Use of a lactobacillus salivarius strain ls.0403 and a preparation derived therefrom for the preparation of a product for improving the state of skin associated with acne

Abstract

The present application relates to the technical field of microorganisms, and specifically discloses a lactobacillus salivarius Ls.0403 and application of a preparation derived from the same in preparation of a product for improving acne-related skin conditions, the strain has a preservation number of CGMCC No.38819; the present application also provides live bacteria of the strain, cell-free culture supernatant, pH-adjusted culture supernatant, bacterial lysate and heat-inactivated bacteria. Experiments prove that the live bacteria of Ls.0403 have a significant inhibitory effect on propionibacterium acnes, staphylococcus aureus and the like; the cell-free culture supernatant, pH-adjusted culture supernatant and bacterial lysate all retain inhibitory activity; the strain has DPPH, ABTS and hydroxyl radical scavenging capacity, the bacterial lysate has detectable total antioxidant capacity and antioxidant enzyme-related activity; the heat-inactivated Ls.0403 can still reduce the expression of chemotactic factor genes such as CXCL2 and CCL20, and improve the expression of epidermis-related genes such as CASP14 and MUC1. The strain and preparation described in the present application can be used in preparation of a drug, medical dressing or functional cosmetic for improving acne-related skin conditions.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to the application of a strain of Lactobacillus salivarius Ls.0403 and its derivatives in the preparation of products that improve acne-related skin conditions. Background Technology

[0002] Acne is a common chronic inflammatory skin disease of the pilosebaceous unit, mainly manifested as comedones, papules, pustules, nodules, skin erythema, and post-inflammatory pigmentation changes. The occurrence and development of acne are closely related to factors such as increased sebum secretion, abnormal follicular keratinization, Propionibacterium acnes-related inflammatory response, abnormal activation of keratinocytes, and changes in the composition of the skin microbiome.

[0003] Propionibacterium acnes is a common microorganism in the pilosebaceous unit of human skin. While it is a component of the normal skin microbiota, it can also induce keratinocytes to produce chemokines and inflammatory mediators under certain conditions, promoting the recruitment of inflammatory cells and exacerbating local skin lesions. Therefore, acne-related interventions should not only aim to completely eradicate Propionibacterium acnes, but also consider the control of abnormal inflammatory responses and the stability of the local skin environment.

[0004] Currently, common interventions for acne include topical or systemic antibiotics, retinoids, benzoyl peroxide, azelaic acid, and other anti-inflammatory agents. These methods can reduce acne-related skin lesions to some extent, but long-term or improper use of antibiotics may increase the risk of bacterial resistance and potentially affect the skin's resident microbiota. Some formulations may also cause skin dryness, irritation, peeling, or decreased adherence. Existing acne care products primarily focus on the direct inhibition of Propionibacterium acnes; however, acne-related skin conditions involve not only bacterial growth but also oxidative stress, keratinocyte inflammation, and abnormal expression of epidermal genes. Selecting active ingredients based solely on a single antibacterial result makes it difficult to comprehensively evaluate their effects on acne-related skin conditions.

[0005] In recent years, lactic acid bacteria, probiotics, and their derived components have attracted attention in acne care, skin inflammation regulation, and the development of skin microbiome-related products. A search revealed Chinese patent application CN118995466A, which discloses a strain of *Lactobacillus salivarius* Lsali-1 (CCTCC NO: M2023348). This strain, derived from the gut of a healthy infant, exhibits good safety (no toxicity factors, non-hemolytic, sensitive to multiple antibiotics), good tolerance to artificial gastric and intestinal fluids, and some antioxidant capacity. This document describes how Lsali-1 can improve antitumor treatment-related diarrhea by inhibiting various pathogenic bacteria, alleviating barrier dysfunction, and inhibiting the expression of pro-inflammatory factors, representing the application of oral probiotics in the field of gut health. However, this document does not address the application of this strain in topical skin care, nor does it address the improvement of acne-related skin conditions. Furthermore, this document only provides application methods for live bacteria strains, without mentioning the preparation and application of non-live bacteria sources such as heat-inactivated bacterial cells, bacterial lysates, or culture supernatants. For topical skin applications, especially in acne-related skin conditions that are often accompanied by skin barrier damage and local inflammation, the survival, colonization, and potential proliferation risks of live bacteria on the skin surface still require further careful assessment.

[0006] Some existing products rely on live bacteria to exert their effects, but the number of live bacteria is easily affected by culture conditions, temperature, oxygen, moisture, storage time, and the formulation matrix. Differences in the number and bioactivity of live bacteria may exist between different batches of products. The survival and persistence of live bacteria on the skin surface are also affected by skin pH, sebum, local moisture, topical products, and the existing skin microbiome. For skin with a damaged barrier or significant inflammation, the risk of proliferation and spread of live bacteria must be further considered before topical application. Therefore, preparing lactic acid bacteria with clearly defined functions as heat-inactivated bacterial cells, bacterial lysates, or other preparations from non-live bacterial sources helps reduce reliance on the survival and colonization of live bacteria.

[0007] Significant differences exist in the antibacterial, antioxidant, and host cell regulatory abilities among different lactic acid bacteria strains. It cannot be simply assumed that all *Lactobacillus salivarius* or all heat-inactivated lactic acid bacteria possess the same effects. Currently, there is a relative lack of technical solutions that simultaneously evaluate live bacteria, culture supernatants, pH-adjusted culture supernatants, bacterial lysates, and heat-inactivated bacteria of specific human *Lactobacillus salivarius* strains and apply them to improve acne-related skin conditions.

[0008] Therefore, this application proposes the application of a strain of Lactobacillus salivarius Ls.0403 and its derivatives in the preparation of products that improve acne-related skin conditions, in order to solve the above-mentioned problems. Summary of the Invention

[0009] The purpose of this invention is to address the problems of limited functionality of active ingredients, poor stability of live bacterial preparations, and insufficient safety for local application in existing technologies for acne-related skin conditions. This invention provides the application of a strain of Lactobacillus salivarius Ls.0403 and its derivatives in the preparation of products that improve acne-related skin conditions.

[0010] The above-mentioned objective of the present invention is achieved as follows:

[0011] One aspect of the present invention provides a strain of *Lactobacillus salivarius* Ls.0403, whose Latin name is *Ligilactobacillus salivarius*, accession number CGMCC No. 38819, accession date May 21, 2026, and deposited at the China General Microbiological Culture Collection Center.

[0012] Furthermore, the Lactobacillus salivarius Ls.0403 was derived from fecal samples from healthy adults.

[0013] The present invention also provides live bacterial cells prepared from Lactobacillus salivarius Ls.0403.

[0014] The present invention also provides a cell-free culture supernatant prepared from Lactobacillus salivarius Ls.0403.

[0015] The present invention also provides a pH-adjusted culture supernatant prepared from Lactobacillus salivarius Ls.0403.

[0016] The present invention also provides a bacterial lysate prepared from Lactobacillus salivarius Ls.0403.

[0017] The present invention also provides heat-inactivated bacterial cells prepared from Lactobacillus salivarius Ls.0403.

[0018] The present invention also provides a composition for improving acne-related skin conditions, the composition comprising at least one of the live bacteria of claim 3, the cell-free culture supernatant of claim 4, the pH-adjusted culture supernatant of claim 5, the bacterial lysate of claim 6, and the heat-inactivated bacteria of claim 7, and one or more skin-acceptable carriers or excipients.

[0019] Furthermore, the skin-acceptable carrier or excipient is selected from one or more of sodium carboxymethyl cellulose, carbomer, hyaluronic acid, sodium hyaluronate, chitosan, polyvinyl alcohol, poloxamer, glycerin, propylene glycol, physiological saline, phosphate buffer, cream matrix, hydrogel matrix, or medical dressing matrix.

[0020] The present invention also provides the use of Lactobacillus salivarius Ls.0403, live cells, cell-free culture supernatant, pH-adjusted culture supernatant, cell lysate, heat-inactivated cells, or combinations thereof in the preparation of products that improve acne-related skin conditions.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The strain of the present invention has a clear and traceable source. The provided Ls.0403 has a clear strain source and preservation information, with the preservation number CGMCC No. 38819. It can be identified and traced through preservation information, 16S rRNA gene sequence and whole genome data, which is beneficial for subsequent strain preservation, production management and quality control of different batches of raw materials.

[0023] 2. This invention exhibits strong comprehensive antibacterial ability among candidate Lactobacillus salivarius strains. In the preliminary screening of 19 candidate Lactobacillus salivarius strains, Ls.0403 showed average inhibition zone widths of 8.80±0.99 mm and 14.48±1.53 mm against Staphylococcus aureus and Propionibacterium acnes, respectively. The combined average inhibition zone width of the two indicator bacteria was 11.64 mm, ranking first among the 19 candidate strains screened. This demonstrates that it is a specific strain with excellent comprehensive antibacterial ability selected after comparison of multiple strains.

[0024] 3. This invention has an in vitro inhibitory effect on a variety of representative bacteria. Ls.0403 forms obvious inhibition zones against Escherichia coli, Staphylococcus aureus, Propionibacterium acnes, and Pseudomonas aeruginosa. Its in vitro antibacterial targets are not limited to a single bacterium.

[0025] 4. The culture supernatant of the present invention has a strong inhibitory effect on Propionibacterium acnes and Staphylococcus aureus. When the volume fraction of cell-free culture supernatant is 25%, the inhibition rates against Staphylococcus aureus and Propionibacterium acnes reach 78.86±2.49% and 81.62±5.84%, respectively; when the volume fraction is increased to 50%, the inhibition rates reach 95.13±0.11% and 91.25±1.00%, respectively, indicating that the culture supernatant of this strain contains active components that can inhibit the growth of the above-mentioned bacteria.

[0026] 5. The culture supernatant of the present invention retains some antibacterial activity after pH adjustment. When the pH of the Ls.0403 cell-free culture supernatant was adjusted to 6.5, and it accounted for 75% of the final total volume of the culture system, the inhibition rates against Staphylococcus aureus and Propionibacterium acnes were 66.53% ± 6.14% and 44.27% ± 1.90%, respectively. These results indicate that the antibacterial effect of the Ls.0403 culture supernatant cannot be entirely explained by the acidic environment, and suggest that other antibacterial components may exist in the culture supernatant.

[0027] 6. The bacterial cell lysate of the present invention exhibits significant anti-Propionibacterium acnes and anti-Staphylococcus aureus activity. When the Ls.0403 bacterial cell lysate accounts for 75% of the final total volume of the culture system, the inhibition rates against Propionibacterium acnes and Staphylococcus aureus are 74.53±4.54% and 50.19±20.14%, respectively. This demonstrates that the active ingredient of Ls.0403 is also present in the bacterial cells or the soluble components after bacterial cell disruption, and can serve as another raw material form independent of live bacterial growth.

[0028] 7. The Ls.0403 provided by this invention exhibits various in vitro antioxidant activities. The highest scavenging rates of the intact Ls.0403 bacterial suspension against DPPH radicals, ABTS radicals, and hydroxyl radicals reached 81.58%±2.55%, 90.44%±2.79%, and 89.41%±3.30%, respectively. The highest T-AOC value of the bacterial lysate reached 75.53%±0.28%, and detectable activities were also observed in T-SOD and GSH-Px related assays. These results demonstrate that both the intact Ls.0403 bacterial suspension and bacterial lysate exhibit excellent activity in different in vitro antioxidant assay systems.

[0029] 8. The bacteria provided in this invention retain keratinocyte regulatory activity even after heat inactivation. After heat inactivation, Ls.0403 significantly reduced the expression of inflammation- or epithelial activation-related genes such as CXCL2, CCL20, EREG, and EGR1 in keratinocytes induced by *Propionibacterium acnes*, with CXCL2 and CCL20 decreasing by approximately 40.95% and 30.68%, respectively, compared to the model group. Simultaneously, it significantly increased the expression of epidermal-related genes such as CASP14 and MUC1, with CASP14 and MUC1 increasing to approximately 6.05-fold and 1.54-fold, respectively, compared to the model group. These results indicate that Ls.0403, even after heat inactivation, retains its ability to reduce keratinocyte inflammation-related responses and promote epidermal-related repair responses.

[0030] 9. This invention has antibacterial, antioxidant and host cell regulatory effects. From bacterial inhibition, bacterial active components, antioxidant effects to host keratinocyte response regulation, this invention forms a multi-level technical effect chain, providing complete experimental evidence for acne-related skin products.

[0031] 10. The heat-inactivated formulation of the present invention is beneficial to the standardization of raw materials and quality control, and avoids the problem of the decrease in the number of live bacteria during storage and use. It can be standardized by CFU-equivalents.

[0032] 11. This invention can reduce the potential risks of local application of live bacteria. By using heat-inactivated bacteria that have been cultured and verified to have no live bacterial growth, the possibility of continuous proliferation and spread of live bacteria in areas of skin inflammation or barrier abnormalities can be reduced.

[0033] 12. This invention has multiple raw material forms and product development routes. Ls.0403 in this invention can be prepared in multiple forms, used alone or in combination, and can be compounded with a variety of topical skin materials to develop into multiple dosage forms, with good potential for formulation development and industrial transformation. Attached Figure Description

[0034] Figure 1 This is a graph showing the in vitro antibacterial screening and comprehensive evaluation results of 19 candidate Lactobacillus salivarius strains against Propionibacterium acnes and Staphylococcus aureus in this invention embodiment;

[0035] Figure 2 This is a graph showing the in vitro antibacterial effect of Ls.0403 live bacteria on Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa and Escherichia coli under different sample bacterial loads in the embodiments of the present invention.

[0036] Figure 3 This is a graph showing the in vitro antibacterial effects of Ls.0403 cell-free culture supernatant, pH 6.5 regulated culture supernatant, and bacterial lysate on Staphylococcus aureus and Propionibacterium acnes in an embodiment of the present invention.

[0037] Figure 4 This is a graph showing the in vitro antioxidant activity detection results of Ls.0403 bacterial suspension and bacterial lysate in the embodiments of the present invention;

[0038] Figure 5 This is a graph showing the test results of Ls.0403's self-aggregation, cell surface hydrophobicity, and acid and bile salt resistance in the embodiments of the present invention;

[0039] Figure 6 This is a graph showing the genome-wide characteristics, functional correlation analysis, and safety-related screening results of Ls.0403 in this embodiment of the invention;

[0040] Figure 7 This is a figure showing the RNA sequencing analysis and real-time quantitative PCR verification results of the effect of heat-inactivated Ls.0403 on the transcriptional response of HaCaT keratinocytes induced by Propionibacterium acnes in this embodiment of the invention.

[0041] Figure 8 This is a schematic diagram of the technical solution for preparing products to improve acne-related skin conditions using Lactobacillus salivarius Ls.0403 and its live cells, cell-free culture supernatant, pH-adjusted culture supernatant, bacterial lysate, and heat-inactivated bacterial cells in this invention. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0043] This invention obtained a human-derived Lactobacillus salivarius strain Ls.0403 through screening. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2026, with accession number CGMCC No. 38819.

[0044] The results of this study indicate that the live Ls.0403 strain of this invention exhibits in vitro inhibitory effects against Propionibacterium acnes, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Cell-free culture supernatant, pH-adjusted culture supernatant, and bacterial lysates of this strain also showed varying degrees of antibacterial activity. The Ls.0403 lysate simultaneously possesses DPPH, ABTS, and hydroxyl radical scavenging capabilities. Even after heat inactivation, this strain was still able to regulate the keratinocyte transcriptional response induced by Propionibacterium acnes, reduce the expression of chemokine-related genes such as CXCL2 and CCL20, and increase the expression of epidermal-related genes such as CASP14 and MUC1.

[0045] The present invention also provides *Lactobacillus salivarius* Ls.0403, live cells prepared from the strain, cell-free culture supernatant, pH-adjusted culture supernatant, cell lysate and heat-inactivated cells, and skin compositions containing the prepareds, and uses them to prepare products that inhibit acne-associated bacteria, regulate keratinocyte inflammatory responses, provide antioxidant effects, and improve acne-associated skin conditions.

[0046] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0047] Example 1: Isolation and culture of Lactobacillus salivarius Ls.0403

[0048] Fecal samples from healthy adults were serially diluted with sterile phosphate buffer and spread onto MRS solid medium. The culture dishes were incubated anaerobically at 37°C for 24–48 h. Colonies exhibiting lactic acid bacteria-like morphology were selected, and pure cultures were obtained by continuous streaking.

[0049] The purified Ls.0403 was inoculated into a culture medium suitable for lactic acid bacteria growth and cultured. The culture medium can be MRS liquid medium, modified MRS medium, food-grade lactic acid bacteria fermentation medium, or a lactic acid bacteria culture medium acceptable for pharmaceutical, cosmetic, and medical device production processes. The culture temperature can be 30-42℃, preferably 35-37℃; the culture time can be 12-48 h, preferably 18-24 h; the culture process is carried out under anaerobic or microaerophilic conditions.

[0050] After cultivation, the bacterial cells were collected by centrifugation and washed 2-3 times with sterile phosphate buffer, physiological saline, or other suitable buffer to reduce culture medium residue. The washed bacterial cells were then resuspended in sterile phosphate buffer, physiological saline, or other suitable solutions to prepare a live bacterial suspension of Ls.0403.

[0051] Example 2: Preparation of cell-free culture supernatant and pH-adjusted culture supernatant of Ls.0403

[0052] Ls.0403 was cultured to the stationary phase, and the culture was centrifuged at 4°C and 5000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm filter membrane to obtain a cell-free culture supernatant containing no intact bacteria.

[0053] A portion of the cell-free culture supernatant was taken, its pH was adjusted to 6.5, and then filtered again through a 0.22 μm filter membrane to obtain the pH-adjusted Ls.0403 culture supernatant. The pH-adjusted culture supernatant can be used to evaluate whether the antibacterial effect of the culture supernatant is entirely dependent on the acidic environment formed during the culture process.

[0054] Cell-free culture supernatant and pH-adjusted culture supernatant can be used directly or further prepared into concentrates, lyophilized powders, spray-dried powders, or other microbial-derived raw materials suitable for skin products.

[0055] Example 3: Preparation of Ls.0403 cell lysates

[0056] Ls.0403 cells were collected by centrifugation and washed twice with sterile PBS. The washed cells were resuspended in a suitable buffer and sonicated at 200 W for 5 s followed by 5 s intervals.

[0057] After cell disruption, the bacterial suspension was centrifuged and the supernatant was collected. The supernatant was then filtered through a 0.22 μm filter membrane to obtain Ls.0403 cell lysate or the soluble components of the disrupted cells. The cell lysate can be used directly or further prepared into a concentrate, lyophilized powder, or composite raw material.

[0058] Example 4: Preparation of thermally inactivated Ls.04O3

[0059] Ls.0403 was cultured to the stationary phase, and the bacterial cells were collected by centrifugation and washed 2-3 times with sterile PBS. The bacterial concentration was adjusted according to the viable cell plate count results, and the bacterial suspension was heat-inactivated.

[0060] The heat inactivation temperature can be 60-100℃, and the treatment time can be 10-120 min. The preferred heat inactivation conditions are 80℃ for 30 min. After heat inactivation, the bacterial suspension should be cooled to room temperature or 4℃.

[0061] A portion of the heat-treated bacterial suspension was spread onto MRS solid medium and incubated anaerobically at 37°C for at least 48 hours. No colony growth was observed after incubation, indicating that the bacteria had been completely inactivated. The amount of heat-inactivated bacteria used was expressed as CFU-equivalents (cluster formation units) of the bacteria before inactivation.

[0062] Heat-inactivated bacterial cells can be prepared as heat-inactivated bacterial cell suspensions, concentrated solutions, lyophilized powders, spray-dried powders, or composite powders containing protectants. They can also be further compounded with skin-specific carriers to form skin preparations.

[0063] Example 5: Screening of candidate Lactobacillus salivarius and in vitro antibacterial effect of live Ls.0403 cells

[0064] Nineteen candidate *Lactobacillus salivarius* strains were screened for in vitro antibacterial activity using an agar cover assay, with *Propionibacterium acnes* and *Staphylococcus aureus* as indicator bacteria. All candidate strains were cultured and sampled under identical conditions, and then covered with soft agar medium containing the corresponding indicator bacteria. After culture, the diameter of the total inhibition zone was measured, and the inhibition zone width R was calculated based on the total inhibition zone diameter and the diameter of the sampled area. The calculation formula is R = (d... inhibition－ d spot ) / 2.

[0065] Nineteen candidate *Lactobacillus salivarius* strains showed varying degrees of in vitro inhibitory effects against *Propionibacterium acnes* and *Staphylococcus aureus*. Figure 1 (A, B). Among them, the average inhibition zone widths of Ls.0403 against Propionibacterium acnes and Staphylococcus aureus were 14.48±1.53 mm and 8.80±0.99 mm, respectively.

[0066] After a comprehensive evaluation of the antibacterial results of the two indicator bacteria, the average inhibition zone width of Ls.0403 was 11.64 mm, ranking first among the 19 candidate Lactobacillus salivarius strains screened. Figure 1 C). The above results indicate that Ls.0403 exhibited strong overall in vitro antibacterial activity in this candidate bacterial screening, therefore this strain was selected for subsequent experiments.

[0067] Based on the above screening, the in vitro inhibitory effect of live Ls.0403 cells on different indicator bacteria was further evaluated. Ls.0403 was prepared at 1×10⁻⁶... 4 2×10 4 and 4×10 4 The CFU / spot sample was inoculated onto MRS agar medium and incubated anaerobically at 37°C for 48 h, followed by covering with soft agar medium containing the corresponding indicator bacteria. Indicator bacteria included Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa, and Escherichia coli. After incubation, the total inhibition zone diameter was measured, and the inhibition zone width R was calculated using the aforementioned formula.

[0068] Ls.0403 showed varying degrees of in vitro inhibitory effects on all four indicator bacteria at the tested sample concentrations. Figure 2 ). In 4×10 4 At a CFU / spot sampling bacterial load, the total inhibition zone diameters of Ls.0403 against Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa, and Escherichia coli were 28.90±2.25 mm, 27.13±1.05 mm, 20.83±1.76 mm, and 35.67±1.15 mm, respectively, with corresponding inhibition zone widths of 8.73±0.25 mm, 7.87±0.29 mm, 5.33±0.38 mm, and 13.17±0.29 mm, respectively.

[0069] The above results indicate that live Ls.0403 cells have in vitro inhibitory effects on Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa, and Escherichia coli, further demonstrating the antibacterial activity of this strain against different indicator bacteria.

[0070] Example 6: Antibacterial effect of Ls.0403 cell-free culture supernatant

[0071] The antibacterial activity of Ls.0403 cell-free culture supernatant was evaluated using a liquid antibacterial assay. Cell-free culture supernatant was added to Staphylococcus aureus and Propionibacterium acnes culture systems at final volumes of 5%, 10%, 25%, 50%, and 75%, with indicator bacteria cultured alone serving as a growth control. OD was measured after culture. 600 The inhibition rate was calculated based on the absorbance of the treatment group and the growth control group.

[0072] Unadjusted pH Ls.0403 cell-free culture supernatant showed in vitro inhibitory effects against both indicator bacteria. When the final volume ratio of the cell-free culture supernatant was 25%, the inhibition rates against Staphylococcus aureus and Propionibacterium acnes were 78.86% ± 2.49% and 81.62% ± 5.84%, respectively. When the final volume ratio increased to 50%, the inhibition rates against the two indicator bacteria reached 95.13% ± 0.11% and 91.25% ± 1.00%, respectively. At a final volume ratio of 75%, the corresponding inhibition rates were 94.79% ± 0.71% and 91.33% ± 0.37%, respectively.

[0073] The above results indicate that the cell-free culture supernatant of Ls.0403 has a strong in vitro inhibitory effect on Staphylococcus aureus and Propionibacterium acnes.

[0074] Example 7: Antibacterial effect of pH-adjusted culture supernatant

[0075] The pH of the cell-free culture supernatant of Ls.0403 was adjusted to 6.5, and added to the Staphylococcus aureus and Propionibacterium acnes culture systems at final volume ratios of 5%, 10%, 25%, 50%, and 75%, respectively. Liquid antibacterial experiments were conducted according to the method described in Example 6.

[0076] The culture supernatant after pH adjustment still showed varying degrees of in vitro antibacterial activity. Figure 3 B). When the final volume ratio of the culture supernatant was adjusted to 75% at pH 6.5, the inhibition rates against Staphylococcus aureus and Propionibacterium acnes were 66.53%±6.14% and 44.27%±1.90%, respectively.

[0077] Compared with the unadjusted culture supernatant, the antibacterial activity of the pH-adjusted culture supernatant decreased, but it still retained some antibacterial effect. These results indicate that the antibacterial effect of the Ls.0403 culture supernatant is related to acidic metabolites, but cannot be entirely explained by the low pH; other antibacterial components may also exist in the culture supernatant.

[0078] Example 8: Antibacterial effect of Ls.0403 bacterial cell lysate

[0079] Ls.0403 bacterial lysate was added to the Staphylococcus aureus and Propionibacterium acnes culture systems at final volume ratios of 5%, 10%, 25%, 50%, and 75%, respectively, and liquid antibacterial experiments were conducted according to the method described in Example 6.

[0080] Ls.0403 bacterial lysate showed varying degrees of in vitro inhibitory effects on two indicator bacteria. Figure 3C). When the final volume ratio of bacterial lysate was 75%, the inhibition rates against Propionibacterium acnes and Staphylococcus aureus were 74.53±4.54% and 50.19±20.14%, respectively.

[0081] The above results indicate that the soluble components obtained after Ls.0403 cell lysis possess certain antibacterial activity, with a particularly significant inhibitory effect against Propionibacterium acnes. This suggests that the active components of Ls.0403 are not only present in the culture supernatant but may also be found in the cell structure or in the soluble components after cell lysis.

[0082] Example 9: In vitro antioxidant activity of Ls.0403

[0083] The DPPH, ABTS, and hydroxyl radical scavenging activities were evaluated using washed Ls.0403 intact bacterial suspensions. The bacterial suspensions were added to the corresponding reaction systems at different final volume ratios, with the final volume ratios for DPPH, ABTS, and hydroxyl radical scavenging experiments being 50%, 66.5%, and 75%, respectively.

[0084] In the DPPH free radical scavenging experiment, the scavenging rates of Ls.0403 bacterial suspension at 50%, 66.5%, and 75% volume fractions were 20.68%±0.56%, 68.31%±3.61%, and 81.58%±2.55%, respectively.

[0085] In the ABTS free radical scavenging experiment, the scavenging rates of Ls.0403 bacterial suspension at 50%, 66.5%, and 75% volume fractions were 56.96%±1.25%, 79.77%±2.45%, and 90.44%±2.79%, respectively, while the scavenging rate of the positive control in the same batch of experiments was 89.96%±0.40%.

[0086] When the hydroxyl radical scavenging activity was evaluated using the TMB method, the scavenging rates of Ls.0403 bacterial suspension at 50%, 66.5%, and 75% v / v were 84.91%±1.28%, 88.14%±0.21%, and 89.41%±3.30%, respectively. The Ls.0403 bacterial suspension also exhibited corresponding hydroxyl radical scavenging activity when detected using the methylene blue method.

[0087] In addition, Ls.0403 bacterial cells were ultrasonically disrupted to prepare bacterial lysates, which were used to detect total antioxidant capacity, total superoxide dismutase-related activity, and glutathione peroxidase-related activity. The T-AOC values ​​of Ls.0403 bacterial lysates at 12.5%, 25%, and 37.5% (v / v) were 35.94% ± 0.98%, 57.76% ± 0.51%, and 75.53% ± 0.28%, respectively. The bacterial lysates also exhibited detectable activities in T-SOD and GSH-Px related assays.

[0088] The above results indicate that strain Ls.0403 exhibits corresponding activities in various in vitro antioxidant assay systems, including DPPH, ABTS, TMB, methylene blue, T-AOC, T-SOD, and GSH-Px, demonstrating that strain Ls.0403 possesses multifaceted in vitro antioxidant capabilities.

[0089] Example 10: Self-aggregation and cell surface hydrophobicity of Ls.0403

[0090] The autoaggregation ability of Ls.0403 and other lactic acid bacteria was evaluated using the static absorbance method. Comparative strains included *Lactobacillus fermentum* LfA27, *Lactobacillus reuteri* Lr6475, and *Lactobacillus plantarum* Lp1-2. After standing for 5 h at room temperature, the average autoaggregation rates of Ls.0403, LfA27, Lr6475, and Lp1-2 were 70.67% ± 1.11%, 11.57% ± 0.97%, 5.57% ± 0.70%, and 21.05% ± 3.61%, respectively. Figure 5 A). Under the experimental conditions, Ls.0403 exhibited strong in vitro self-aggregation ability.

[0091] The hydrophobicity of cell surfaces of different bacterial strains was evaluated using the microbial adhesion solvent method, with dodecane, xylene, and chloroform used as detection solvents, respectively. Figure 5 B). The average hydrophobicity of Ls.0403 in dodecane, xylene, and chloroform systems were 49.85%±9.39%, 54.98%±5.66%, and 60.14%±12.53%, respectively. Other comparative strains exhibited varying degrees of cell surface hydrophobicity in different solvent systems.

[0092] The above results indicate that Ls.0403 has a strong in vitro self-aggregation ability and exhibits significant cell surface hydrophobicity in different solvent systems. It can be used to characterize the physicochemical properties of the cell surface of this strain and provide a basis for further research on the active ingredients derived from the cell surface.

[0093] Example 11: Environmental tolerance of Ls.0403

[0094] Ls.0403 cells were cultured in MRS media with different pH values ​​and bile salt concentrations. Samples were taken at 0, 2, 4, and 6 h, and the number of cells was determined by plate counting to evaluate the survival of Ls.0403 cells under different environmental conditions.

[0095] In the acid tolerance test, the bacterial counts of Ls.0403 after culturing for 0, 2, 4, and 6 h at pH 2.5 were 7.24±0.11, 5.98±0.11, 4.89±0.30, and 4.22±0.08 log10 CFU / mL, respectively; at pH 3.5, they were 6.51±0.02, 6.95±0.13, 6.89±0.25, and 7.07±0.05 log10 CFU / mL; at pH 5.5, they were 7.32±0.06, 7.62±0.06, 8.37±0.05, and 9.44±0.03 log10 CFU / mL; and at pH 6.5, they were 7.29±0.06, 8.15±0.02, 8.36±0.11, and 9.59±0.13 log10 CFU / mL. The values ​​of CFU / mL were 7.24±0.15, 8.17±0.05, 9.01±0.05 and 9.21±0.02 log10 CFU / mL at pH 7.5, and 6.57±0.07, 6.93±0.00, 6.98±0.27 and 9.00±0.16 log10 CFU / mL at pH 8.5.

[0096] The results show that, under pH 2.5 conditions, the bacterial count of Ls.0403 decreased with prolonged culture time, but viable bacteria could still be recovered after 6 h of culture; under pH 3.5 conditions, the bacterial count remained generally stable; and under pH 5.5-8.5 conditions, the bacterial count after 6 h of culture was higher than the initial level.

[0097] In the bile salt tolerance test, the bacterial counts of Ls.0403 after culturing for 0, 2, 4, and 6 h under 0.9% bile salt conditions were 6.22±0.03, 7.47±0.01, 8.12±0.05, and 9.36±0.04 log10 CFU / mL, respectively; under 2% bile salt conditions, they were 6.19±0.03, 7.88±0.01, 8.08±0.02, and 9.32±0.09 log10 CFU / mL; under 3% bile salt conditions, they were 6.41±0.06, 7.91±0.01, 7.82±0.06, and 9.17±0.06 log10 CFU / mL; and under 5% bile salt conditions, they were 7.31±0.04, 6.80±0.11, 6.88±0.15, and 9.28±0.09 log10 CFU / mL. CFU / mL; under 6% bile salt conditions, the values ​​were 6.04±0.05, 6.06±0.01, 6.44±0.02, and 7.07±0.06 log10 CFU / mL, respectively. Figure 5 D).

[0098] The results showed that the overall bacterial count of Ls.0403 increased during culture under 0.9%-3% bile salt conditions; under 5% bile salt conditions, the bacterial count decreased in the early stage, but still reached 9.28±0.09 log10 CFU / mL after 6 h of culture; under 6% bile salt conditions, bacterial growth was somewhat limited, but 7.07±0.06 log10 CFU / mL of viable bacteria could still be recovered after 6 h of culture.

[0099] In summary, Ls.0403 exhibited a certain degree of tolerance to the in vitro environment within the tested pH and bile salt concentration ranges, and was able to maintain a certain survival rate even under strong acid and high salt concentration conditions.

[0100] Example 12: Genome-wide identification and functional analysis of Ls.0403

[0101] The entire genome of Ls.0403 was sequenced and assembled. The total assembled genome length of Ls.0403 was 2,120,621 bp, with a GC content of 33.08%. A total of 2,163 protein-coding genes were predicted, with a total coding sequence length of 1,874,976 bp and a coding density of 88.42%. In addition, 77 tRNA genes, 22 rRNA genes, and 5 sRNA genes were predicted in the genome. The estimated sequencing depth was 44.5-fold, and the BUSCO integrity was 99.2%, indicating that the genome has high integrity.

[0102] Genome-wide average nucleotide identity analysis supports the attribution of Ls.0403 to *Lactobacillus salivarius*. Functional annotation identified genes associated with carbohydrate utilization, redox homeostasis, acid-base stress, osmotic stress, cell surface structure, and secondary metabolite synthesis potential.

[0103] AntiSMASH analysis predicted a gene cluster related to the synthesis of secondary metabolites associated with lasso peptides and type III polyketide synthases. These results suggest that Ls.0403 possesses the genetic basis for producing potential bioactive components, but specific bioactive substances require further isolation and identification.

[0104] Example 13: Genome-wide safety-related screening of Ls.0403

[0105] Antimicrobial resistance-related genes in Ls.0403 were screened using the ARDB and CARD databases. Analysis showed no match in the ARDB database, but one vanT-like homology annotation was detected in the CARD database.

[0106] Virulence-related genes were screened using the VFDB database. The detected homology annotations mainly belonged to unclassified categories, capsule-related categories, and other low-quantity categories. Since these results are derived from sequence homology analysis, they are primarily used for preliminary genomic-level evaluation of the strain.

[0107] Mobile genetic element analysis revealed genomic islands, prophage regions, CRISPR sequences, insertion sequences, plasmid-related annotations, and integron-related annotations. These genome-wide results provide a foundation for strain identification, functional analysis, and subsequent phenotypic safety assessment of Ls.0403.

[0108] Example 14: Regulation of keratinocyte response induced by heat-inactivated Ls.0403

[0109] A cell response model induced by Propionibacterium acnes was established using HaCaT human keratinocytes. A blank control group, a Propionibacterium acnes model group, and a heat-inactivated Ls.0403 treatment group were set up. Heat-inactivated Ls.0403 was prepared according to Example 4, and culture confirmed the absence of viable bacterial growth. After HaCaT cells were cultured to approximately 80% confluence, they were injected with 1×10⁻⁶ cells / cells. 8 The cells were pretreated with CFU-equivalents / mL heat-inactivated Ls.0403 for 6 h, followed by stimulation with Propionibacterium acnes with OD600 of 1.0 for 6 h, and then RNA sequencing and real-time quantitative PCR were performed.

[0110] Principal component analysis of RNA sequencing revealed different overall transcriptional characteristics in the three groups. Compared with the blank control group, the *Propionibacterium acnes* model group showed 608 upregulated genes and 533 downregulated genes, including inflammation-related genes such as IL1β, IL1α, CXCL8, CXCL3, CXCL2, CCL20, and TNF. Compared with the model group, heat-inactivated Ls.0403 treatment resulted in 34 upregulated genes and 11 downregulated genes, with decreased expression of CXCL2, CCL20, EREG, and EGR1, and increased expression of CASP14 and MUC1.

[0111] Real-time quantitative PCR further confirmed the above changes. Compared with the model group, after heat inactivation of Ls.0403, the relative expression levels of CXCL2, CCL20, EREG and EGR1 decreased from 3.026±0.108, 3.327±0.325, 4.664±0.573 and 7.383±0.224 to 1.787±0.299, 2.306±0.279, 3.413±0.375 and 6.165±0.167, respectively, representing decreases of approximately 40.95%, 30.68%, 26.82% and 16.50%; while CASP14 and MUC1 increased from 0.243±0.122 and 3.059±0.365 to 1.472±0.143 and 4.699±0.216, respectively, approximately 6.05 times and 1.54 times that of the model group. All of the above differences were statistically significant.

[0112] The above results indicate that even after Ls.0403 was treated at 80℃ for 30 min and lost its proliferative capacity, it could still selectively regulate the transcriptional response of keratinocytes induced by Propionibacterium acnes, reduce the expression of some chemokines and epithelial activation-related genes, and increase the expression of some epidermal-related genes.

[0113] Example 15: Hydrogel for improving acne-related skin conditions

[0114] By weight percentage, 1 × 10⁻⁶ of the heat-inactivated Ls.0403 (calculated as CFU-equivalents) prepared in Example 4 was taken. 8 A hydrogel was prepared by mixing CFU-equivalents / g with 1% sodium carboxymethyl cellulose, 5% glycerin, and the remainder deionized water, and stirring until homogeneous. This hydrogel can be used to inhibit Propionibacterium acnes, reduce keratinocyte inflammation, and improve acne-related skin conditions.

[0115] Example 16: Lotion for improving acne-related skin conditions

[0116] By weight percentage, 1% of the lyophilized Ls.0403 cell-free culture supernatant prepared in Example 2 was mixed with 5% squalane, 3% caprylic / capric triglyceride, 2% glyceryl stearate, 1% cetearyl alcohol, 5% glycerol, 0.5% sodium hyaluronate, and the remainder deionized water, and emulsified to obtain an emulsion. This emulsion can be used to inhibit acne-related bacteria, provide antioxidant effects, and improve acne-related skin conditions such as erythema, excessive sebum secretion, and local inflammation.

[0117] Example 17: Lyophilized powder for improving acne-related skin conditions

[0118] The Ls.0403 bacterial cell lysate prepared in Example 3 was mixed with an appropriate amount of lyophilization protectant (such as trehalose or mannitol) and freeze-dried to obtain a lyophilized powder. It was reconstituted with physiological saline before use and can be used for topical skin care.

[0119] Example 18: Application Case

[0120] The *Lactobacillus salivarius* Ls.0403, live cells prepared therefrom, cell-free culture supernatant, pH-adjusted culture supernatant, bacterial lysate, and heat-inactivated bacterial cells, as well as a skin composition containing the prepared products, described in the above embodiments of the present invention, can be used to prepare products that improve acne-related skin conditions. This product can inhibit acne-related or skin-related bacteria such as *Propionibacterium acnes* and *Staphylococcus aureus*, and can also reduce the expression of inflammation- or epithelial activation-related genes such as CXCL2, CCL20, EREG, and EGR1 in keratinocytes, while increasing the expression of epidermal-related genes such as CASP14 and MUC1. This product can also provide antioxidant effects, improving acne-related skin erythema, excessive sebum secretion, local inflammation, rough skin, and other acne-related skin conditions. In this embodiment, the product can be prepared as a pharmaceutical, hygiene product, medical dressing, functional cosmetic, or skin care product.

[0121] In summary, this invention comprehensively verified the multiple efficacies of *Lactobacillus salivarius* Ls.0403 and its preparations from various sources in improving acne-related skin conditions through systematic in vitro and cell experiments. At the strain screening level, this invention selected Ls.0403 from 19 candidate *Lactobacillus salivarius* strains through comparative screening. Its average inhibition zone widths against *Staphylococcus aureus* and *Propionibacterium acnes* were 8.80±0.99 mm and 14.48±1.53 mm, respectively, with a comprehensive value of 11.64. The strain ranked first among 19 candidate bacteria, indicating that it is not the conventional *Lactobacillus salivarius* in this field, but a specific strain with excellent comprehensive antibacterial properties selected through parallel comparison of multiple strains. Regarding antibacterial activity, live *Ls.0403* cells formed significant inhibition zones against *Propionibacterium acnes*, *Staphylococcus aureus*, *Escherichia coli*, and *Pseudomonas aeruginosa*. Its cell-free culture supernatant, at a final volume ratio of 50%, showed inhibition rates of 91.25% and 95.13% against *Propionibacterium acnes* and *Staphylococcus aureus*, respectively. Even after pH adjustment to 6.5, significant antibacterial activity was retained. The bacterial lysate also exhibited anti-*Propionibacterium acnes* activity (74.53%), demonstrating that the antibacterial active ingredients of this strain exist both in the culture supernatant and within the bacterial cells, and can be developed into various forms of non-live bacterial raw materials. Regarding antioxidant activity, the *Ls.0403* bacterial suspension showed resistance to DPPH, ABTS, and... The highest hydroxyl radical scavenging rates were 81.58%, 90.44%, and 89.41%, respectively, and the total antioxidant capacity of the bacterial lysate reached 75.53%, indicating that this strain has multiple antioxidant functions. Most importantly, after being heat-inactivated at 80℃ for 30 min and confirmed to have no viable bacterial growth by culture, Ls.0403 could still significantly downregulate the expression of inflammation and epithelial activation-related genes such as CXCL2 (down 40.95%), CCL20 (down 30.68%), EREG (down 26.82%), and EGR1 (down 16.50%) in keratinocytes induced by Propionibacterium acnes, while upregulating the expression of epidermal differentiation-related genes CASP14 (up to 6.05 times that of the model group) and MUC1 (up to 1.54 times that of the model group), proving that its keratinocyte regulation function is completely independent of bacterial survival, colonization, or proliferation. The above multi-level experimental data systematically demonstrate that Ls.0403 has the advantage of selective strain screening, achieves multi-mechanism synergy of "antibacterial, antioxidant, host cell inflammation regulation, and epidermal barrier-related gene upregulation" at the functional level, and breaks through the limitations of traditional live bacteria preparations at the formulation level. It provides a complete experimental basis and sufficient technical support for the development of acne-related topical skin products with higher safety and more controllable quality.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of *Ligilactobacillus salivarius* Ls.0403, characterized in that, The strain was deposited on May 21, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 38819.

2. The *Lactobacillus salivarius* Ls. O403 according to claim 1, characterized in that, The Lactobacillus salivarius Ls.0403 was isolated from fecal samples from healthy adults.

3. Live bacteria prepared from Lactobacillus salivarius Ls.0403 as described in claim 1 or 2.

4. Cell-free culture supernatant prepared from Lactobacillus salivarius Ls.0403 as described in claim 1 or 2.

5. The pH-adjusted culture supernatant prepared from Lactobacillus salivarius Ls.0403 as described in claim 1 or 2.

6. The bacterial lysate prepared from Lactobacillus salivarius Ls.0403 as described in claim 1 or 2.

7. Heat-inactivated bacterial cells prepared from Lactobacillus salivarius Ls.0403 as described in claim 1 or 2.

8. A composition for improving acne-related skin conditions, characterized in that, The composition comprises at least one of the live bacteria of claim 3, the cell-free culture supernatant of claim 4, the pH-adjusted culture supernatant of claim 5, the bacterial lysate of claim 6, and the heat-inactivated bacteria of claim 7, as well as one or more skin-acceptable carriers or excipients.

9. The composition according to claim 8, characterized in that, The skin-acceptable carrier or excipient is selected from one or more of the following: sodium carboxymethyl cellulose, carbomer, hyaluronic acid, sodium hyaluronate, chitosan, polyvinyl alcohol, poloxamer, glycerin, propylene glycol, physiological saline, phosphate buffer, cream matrix, hydrogel matrix, or medical dressing matrix.

10. The use of Lactobacillus salivarius Ls.0403 of claim 1, the live bacteria of claim 3, the cell-free culture supernatant of claim 4, the pH-adjusted culture supernatant of claim 5, the bacterial lysate of claim 6, the heat-inactivated bacteria of claim 7, or the composition of claim 8 in the preparation of a product for improving acne-related skin conditions.

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