect the supernatant, filtered through a 0.22μm filter membrane for sterilization, and frozen at -20℃ for later use (the fermentation filtrate contains RC-19 metabolites).

[0038] Emulsion preparation: (1) Oil phase preparation: Stearic acid and squalane are added to the oil phase pot and heated and stirred at 75°C until completely melted; (2) Aqueous phase preparation: Deionized water, glycerol and sodium hyaluronate are added to the aqueous phase pot and heated and stirred at 75°C until sodium hyaluronate is dissolved; (3) Emulsification: The oil phase is slowly added to the aqueous phase and emulsified in a homogenizer (2000 rpm) for 5 min. When the temperature is reduced to 45°C, RC-19 fermentation filtrate, triethanolamine, methylparaben and flavoring are added and stirred for 10 min; (4) Standing and cooling: Refrigerate at 4°C for 24 h and test the pH value (5.5-6.5), viable count (≤100 CFU / g, to avoid contamination by other bacteria) and stability (centrifugation test: no stratification after centrifugation at 3000×g for 10 min).

[0039] Quality evaluation: pH value: 5.8±0.2 (matches the slightly acidic environment of the skin); viable bacteria count: Not detected (fermentation filtrate has been sterilized to ensure product safety); Stability: No stratification or discoloration was observed during the 45℃ constant temperature accelerated test (2 weeks); no emulsion breakage was observed after 3 cycles of freezing at -15℃ and thawing at -25℃. Human Trial: Sixty healthy subjects (aged 20-40 years, with a skin dryness score ≥3 out of 10) were randomly assigned to either the experimental group or the placebo group. The placebo group received a "simulated lotion" without *Lactobacillus reuteri* RC-19 fermentation filtrate; all other ingredients, proportions, and physical properties (appearance, texture, odor) were identical to the experimental group to ensure blinding (neither the subjects nor the researchers could distinguish between them). The experimental group applied the lotion to the flexor surface of their forearms morning and evening for four weeks. The placebo group applied the "simulated lotion" to the flexor surface of their forearms morning and evening for four weeks. Skin moisture content (MC value) was measured using a Corneometer CM825. Results showed:

[0040] The MC value in week 1 increased from (35±5)% to (45±6)% (p<0.05 vs baseline); In week 4, the MC value rose to (55±7)%, an improvement of 57.1%±8.3% from baseline (p<0.01), which was significantly better than the placebo group (which improved by only 12.3%±3.5%).

[0041] In summary, this invention successfully isolated and preserved a strain of *Lactobacillus reuteri* RC-19 (CCTCC NO: M20251292) with significant moisturizing effects. This strain effectively improves skin hydration by promoting the secretion of hyaluronic acid (HA) by human keratinocytes and enhancing the barrier function of an in vitro reconstructed epidermal model (increasing TEER and upregulating Occludin / Claudin-1). A topical lotion based on its active metabolites was clinically validated in humans; continuous use for 4 weeks increased skin moisture content by 57.1% compared to baseline, significantly superior to the placebo group. This invention reveals for the first time the application potential of *Lactobacillus reuteri* RC-19 in the field of skin hydration, providing an endogenous and long-lasting probiotic solution for improving dry and sensitive skin, combining scientific merit with practical application.

[0042] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of *Lactobacillus reuteri* ( Limosilacto bacillus reuteri RC-19, characterized in that, Its accession number is China Center for Type Culture Collection (CCTCC) NO:M20251292 (accessed on June 6, 2025), and its classification name is *Lactobacillus reuteri* RC-19. Limosilacto bacillus reuteri RC-19).

2. The use of the *Lactobacillus reuteri* RC-19 of claim 1 in the preparation of topical formulations that improve the hydration of human skin.

3. The application according to claim 2, characterized in that, The topical formulation works through at least one of the following mechanisms: (1) Promotes the secretion of hyaluronic acid (HA) by keratinocytes in the skin; (2) Enhance the skin barrier function.

4. The topical preparation according to claim 2, characterized in that, The topical preparation is an emulsion, cream, gel, or mask.

5. The topical preparation according to claim 4, characterized in that, The emulsion contains the following ingredients by weight percentage: 0.1-5% fermentation filtrate of *Lactobacillus reuteri* RC-19; Sodium hyaluronate 0.01-0.5%; Squalane 0.5-3%; Glycerin 2-8%; Emulsifier 0.5-2%; Preservative 0.1-0.5%; The remainder consists of deionized water and pH adjuster.

6. A composition, characterized in that, It contains *Lactobacillus reuteri* as described in claim 1. Limosilacto bacillus reuteri RC-19 and pharmaceutically acceptable carriers.

7. The use of the composition of claim 6 in the preparation of a topical formulation for improving the hydration of human skin.

Images