Introduction

The skin covers a surface area of approximately 1.5–2.0 m2 in an adult human. However, given its complexity, which includes nearly 5 million appendages, the total surface area increases significantly to approximately 25 m2, making it the largest organ in the body1,2,3. The skin microbiome plays a key role in maintaining skin health and homeostasis. Commensal microorganisms compete with potential pathogens for colonization sites and nutrients, inhibiting pathogen overgrowth4,5. In addition, the skin microbiome plays a role in controlling local immune response (innate immunity), influencing inflammation-related pathways and defense against microorganisms6,7. Additionally, recent studies focus on the importance of microbiome in skin cancer research and interactions between the immune system and skin microbiota in health and disease (including cancer)8,9,10,11.

An imbalance in the skin microbiome, known as dysbiosis, can cause a range of skin conditions6. Skin barrier dysfunction is linked to various dermatological issues, including acne vulgaris, atopic dermatitis, psoriasis, wound infections, and skin aging12,13,14,15,16,17.

In skin dysbiosis cases where microbiome diversity is disturbed, substances from pre- and probiotic agents can be used as modulators to restore microbial balance1.

Pro- and prebiotics have been studied for their ability to enhance skin barrier function and potentially alter the effects of aging. Probiotics introduce beneficial bacteria to the skin, thereby enhancing its integrity and boosting natural defenses. Prebiotics, in turn, feed these beneficial microorganisms, supporting a balanced microbiome that promotes overall skin health18. Together, these biotic components work together to form a synergistic skincare strategy designed to keep skin resilient, youthful, and moisturized with advanced bioactive compounds2.

Microbiome balance became particularly important during the coronavirus pandemic, where the requirement to wash and disinfect hands was one of the crucial elements of the sanitary regime, resulting in nondiscriminatory removal of pathogens and other skin microbes. This resulted in the increased recognition of topically applied probiotics to address this newly emerged need19,20 As probiotics are widely used in food and pharmaceutical industries to regulate gut microbiome and combat antibiotic resistance21,22,23, the acquired knowledge and manufacturing technology allows straightforward transfer and development of formulations addressing skin microbiome maintenance.

The use of probiotics in topical preparations can replenish skin microbiome affected by inflammation, in conditions, such as atopic dermatitis, and improve dry or sensitive skin24. Restoring skin bacterial flora balance may translate into reintegration of the skin barrier and a local reduction in inflammation associated with the multiplication of pathogenic bacteria.

To create a formulation containing live probiotic bacteria, a carrier must be used to enclose microorganisms. Selecting the right coating material is crucial for the stability of the obtained particles. This carrier can be alginate–tapioca microspheres, which are not only biocompatible but also biodegradable and non-toxic systems to deliver active substances25,26. Furthermore, the use of tapioca flour as an additional co-encapsulation agent increases the viability of the microorganisms encapsulated within the microspheres, while the hyaluronic acid coating seals their structure, as confirmed in our previous publication27. These studies focused primarily on optimizing the formulation’s composition and characterizing its physicochemical properties. In contrast, the present study focuses on confirming its biological activity, particularly its positive impact on the skin microbiome—a novel aspect not explored in previous research.

Therefore, the aim of the study was to test the antimicrobial activity of L. casei ATCC 393 against Staphylococcus epidermidis and Staphylococcus aureus encapsulate the probiotic strain in hyaluronic acid-coated alginate-prebiotic (tapioca flour) microspheres, and introduce them in a topical formulation. Further studies included verification of formulation biosafety and efficacy in relation to the skin microbiome. In vitro methods were used to assess the potential use of formulations.

Materials and methods

Materials

The study used microorganism strains obtained from the following three centers: The American Type Culture Collection (ATCC), the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), and the Polish Collection of Microorganisms (PCM). The strains included: A probiotic strain — Lactobacillus casei ATCC 393; symbiotic/commensal strains — Staphylococcus epidermidis ATCC 49,134, Micrococcus luteus DSM 1790, Staphylococcus capitis subsp. urealyticus DSM 6717, and Staphylococcus hominis DSM 20,329; and pathogenic strains — Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10,231, and Cryptococcus neoformans PCM 2999. During the study, de Man-Rogosa-Sharpe (MRS) broth and tryptic soy broth (TSB) were used for the propagation of L. casei and other bacteria, respectively. The bacterial stock cultures were prepared in glycerol (20%) and stored at − 70 °C. The tested strains of yeast-like fungi were grown and stored on Sabouraud dextrose agar slants at 4 °C and transferred to the fresh medium every 4–8 weeks. All culture media and survival-assessing media (TSB, tryptic soy agar (TSA), MRS broth and agar, Baird Parker agar (BPA), Sabouraud dextrose agar (S), MacConkey agar, FTO agar, Levine eosin-methylene blue agar, cetrimide agar, and Eugon LT 100 broth) as well as their ingredients were purchased from Biomaxima (Poland). Alginic acid sodium salt from brown algae and sodium citrate were purchased from Sigma Aldrich (Poland). Tapioca flour was bought from Green Essence (Poland). Calcium chloride was bought from Avantor Performance Materials Poland S.A. Caprylic/capric triglycerides, ECO-tween 80, and sodium hyaluronic acid (0.05–0.1 MDa) were kindly provided by Croda (Poland) and Alfa Sagittarius (Poland). Avocado oil was bought from Ecospa (Poland). Sodium levulinate, sodium anisate, and sodium benzoate were obtained from Evonik Industries AG. Xanthan gum was bought in Warchem (Poland). Emulsifiers (glyceryl stearate, polyglyceryl-6 palmitate/succinate, cetearyl alcohol, sorbitan stearate, and sucrose cocoate) were kindly supplied by Croda (Poland). Deionized water was used as a solvent.

Effect of L. casei on S. aureus and S. epidermidis

The activity of metabolites of the probiotic strain L. casei was assessed using pathogenic (S. aureus) and commensal (S. epidermidis) test microorganisms, which are the most common component of the human microbiome.

Time-kill test

Time-kill assay was conducted according to Prabhurajeshwar and Chandrakanth28 and Abdelmaksod et al.29 by co-culturing each of the tested bacteria (S. aureus and S. epidermidis) with L.casei. In brief, L. casei was cultured in the MRS broth, and S. aureus and S. epidermidis were cultured in the TSB broth at 37 °C for 18 h. The bacterial cells were collected by centrifugation (4000 rpm, 10 min at 4 °C), washed twice, and re-suspended in PBS to obtain appropriate concentrations. Test strain suspensions, with a density of 0.5 McFarland (∼ 1 × 108 CFU ml−1), were mixed together in 1:1 ratio, added to the MRS broth, and incubated at 37 °C for 5 and 24 h. The number of viable cells in suspensions (log CFU/ml) was assessed using serial dilutions on MRS agar for L. casei (37 °C, 48 h) and BPA for S. aureus and S. epidermidis (37 °C, 48 h). All tests were done in duplicates.

Well diffusion assay

L. casei activity against S. aureus and S. epidermidis was determined by agar-well diffusion method in accordance with the previously described methodology28 with slight modifications. The supernatants of 24 h L. casei culture, grown on the MRS broth at 37 °C, were assessed and prepared according to the procedure described by Chen et al.30 Exponential cultures of the test microorganisms (S. aureus, S. epidemidis) were diluted to a suitable turbidity and used to inoculate a melted and cooled TSA to a final concentration of ∼106–107 CFU/mL on plates. Then, 6 mm diameter wells were punctured in the agar medium (TSA) containing S. aureus and S. epidermidis, and 100 ul of L. casei cell-free supernatant were added to them.

The diameter of the growth inhibition zone was assessed after 24 h of incubation at 37 °C. When the zone of inhibition was larger than 1 mm, L. casei was found to possess activity against S. aureus and S. epidermidis31. All tests were done in duplicates.

Encapsulation of probiotics

Probiotic microspheres were prepared according to the modified methodology described by Łętocha et al.32 and in the patent PL 246,67433. Tapioca flour was used as the co-encapsulation material. After encapsulation, the microspheres were coated with hyaluronic acid according to the methodology of Łętocha et. al.27 The encapsulation process is shown in Fig. 1. Then, the probiotic microspheres were used as the active ingredient of the topical formulation.

Fig. 1
figure 1

Schematic description of encapsulation process.

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Preparation of probiotic formulation (Base formulation – E1 and formulation with microspheres – E2)

The emulsions were prepared according to the methodology described by Łętocha et al.34 Briefly, both phases (oil and water with emulsifiers) were heated to reach 70 °C and then combined and mixed in a mechanical mixer (IKA C-MAG HS 7) at 600 rpm for 15 min. In case of formulation E2, the probiotic microspheres were added after cooling to a temperature below 40 °C and mixed until the formulations reached a temperature of 25 °C. Table 1 shows the compositions of the base formulation and formulation containing microspheres. The E2 formulation is protected by a patent application (P. 445,990)35.

Table 1 Compositions of the topical formulations.
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Physicochemical properties and viability of probiotics in the formulations

The physicochemical properties of the formulations were assessed according to the methodology described by Łętocha et al.34 Briefly, the stability of the obtained emulsions was assessed by centrifugation test and thermal stability test. The pH values ​​were measured with a Mettler Toledo seven easy pH meter equipped with an Inlab 410 glass electrode. The average droplet size in the products was measured using an optical microscope (Motic) equipped with a digital camera and connected to digital image processing software (Motic Images Plus 2.0). The rheological study of the formulations were carried out using Brookfield model R/S plus equipped with the cone/plate (C-25–2) system in the shear rate range of 1–100 s−1 at 25 °C for 60 s. The assessment of the viability of L. casei in microspheres in the developed formulations was conducted according to the methodology described by Lasta et al.36 with slight modifications. The viability of probiotic bacteria was checked immediately after introducing the microspheres into the cosmetic formulation. Briefly, 1 g of formulations containing microspheres with an initial density of 10.50 log CFU/g was diluted in 9 mL of 0.2 mol·L-1 sterile sodium citrate solution (pH 6.0), followed by shaking at 2000 rpm for 5 min. Subsequently, 0.1 mL of serial dilutions were plated on MRS agar and incubated at 37 °C for 72 h. Viable cells were enumerated as the number of colonies obtained after 72 h incubation at 37 °C in aerobic conditions. All tests were performed in triplicates, maintaining the principles of sterility. The results are presented as the logarithm of colony-forming units per gram (log CFU g–1).

In vitro studies

Cell culture

For cell culture experiments two human skin-derived cell lines were selected: Human keratinocyte-like HaCaT – 300,493 (CLS Cell Lines Service GmbH) and human skin fibroblast HSF – CRL-2522 (ATCC). The cells were cultured in DMEM (ThermoFisher) supplemented with 10% FBS (Thermo Scientific), 100 U/ml penicillin (Thermo Scientific), and 100 ug/ml streptomycin (Thermo Scientific) at 37 °C/5% CO2. Cells were passaged twice a week using trypsin. Before each experiment, cells were reseeded into the media without antibiotics added (seeding density details are mentioned for each experiment).

Cell viability assay

A day prior to the experiment HaCaT and HSF cells were seeded into 96-well plates (50,000 and 30,000 cells/well, respectively). On the day of the experiment respective dilutions of formulation in DMEM were applied into the cell-containing wells and plate was incubated for 24 h under 37 °C/5% CO2. Subsequently medium was removed; cells were then washed with PBS and incubated for 20–40 min at 37 °C/5% CO2 with 0.5 mg/ml thiazolyl blue tetrazolium bromide (Sigma-Aldrich) diluted in DMEM (200 µl/well). Then, supernatant was removed, and precipitated formazan crystals were dissolved in 120 µl of 100% isopropanol/5 mM hydrochloric acid (POCH, Poland). The samples were transferred to a new, transparent 96-well plate (90 µl from each well) and absorbance at 570 nm was measured using Hidex Sense microplate spectrophotometer (Hidex). The decrease or increase in absorbance in comparison to the DMEM-only treated control sample was interpreted as a decrease/increase in cell viability.

Proliferation

A day before the experiment HaCaT and HSF cells were seeded in 96-well plates at 5,000 cells/well or 10,000 cells/well, respectively. On the day of the experiment respective dilutions of formulation in DMEM with 2% FBS were applied on the cells. After 48 h samples were analyzed similarly as for the viability assay described above, with HaCaT and HSF incubated with thiazolyl blue tetrazolium bromide (Sigma-Aldrich) solution for 20 min and 1 h, respectively. Absorbance of the 100% isopropanol/5 mM HCl-dissolved formazan crystals was measured at 570 nm in a new, transparent 96-well plate using Hidex Sense microplate reader (Hidex). Results were compared to DMEM-only treated control and increase/decrease in absorbance was interpreted as enhancement or decrease of cell number, respectively.

Effect of formulation on cytokine release

To prepare the cell-conditioned media, cells were seeded into the 48-well plates a day prior to the experiment (at a density of 100,000 cells per well (HaCaT ) and 50,000 cells per well (HSF cells). After overnight incubation at 37 °C/5% CO2, confluent cultures were stimulated with different formulation concentrations prepared in DMEM. Subsequently, cell media were collected after 24 h and stored in -20 °C for further analysis.

ELISA

The levels of IL-6 and IL-8 released into the cell culture media were measured using commercial ELISA kits (R&D) according to the manufacturer’s instructions. Briefly, a half-area, high binding, 96-well plates (Greiner) were coated with appropriate coating antibodies diluted (1:120) in PBS and left overnight at room temperature (20 ± 2 °C). Following day, wells were washed with washing buffer (0.05% Tween in PBS) and blocked for 1 h at room temperature (20 ± 2 °C) with 150 µl of 1% BSA diluted in PBS. Again, the wells were washed and 50 µl of samples were added to the individual wells and incubated further at room temperature (20 ± 2 °C) for 2 h. Subsequently wells were again washed and incubated with biotin-conjugated primary antibodies (1:60) for 2 h. Upon penultimate wash, the wells were incubated with streptavidin-HRP (1:40) for 30 min and finally washed again (5x) and developed. TMB substrate solution (50 µl of TMB substrate reagent set, BD) was added to each well and incubated for up to 5 min, before the reaction was stopped with 25 µl of 2N H2SO4 (POCH). Then sample absorbance was measured at 450 nm and 570 nm (wavelength correction for plate imperfection) using Hidex Sense microplate reader (Hidex) and recalculated using the prepared calibration curve.

Scratch assay

For the wound healing assay, inserts (Ibidi) were placed in a 12-well plate, prior to HaCaT cells (600,000 cells/well) being seeded around and in the chambers of insert in DMEM containing 10% FBS. Cells were grown overnight under 37 °C/5% CO2 incubation, the inserts were removed, forming the cell-free wound area. The medium was removed and cells were stimulated with formulations at concentrations of 0.5% and 2% (diluted in DMEM containing 2% FBS) for 15 h. The cell migration/growth into the scratch was followed using a cell imaging system (at 4 × magnification) (EVOS FL, Thermo Scientific) and images at time 0 and after 15 h were collected. Images were analyzed with TScratch software (19,450,233, CSE Lab, ETH Zurich).

Effect of a topical formulation containing L. casei on skin microbiome

This experiment was carried out according to the methodology proposed by Jałosińska37 with modifications. For this purpose, 5 g portions of the formulation (E2) containing encapsulated. L. casei microspheres were inoculated with the suspensions of the reference strains listed in the Materials section and mixed thoroughly. The initial density of the inoculation suspensions was 1 × 106–1 × 109 CFU/ml and was checked by deep inoculation on TSA or Sabouraud dextrose agar for bacteria and yeast-like fungi, respectively. Then, 1 g of formulation was weighed under aseptic conditions and added to 9 ml of sterile sodium citrate solution (2 mol∙L-1, pH = 6) to break the alginate capsules with probiotic bacteria. After a series of dilutions, viability of the test microorganisms introduced into the topical formulation was determined by the deep plate method using the following selective media: Baird Parker agar (S. aureus, S. epidermidis, S. capitis subsp. urealyticus, and S. hominis), Sabouraud dextrose agar (C. albicans and C. neoformans), MacConkey agar (E. coli), FTO agar (M. luteus). Incubation was carried out under the following conditions: Bacteria: 37ºC, 48 h and yeast-like fungi: 28ºC, 48 h. The viability of the test microorganisms in the formulation with probiotics were determined immediately after introducing the microbial suspensions into the formulation as well as after incubation at 37 °C for 2 and 24 h. A similar procedure was used for the topical formulation lacking probiotic microcapsules (E1). The experiment was repeated twice. The viability determinations are presented as the log number of CFU in 1 g of given formulation. These values were used to calculate the average increase or decrease in reference microorganisms introduced in topical formulations after a specified time.

Statistical analysis

The results were analyzed with GraphPad Prism (GraphPad Prism, ver. 10.0.2, GraphPad Software LLC., Boston, MA, USA) software and are presented as mean ± SD. Statistical significance was evaluated using built-in one-way ANOVA. The results were considered statistically significant if p value ≤ 0.05. *: p = 0.05–0.011; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001.

Results

Effect of L. casei on S. aureus and S. epidermidis

Time kill test

The effect of L. casei—a probiotic strain—on the component species of the human skin microbiome, i.e. S. epidermidis—a symbiotic strain and S. aureus—a pathogenic strain was evaluated (Table 2). Staphylococcus viability was assessed on the selective agar medium—BPA, and lactic acid bacteria viability was determined on the selective agar medium—MRS.

Table 2 The effect of the L. casei strain on S. aureus and S. epidermidis strains that are part of the skin microbiome.
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The data presented in Table 2 shows that the presence of L. casei in the culture medium resulted in a reduction in S. aureus strain cells by 0.81 and 0.99 log after 5 and 24 h, respectively (Mixture 1, BPA). S. epidermidis strain count increased by 0.13 and 0.59 log after 5 and 24 h (Mixture 1, BPA). Additionally, it was found that incubating L. casei with S. aureus and S. epidermidis for 5 h and 24 h did not adversely affect the probiotic strain. When L. casei was incubated with S. aureus, an increase in probiotic cell concentration was found in a range of 0.94–3.36 log (Mixture 2, MRS medium). when L. casei was incubated with S. epidermidis, probiotic cell concentration increase ranged from 0.94 to 2.21 log (Mixture 2, MRS medium). Taken together, L. casei metabolites were found to reduce S. aureus growth without practically affecting the growth of S. epidermidis. Contrarily, neither S. epidermidis nor S. aureus limited the growth of the probiotic bacteria.

Well diffusion assay

L. casei inoculation into agar wells induced the formation of radial inhibition zones, limiting the growth of S. aureus. The zones exceeded 5 mm in diameter (Fig. 2). Simultaneously, in accordance with time kill findings, no growth inhibition zones were observed on S. epidermidis plates. This indicates that L. casei has no negative impact on the growth of this strain in vitro. As S. epidermidis is a commensal species that play an important role in maintaining human skin microbiome composition quantitatively and qualitatively, the L. casei formulation would be beneficial in this regard.

Fig. 2
figure 2

Effect of L. casei on human skin isolates—S. aureus (a) and S. epidermidis (b).

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Physicochemical properties and viability of probiotics in topical formulations

The thermal and centrifugal stability of the obtained emulsions were assessed. Additionally, the average droplet size and emulsion pH were evaluated, followed by rheological analysis. The physicochemical properties of the base formulation—E1 and the formulation containing probiotic microspheres—E2 are listed in Table 3. The analyzed emulsions were found to be stable (the stability of both products is marked as “ + ”), and were characterized at pH 5.8. The presence of probiotic microspheres resulted in 10% decrease in the mean droplet size and marginal reduction in measured viscosity. The viability of L. casei bacteria within the microspheres following incorporation into the formulation was measured at 7.80 log CFU/g, indicating a reduction of 2.70 log compared to the initial bacterial load within the microspheres.

Table 3 Physicochemical properties and viability of probiotics in prepared emulsions.
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These results confirm that stable O/W emulsions were obtained, with a uniform, smooth consistency and a pH value corresponding to the physiological pH of the skin, and containing viable probiotic bacteria.

In vitro studies

To examine the cytotoxicity of the prepared topical formulations, cellular models having two skin cells types—keratinocytes and fibroblasts—were implemented. Immortalized normal human keratinocyte-like (HaCaT) (Fig. 3) and normal human fibroblast (CRL-2522) (Fig. 4) cells were selected for the study. These lines are popular models to assess cytotoxicity of materials used as topical formulations. To verify the effect of the prepared emulsion on the viability of these cells, highly confluent cultures (~ 90%) were incubated with serial dilutions of each preparation for 24 h, and the results were evaluated by MTT cell viability assay.

Fig. 3
figure 3

HaCaT cell viability test (MTT) in the (A) base formulation and (B) containing probiotic microspheres. Results were calculated as the percentage of control cells incubated in the presence of DMEM alone and presented as mean ± SEM. *—p = 0.05–0.011; ** — p ≤ 0.01; ***—p ≤ 0.001; ****—p ≤ 0.0001. The green bar represents the unstimulated control.

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Fig. 4
figure 4

HSF cell viability test (MTT) in the (A) base formulation and (B) containing probiotic microspheres. Results were calculated as the percentage of control cells incubated in the presence of DMEM alone and presented as mean ± SEM. *—p = 0.05–0.011; ** — p ≤ 0.01; ***—p ≤ 0.001; ****—p ≤ 0.0001. The green bar represents the unstimulated control.

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Results indicate no negative effect of the formulation on HaCaT cell viability up to a concentration of 2.5%. The 5% concentration resulted in a ~ 25% decrease in cell viability (IC25%), and an IC50% was achieved at concentrations of approximately 10% for both formulations. It should be noted that the base formulation and probiotic microsphere-containing emulsion exhibited similar effects, indicating no negative effect of microspheres on cell viability.

No negative effect of the formulation on fibroblasts was observed up to a concentration of 5%, and IC50% was achieved at approximately 10% concentration for both formulations. Moreover, for 1.25–5% concentrations, an increase in HSF cells viability was observed.

The effect of both formulations on HaCaT (Fig. 5) and HSF (Fig. 6) cell (cultured at low-confluency in a medium containing 2% FBS) proliferation was determined. Cell viability was determined 48 h after stimulation using MTT assay.

Fig. 5
figure 5

Effect on the proliferation of HaCaT cells (MTT) in the (A) base preparation and (B) the preparation containing probiotic microspheres. Results were calculated as the percentage of control cells incubated in the presence of DMEM + FBS and presented as mean ± SEM. *—p = 0.05–0.011; ** — p ≤ 0.01; ***—p ≤ 0.001; ****—p ≤ 0.0001. The green bar indicates the unstimulated control.

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Fig. 6
figure 6

Effect of (A) base preparation and (B) preparation containing probiotic microspheres on HSF cell proliferation (MTT). Results were calculated in comparison to control cells incubated in DMEM + FBS medium and presented as mean ± SEM. *: p = 0.05–0.011; **: — p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. The green bar indicates unstimulated control.

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HaCaT cells stimulated with formulation concentrations above 0.08–0.16% (Fig. 5) exhibited decreased proliferation. However, at lower concentrations of the stimulants, a minor induction in proliferation was observed for both formulations.

A similar relationship was observed in HSF cells. Here, a slight induction in proliferation was observed up to 0.08% of the basic formulation and up to 0.16% of the formulation containing encapsulated probiotic microspheres. Human fibroblast proliferation was inhibited at higher concentrations, as basic formulation above 0.16% and probiotic formulation above 0.31% reduced the observed cell viability.

The formulation containing encapsulated probiotic microspheres was intended for topical application. Thus, the encapsulated ingredient was intended to affect the outer layer of the epidermis, lacking proliferating cells, while retaining the positive effect of the formulation on the skin microbiome. Formulation concentrations that were considered non-cytotoxic were selected for further in vitro testing, including cytokine release profile as measured by ELISA and wound healing effect. Both formulations stimulated the release of pro-inflammatory cytokines (IL-6 and IL-8) in HaCaT and HSF cells (Fig. 7A and 7B, respectively).

Fig. 7
figure 7

Cytokine production in (A) HaCaT and (B) HSF cells stimulated with base formulations and those containing encapsulated probiotic microspheres for 24 h. IL-6 and IL-8 cytokine levels in cell media after stimulation with 0.5% of the two formulations. Graphs represent data from three independent experiments, with triplicates in each. Statistical significance was estimated using GraphPad Prism using built-in ANOVA test (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

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In HaCaT-conditioned media treated with 0.5% formulation, IL-6 and IL-8 levels were ~ 50% higher than in unstimulated cells. In case of fibroblast cells stimulation with formulations resulted in less than 40% increase in IL-6 release similar for both empty formulation and one containing microspheres. Moreover, IL-8 levels increased but stimulation with formulation containing encapsulated probiotic microspheres had a less pronounced effect compared to the base formulation, suggesting anti-inflammatory effect of L. casei. The increased levels of proinflammatory cytokines upon stimulation with tested formulations indicate their weak irritating nature. It must be emphasized, however, that the observed IL-6 and IL-8 levels were modest even post stimulation and were far from a typical response which those types of cells might produce under proinflammatory stimuli.

Although the formulations are ultimately intended for use on healthy, intact skin, we decided to exclude any potential negative effects on the regeneration of micro-injuries within the superficial skin layer, specifically involving the keratinocyte layer. Base formulation and formulation containing encapsulated probiotic microspheres exhibited regenerative properties in wound healing assay (Fig. 8).

Fig. 8
figure 8

(A) Impact of base formulation and formulation containing encapsulated probiotic microspheres on wound healing in HaCaT cell scratch assay model. After ibidi insert removal, confluent HaCaT cells were stimulated with 0.5 and 2% of E1 and E2 formulations. Wound closure was observed using EVOS FL microscope under 4X objective lens after 15 h of stimulation. (B) Control at time "0", (C) Control after 15 h, (D) E1 at a concentration of 0.5%, (E) E1 at a concentration of 2.0%, (F) E2 at a concentration of 0.5% and (G) E2 at a concentration of 2.0%. Images were analyzed with TScratch software. The wound area closure in each respective image was measured and presented as the percentage of the unstimulated control at incubation time point 0 h.

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As shown in Fig. 8, the open wound area was approximately 30% after 15 h of incubation in unstimulated control. However, wound closure reached approximately 90% in the cells incubated with 0.5% base formulation (E1) for 15 h. Simultaneously, a virtually complete wound closure was observed in the cells incubated with 0.5% formulation containing encapsulated probiotic microspheres (E2). Cells incubated with 2% concentration of both formulations exhibited similar wound closure as that in the control. Thus, higher concentrations of both formulations may limit cell proliferation, effectively limiting the wound healing response. Thus, the formulation containing encapsulated probiotic microspheres have a potential of being a preparation with pro-regenerative properties.

Effect of topical formulation containing L. casei on skin microbiome

The impact of the formulation containing encapsulated L. casei (E2) microspheres on seven commensal microorganisms of the natural skin microbiome and pathogens was assessed. A formulation without a probiotic strain was used as a control (E1) (Table 4).

Table 4 Assessment of the impact of a topical formulation on reference microorganisms occurring in the natural microbiome of human skin.
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For this purpose, the formulations (E2 and E1) were inoculated with one of the selected strains, and their growth was assessed after 2 and 24 h of incubation. The growth of skin commensals (S. epidermidis, M. luteus, S. hominis, and S. capitis subsp. urealyticus) moderately increased after 2 h (0.16–0.50 log) and 24 h incubation (0.24–0.59 log) with E2 formulation. The effect, however, was similar to the one observed in the control E1 formulation (0.06–0.35 log after 2 h and 0.42–-0.73 log after 24 h), suggesting that the presence of L. casei microspheres in the E2 formulation did not affect the growth of these commensal strains. In contrast, the growth of pathogenic strains (E. coli, S. aureus, C. albicans, and C. neoformans) in the E2 formulation was inhibited to some degree after 2 h (-0.20–1.38 log) and 24 h (-0.30–2.82 log) compared to the control E1 formulation (-0.06–0.5 log and -0.16–1.25 log after 5 and 24 h, respectively). Among the tested pathogenic strains, C. neoformans (-2.82 log reduction after 24 h) and S. aureus (-1.60 log reduction after. 24 h) had the highest sensitivity to the E2 formulation, whereas C. albicans growth was affected to a minor degree (-0.30 log reduction after 24 h).

Disscusion

Human skin microbiota plays an important role in maintaining skin health and potentially preventing premature aging “Human skin is inhabited mainly by four types of bacteria: Actinobacteria (52%), Firmicutes (24%), Proteobacteria (16%), and Bacteroidetes (6%)”3,38. It is estimated that 50% of the bacteria living on the skin are S. epidermidis, which inhabits the upper areas of the hair follicle openings. In addition, S. saprophyticus, S. hominis, S. warneri, S. haemolyticus, and S. capitis live on the skin surface together with Corynebacterium (C. jeikeium), Propionibacterium (P. acnes), and Micrococcus (M. luteus M. varians, M. lylae, M. sedentarius, M. roseus, M. kristinae, and M. nishinomiyaensis)39. The above-mentioned bacteria are commensal and play an important role in maintaining skin microbiome composition quantitatively and qualitatively, as they condition the growth of other microorganisms40,41. Further, pathogenic bacteria, such as S. aureus, Group A streptococci (Streptococcus pyogenes), aerobic corynebacteria (Corynebacterium spp.), and gram-negative rods (P. aeruginosa), may be present on the skin. These bacteria constitute undesirable flora that cause skin infections in the event of disturbances in physicochemical conditions, in states of immune deficiency, antibiotic treatment, skin disruption, or in the presence of foreign bodies. The dominant fungal species inhabiting the skin belong to the genus Malassezia, which constitute 1–22% of the human microbiome. The genus Malassezia includes 14 species, 9 of which can colonize human skin: “M. furfur, M. sympodialis, M. globosa, M. restricta, M. slooffiae, M. yamatoensis, M. obtusa, M. dermatis, and M. japonica.”12,42,43 In addition, fungi from genera Penicillum (P. chrysogenum and P. lanosum), Aspergillus (A. candidus, A. terreus, and A. versicolor), Alternaria, Candida (C. tropicalis, C. parapsilosis, C. orthopsilosis), Chaetomium, Chrysosporium, Cladosporium, Mucor, Debaryomyces, Cryptococcus (C. flavus, C. dimmennae, and C. diffluent), Trichophyton, and Rhodotorula are present42. Further, yeast-like fungi from the genera Candida and Cryptococcus as well as dermatophytes from the genera Microsporum, Epidermophyton, and Trichophyton can colonize the skin and under favorable development conditions, they can cause candidiasis, cryptococcosis, and dermatophilosis, respectively42,44. Moreover, the skin is inhabited by viruses, mainly those containing double-stranded DNA, polyomaviruses—Polyomaviridae, and papillomaviruses—Papillomaviridae, and mites, such as Demodex45,46,47,48.

The use of probiotics for therapeutic purposes on the skin is an attractive idea as it may constitute a convenient and effective treatment for some inflammatory skin diseases and dry or sensitive skin. Probiotics produce a broad variety of antimicrobial metabolites, including organic acids, diacetyl, acetoin, hydrogen peroxide, and bacteriocins49. These activities maintains microbial balance by controlling the growth of other microorganisms, most importantly, inhibition of the growth of pathogenic bacteria48,50,51. Probiotics help maintain the balance of microbiome in a stable ecological environment and boost the population of beneficial bacteria by stimulating the growth of native microbial communities52. In addition, probiotics may regulate skin balance by competitive exclusion, which is based on a natural competition and ecological spaces. This mechanism promotes the growth of beneficial bacteria while limiting the colonization of harmful bacteria53. Probiotics are defined as “live microorganisms that when administered in adequate amounts confer a health benefit to the host”54. The species of lactic acid bacteria (Lactococcus, Lactobacillus, Streptococcus, and Enterococcus) and Bifidobacterium55,56 are among the best known and widespread probiotics.

The genus Lactobacillus is considered the oldest documented probiotic. It includes gram-positive bacteria from the LAB group. These rod-shaped bacteria comprise about 183 known species (including L. casei used in this study) and are commonly applied in different industrial food processes57. LAB group microorganisms may process different carbon sources, including simple carbohydrates, to produce lactic acid58. In addition, they release bacteriocins and secondary metabolites (e.g., bacteriocins, exopolysaccharides, and enzymes) that inhibit the growth of other microorganisms, maintaining the commensal composition of the microbiome. All these factors contribute to the antimicrobial mode of action of probiotics59,60. There is an increasing number of studies on the use of probiotics but most of them focus only on assessing their impact on intestinal microbiome23,61.

The literature available on probiotics and pathogenic and commensal microorganisms residing directly on human skin is still limited. Similarly, the information on the application of probiotics as a treatment to alleviate skin diseases is limited. Therefore, our study aimed to assess the effect of L. casei and its metabolites on the growth of strains representing commensal and pathogenic microorganisms, respectively, that are part of the human skin microbiome i.e. S. epidermidis and S. aureus.

The activity of probiotic strains in relation to other microorganisms was assessed using "time-kill" and “well diffusion” tests62. The presence of L. casei and its metabolites in the culture medium limited the growth of S. aureus but did not affect S. epidermidis. Our findings regarding the effect of L. casei on S. aureus are consistent with the findings of Koohestani et al.,63 who demonstrated the antibacterial properties and potential of L. casei cell-free supernatant in removing biofilm from polystyrene and glass surfaces. Saidi et al.64 indicated not only the activity of cell-free supernatants of the L. casei conditioned medium but also antimicrobial effect of their acetate extracts against S. aureus, for which the minimal inhibitory concentration (MIC) and minimal killing concentration values were determined at 4 mg/ml and 8 mg/ml, respectively. Moreover, it was found that acetate extracts from the L. casei supernatants at sub-MIC concentration significantly reduced the surface hydrophobicity of the tested materials and consequently limited biofilm formation. Further, the activity of Lactobacillus metabolites against Staphylococcus was evidenced by Mohamed et al.,65 who showed that cell-free supernatants of L. acidophilus, L. plantarum, L. helveticus, and L. rhamnosus exhibited activity against various strains of S. aureus and S. epidermidis isolated from the conjunctiva of inflamed eyes. Besides, it was found that isolates identified as S. aureus were more sensitive to the inhibitory effect of CFSM probiotics than S. epidermidis isolates. Our studies align with the described activity of L. casei metabolites against S. aureus; however, they do not indicate the L. casei activity against S. epidermidis. This may be due to the production of various metabolites, such as bacteriocins, by lactic acid bacteria, including those of the genus Lactobacillus. These metabolites differ not only in molecular weight, genetic origin, and biochemical properties but also in activity and specific spectrum of action towards individual microorganisms66.

Li et al.67 indicated that different Lactobacillus strains represented by L. rhamnosus, L. reuteri, L. plantarum Lactobacillus jensenii and L. casei can reduce the level of production proinflammatory mediators, such as interleukins – IL-6 and IL-1β, tumor necrosis factor-α – TNF-α, and others in the inflamed tissues of mice with symptoms of colitis. Lee et al.68 reported that L. casei can significantly reduce TNF-α and IL-6 levels. Ayyanna et al.69 showed a decrease in the expression of proinflammatory cytokine genes in the paw tissues of rats treated with probiotics L. mucosae AN1 and L. fermentum SNR1.

In a study by Khmaladze et al.,70 the effect of the probiotic strain L, reuteri DSM 17,938 in skin topical applications was investigated, using two models: First – ex vivo focusing on anti-inflammatory and skin barrier effects and second – in vitro focusing on antimicrobial activity. The studies showed that the use of both live bacteria and L. reuteri DSM 17,938 lysate reduced the pro-inflammatory effects of IL-6 and IL-8. In addition, the live form of L. reuteri DSM 17,938 showed antibacterial activity against pathogenic skin microorganisms (S. aureus, S. pyogenes M1, C. acnes AS12, and P. aeruginosa), while the lysate did not show such activity. Research indicate that L. reuteri DSM 17,938 may be beneficial for overall skin health, improving barrier function, or treating unhealthy, inflammation-prone skin due to its antibacterial, anti-inflammatory, and skin barrier-enhancing effects, but the mechanisms of such action were not explained. In view of the above results, the next stage of the research included in vitro studies of formulations differing in the absence (E1) and presence (E2) of L. casei microspheres. The studies conducted using keratinocytes and skin fibroblasts indicate similar, relatively low cytotoxicity of both formulations, regardless of the L. casei presence. In the cytotoxicity analysis, the IC50% determined for both cell lines was about 10%, indicating good tolerance of the tested preparations, as other topical formulations, e.g. in the form of cream or serum, reach 50% cytotoxicity at much lower concentrations71. However, it should be noted that the studies include an incubation time of 24 h. If the formulation is used as a topical preparation, the contact time with the skin will be much shorter (maximum upto several hours). Despite the minor stimulatory effect on pro-inflammatory cytokine production, the formulation may be considered safe. The observed levels of IL-6 and IL-8 in HaCaT media were modest and far from maximal capacity of cell production and typical amounts observed upon stimulation with natural and artificial proinflammatory stimulators, such as LPS and others, which stimulate the release of ng/ml concentrations of these cytokines under similar experimental conditions72. In HSF cells effect of formulations was even less pronounced and did not exceed the release of 100 pg/ml of both IL-6 and IL-8, even as fibroblasts are considered more responsive cells in case of cytokine production with levels easily reaching 8 ng/ml73. Additionally, moderate levels of IL-8 and CXCL-1 are observed in wound healing and are considered beneficial for this process as indicated by Albuquerque-Souza et al.74, where L. casei showed positive effect on re-epithelization of the oral epithelium infected by Porphyromonas gingivalis. Moreover, the formulations are designed for topical usage on fully grown skin, which differs significantly from single-layer in vitro cell cultures in terms that it is characterized by stratified 3-dimensional organization, where the upper layer of keratinized epidermis limits the penetration of applied ingredients into the skin interior. Considering all of the above, although the inflammatory stimulation by our formulations is unlikely, to fully exclude a minimal pro-inflammatory or irritant effect, our formulation require dermatological testing, as expected for any other similar product. In cell proliferation assay, both E1 and E2 formulations showed a minor pro-proliferative effect on both cell lines upon stimulation with very low concentration of E1/E2 (HaCaT up to 0.04% and HSF up to 0.16%). This L. casei-independent effect might be attributed to the use of avocado oil in the formulation, which has a confirmed proliferative effect. Avocado oil (Persea americana) is rich in nourishing waxes, proteins, and vitamins (A, D, E) as well as minerals, and is a great source of enrichment, especially for dry, damaged or chapped skin75. Wang et al.76 investigated the effect of natural avocado and olive oils on HaCaT cell proliferation and differentiation and concluded that certain concentrations of avocado and olive oils (3%) can promote HaCaT cell proliferation and differentiation; avocado oil has a greater ability to accelerate HaCaT cell growth and multiplication, and olive oil improves HaCaT cell differentiation.

“Scratch assay” indicate that the microsphere formulation may exhibit the potential of being a preparation with regenerative properties. Our formulations applied at the optimized concentration (0.5%) enhanced observed wound healing in this simplified model. Moreover, a tendency to improve effect in presence of L. casei was noticed, which might be at least partially attributed to postbiotic effect of L. casei metabolites diffusing from the microspheres. L. casei metabolites, such as lactic acid, acetic acid, citric acid, and succinic acid, have previously been implicated in wound healing studies and have been suggested to be responsible for reducing P. aeruginosa adhesion77. The use of probiotics in the topical preparations may be therefore an alternative/supplemental to the care of skin affected by inflammation, including atopic dermatitis, dry, or sensitive skin. Atopic dermatitis is associated with S. aureus growth, which stimulates the production of kallikreins, leading to pro-inflammatory stimulation of the skin78. Therefore, a formulation containing probiotic microspheres may be particularly effective in limiting S. aureus growth. Restoring skin bacterial flora balance may translate into the restitution of the proper skin barrier and reduction in inflammation associated with the disruption of skin integrity and multiplication of pathogenic bacteria. However, it should be noted that the relatively simple scratch assay model used in our study, which involves only a single type of skin cells, constitutes a significant limitation. The wound healing process is complex and involves not only keratinocytes but also fibroblasts and immune cells, which interact through paracrine signaling. To better investigate the potential effects of the tested formulations on wound healing, more advanced in vitro models or preferably in vivo systems should be employed.

Further, our research included an assessment of the impact of the E2 topical formulation containing encapsulated L. casei microspheres on the microorganisms included in the skin microbiome. In addition to S. epidermidis and S. aureus, the effect of L. casei on the growth of natural microbiota and pathogenic strains, namely, M. luteus, C. neoformans, C. albicans, E. coli, S. capitis urealyticus, and S. hominis, was evaluated.

It was shown that in the case of skin commensals, such as S. epidermidis, M. luteus, S. hominis, and S. capitis subsp. urealyticus, 2-h and 24-h contact with E2 preparation induced a minor increase in the identified numbers of tested strains and the effect did not exceed 0.59 log. Contrarily, 2-h and 24 h contact of E2 formulation with pathogenic strains, namely, E. coli, S. aureus, C. albicans, and C. neoformans, resulted in the growth retardation reaching -2.82 log.

Currently, there is no information on the use of topical formulations containing encapsulated bacteria of the Lactobacillus genus to combat pathogenic bacteria and maintain skin microbiome homeostasis in the available literature. There are reports, however, describing several studies using in vitro and in vivo models, which demonstrate that the mere consumption of probiotics and their topical application leads to a dramatic improvement in the health of both intestinal mucosa79 and skin, indicating health benefits resulting from their use70,80. For example, Kim et al.80 showed that oral administration of live or heat-inactivated Lactobacillus sakei probio 65 improved the skin condition and reduced the frequency of scratching, contributing to the alleviation of inflammation and skin lesions, which mimic atopic dermatitis and may constitute a new, effective anti-inflammatory agent. Nonetheless, these research models used the bacterial culture and conditioned medium and not encapsulated formulations.

Other studies have reported the use of microencapsulated L. reuteri probiotics in a binary matrix consisting of inulin and maltodextrin to inhibit the growth of Listeria monocytogenes, a bacterium responsible for food poisoning81, and Erginkaya et al.82 confirmed the activity of L. rhamnosus encapsulated in inulin-coated microcapsules against clinical isolates of vancomycin-resistant Enterococcus faecalis (VREF).

Conclusions

The aim of the study was to design a new probiotic formulation based on the L. casei ATCC 393 strain, for which the inhibitory effect against S. aureus was determined in time-kill and well-diffusion assay studies. The probiotic strain was encapsulated in previously optimized alginate-prebiotic microspheres (tapioca flour) and coated with hyaluronic acid and then introduced into a topical formulation. In vitro studies show that the formulation containing microspheres may have potential of being a preparation with pro-regenerative properties. The presented data confirm the safety of the developed topical formulation and indicate their antibacterial and potential normalizing effects on the skin microbiome. This is, therefore, the first report in our knowledge, demonstrating the use of encapsulated bacterial microspheres and their introduction into a formulation for local application on the skin.