Clinical effect of Pediococcus acidilactici PMC48 on hyperpigmented skin

The excessive production and accumulation of melanin in the epidermal skin layer can result in skin hyperpigmentation and darkening. Current technologies for regulating melanin are based on inhibiting melanin biosynthesis. They have low effectiveness and safety issues.


| INTRODUC TI ON
As an irregular light-absorbing polymer, melanin contains indoles and other intermediate products derived from the oxidation of tyrosine. 1e chemical pigment melanin is found in both plant and animal kingdoms. 2It is primarily produced by the oxidation of L-tyrosine in the presence of tyrosinase enzyme. 3Melanin is the primary pigment found in vertebrates' surface structures. 1 It remains active only in specialized cells such as retinal pigment epithelium and melanocytes. 4Melanin has a great variety of functions.For instance, it can work as an energy transducer, attach to various drugs and chemicals, and affect cellular integrity. 5It also has an antiviral activity in vitro against the human immunodeficiency virus. 6Spatial distribution and concentration of chromophore melanin are also exclusively responsible for human skin coloration. 7in pigmentation has traditionally been considered the most critical photoprotective factor since melanin can act as an ultraviolet absorber, an antioxidant, and a radical scavenger. 8Melanin in moderate amounts has beneficial effects on the body.However, excessive amounts of melanin can lead to pigmentation disorders, including melasma, age spots, freckles, lentigo, naevus, chloasma, and melanoma. 9An excessive amount of melanin in the skin is defined as hyperpigmentation or melanoderma. 10It is a common dermatological problem that significantly affects a patient's appearance and quality of life. 11Therefore, regulating melanogenesis is a promising therapeutic strategy to treat abnormally pigmented skin. 12e chemical-based skin-whitening substances, including hydroquinone and derivatives, phenolic amines, biphenyl, chalcone agents, and mercury compounds, have been used in cosmetic formulations for a long time. 13But these substances have multiple adverse effects on consumers, 14 and natural melanin inhibitors are attracting masses of people. 15Although, in recent years, various tyrosinase inhibitor natural compounds (kojic acid, arbutin, azelaic acid, resveratrol, glycolic acid, and epigallocatechin gallate) have been reported that interfere with novel melanin synthesis in melanocytes. 9There is a need for an alternative drug to solve skin-related problems effectively.In this context, probiotics can be a potential option, as their beneficial effects on the skin have already been demonstrated. 16obiotics are beneficial microorganisms when administered in adequate amounts to a host. 17Probiotics work through various mechanisms, including enhancing the host's immunity, helping digest foods, and absorbing or lowering cholesterol levels. 18Also, probiotics are effective against cancer, hypertension, anxiety, depression, and obesity, 15 and some probiotics treat skin diseases such as acne, rosacea, psoriasis, and atopic dermatitis. 19Studies have demonstrated the applications of some probiotic strains, such as Lactobacillus helveticus, Bacillus subtilis, and Leuconostoc mesenteroides, as skin-whitening agents by inhibiting melanin synthesis. 20e melanin-decomposing probiotics have substantial value as therapeutic agents against hypermelanosis due to their unique mechanism of action, which differs from existing melanin synthesis inhibitors.Previously, we showed that Pediococcus acidilactici PMC48 isolated from sesame leaf kimchi has an excellent direct melanin-degrading effect in agar well diffusion and tube broth assays. 9It also has a tyrosinase-inhibiting effect based on L-DOPA (L-3,4-dihydroxyphenylalanine).Furthermore, Pediococcus acidilactici PMC48 showed a significant whitening effect on melanocytes (B16F10 cells).
These data led us to hypothesize that PMC48 might possess a clinical effect on hyperpigmented skin.Thus, this study aimed to investigate the skin-whitening effect of this strain by arranging an 8-week clinical trial with 32 participants.First, the PMC48 formulation was tested for stability and microbial safety.Then, it was applied to the artificially UV-induced tanned skin of the participants.
Before conducting this study, the irritation potential of PMC48 was assessed by a skin patch test.Visual evaluation, skin brightness, melanin index, and moisture content were analyzed during the clinical trial.This study aimed to evaluate the potential role of P. acidilactici PMC48 as a probiotic strain in medicines and cosmetics for skin treatment.

| P. acidilactici PMC48 strain
Pediococcus acidilactici PMC48 strain isolation and identification were previously reported by our team. 9In this study, the growth conditions of PMC48 were standardized through optimization processes to get the uniform quality of the PMC48 supernatant.Briefly, the stocked PMC48 strain was cultured in MRS broth (BD Difco, USA) and incubated at 37°C for 18 h.Bacterial growth (OD 600 nm ) was determined using a spectrophotometer (DR 1900, HACH, USA), and the culture was centrifuged at 4000 rpm for 30 min.After that, the supernatant was collected, filtered using a 0.22μm membrane filter (Millipore, USA), and finally stored at −80°C until the subsequent use.

| Preparation of a test skin toner using PMC48 culture supernatant
Using PMC 48 culture supernatant, a skin toner was prepared to investigate its effectiveness on hyperpigmented skin.The composition of the toner was as follows: purified water (90.55%),glycerin (5%), PMC48 culture supernatant (2.5%), 1,2-hexanediol (1.5%), sodium citrate (0.4%), and citric acid (0.05%).In this toner formulation, glycerin was added to increase the skin's moisture content.Sodium citrate and citric acid were employed as a buffer to adjust the pH of the formulation.In the control group of the clinical trial, the same formulation as above but without the PMC48 culture supernatant was used.

| Microbial safety analysis of the prepared formulation
The microbial safety analysis of the formulated product was carried out to avoid health-damaging consequences to the users and to test the quality of the product. 21In this test, formulated PMC48, PMC48 with distilled water and PMC48 supernatant (only) were used.
Microbial contamination was tested using Tryptic Soy Agar (TSA) medium (BD Difco, USA) and Potato Dextrose Agar (PDA) medium (BD Difco, USA).TSA medium was used to determine total aerobic microbial count (TAMC).PDA was used to check the test substance's total yeast and mold count (TYMC).100 μL of the test product was inoculated and spread onto TSA and PDA plates, followed by incubation at 35°C and 25°C for 72 and 120 h, respectively. 22After incubation, plates were examined for microbial growth.

| Thermal stability tests
The thermal stability of the product was tested at different temperatures as described previously. 23The formulated product was stored at chilled temperature (4°C), room temperature (23 ± 2°C), elevated temperature (45°C), and a cycle of temperatures of 4°C → 25°C → 45°C.In addition, to evaluate the product stability under the thermal stresses, phase, odor, and color under different temperatures were observed after 1, 3, 7, 14, 21, and 28 days.

| In vitro cytotoxicity assay
Effects of formulations containing PMC48 culture supernatant on a murine macrophage cell line RAW 264.7 (KCLB 40071) were evaluated using an in vitro cytotoxicity assay.The viability of RAW 264.7 cells (KCLB 40071) after treatment with a formulation containing PMC48 culture supernatant was evaluated using an EZ-cytox cell viability assay kit.Monolayers of RAW 264.7 cells maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) (HyClone, USA) were seeded (1 × 10 4 cells/mL) into a 96-well plate and incubated at 37°C overnight with a humidified 5% CO 2 atmosphere.After reaching approximately 70%-80% confluency, cells were washed with warmed 1× PBS.The formulated product of PMC48 at different concentrations (1.5-100 μg/mL) was then added to wells containing fresh media.After 24 h of incubation, 20 μL of WST solution was added to each well, followed by incubation at 37°C for 2 h.Cytotoxicity was then evaluated by measuring the absorbance at 570 nm using a Victor Nivo Multiplate reader.

| Patch test
Before the clinical trials, a skin irritation test was conducted on 32 Korean adults to rule out skin irritation potential.This test used three samples: a formulated product containing 2.5% PMC48 culture supernatant, a formulated product containing distilled water instead of PMC48 supernatant, and a PMC48 supernatant (100%) only.For this test, test substances were first loaded into a clear patch chamber (IQ Ultimate, Sweden).After cleaning test sites with sterilized wet cotton swabs, these patches were applied to the participants' backs.During the experimental period, all individuals were told to stay away from water, strenuous activity, and itching.After  (5) those who were judged to be unsuitable for this study by the investigator.This study was conducted following the Helsinki Declaration of 1964. 24

| Study design for the clinical trial
The effect of PMC48 culture supernatant on an artificially induced tanned skin was evaluated in an 8-week clinical trial.First, the minimal erythema dose (MED) was determined for each potential participant.Based on MED results, participants were finally selected.
MED is the amount of UV radiation that produces minimal erythema (sunburn or redness) on an individual's skin.It depends on skin sensitivity. 25However, for determining the MED, each participant's forearm (5-10 cm from the wrist) was exposed to UV radiation on day 9 using a multiport UV photonic simulator (XUV-3006, DTX, Korea).

MED was determined on Day 8. Based on MED values, participants
were classified according to the Fitzpatrick skin type classification system. 26Those who belonged to skin types II, III, and IV (type II, usually burn and less than average tan, MED of 25 ~ 35; type III, mild burn and about average tan, MED of 30 ~ 50; type IV, rarely burn and more than average tan, MED of 45 ~ 60) were finally selected for the clinical trial.
After determining the MED, the test site was observed for abnormal skin reactions from Day −7 to Day 0. Next, the formulated product containing PMC48 culture supernatant was applied twice a day (morning and evening) to the tanned skin from Day 0 until the end of the experiment.The skin was observed once every week after 1 week for 8 weeks.During this experimental period, the pigmented site was observed by 30 experts for visual evaluation based on a point scale from 0 to 10 (zero, light; 9 ~ 10, dark).
The brightness of the test area was also assessed using a chromometer (Konica Minolta, Japan).It is a device that can express colors in numbers for precise color communication. 27The measuring part of the instrument was positioned on the artificial pigmentation site.Then, L*, a*, and b* values were measured (L*, the luminance parameter, a* and b*, chromaticity coordinate).L* value was used to evaluate the skin brightness, with a higher value indicating a higher skin brightness. 28The L*, a*, and b* values of the test site were measured three times for each site at intervals of 2 weeks during the experimental period.
Skin melanin index (MI) in the artificial pigmented site was measured using a Mexameter (Courage and Khazaka, Germany), which could be used to calculate melanin levels based on light absorption and reflection measurements. 29First, the test site was touched vertically by a probe that could emit three different wavelengths of light.The amount of light the skin reflected was then measured with a receiver.A physical component that absorbs more light has more melanin.It is darker in color than one that reflects more light.Body areas that reflect more light are lighter in color and contain less melanin.This measurement was carried out three times with an interval of about 5 s.
The moisture content of the pigmented spot was checked using a corneometer (Courage and Khazaka, Germany) following the previously described method. 30The moisture content was measured by vertically positioning the corneometer probe onto the test site.The test was repeated thrice after 5 s.The depth of measurement was 10 ~ 20 μm below the stratum corneum.

| 16S rRNA-based amplicon sequencing
To check microbial composition after treatment, skin samples were collected from the antebrachial region (3 × 3 cm 2 test site) using an NBgene-SKIN kit (Noble Bio, Korea) following the manufacturer's protocol and stored at −80°C until the subsequent use. 31DNA was extracted from each skin sample for next-generation sequencing using the bead-beating method. 32The V4 region of the 16S rRNA was amplified using 5 μM of each (Illumina 16S 515f and 806r uni- Five microliters of purified amplicon PCR products were used for index PCR with a Nextera XT DNA Library Prep Kit (Illumina, USA) following the recommended protocol.Indexed PCR samples were purified using AMPure beads.The concentrations of all samples conditions during the study period.The above results showed that the prepared formulation was physically stable throughout the storage period of 100 days.

| Tyrosinase inhibition activity of PMC48 culture supernatant
The inhibitory effect of PMC48 culture supernatant on melanin biosynthesis was evaluated using L-DOPA as a representative tyrosinase (melanin synthase) inhibitor with arbutin as a positive control.As shown in Figure 1, the PMC48 supernatant had an inhibitory effect on melanin biosynthesis in a dose-dependent manner.At 0.00%-12.5% concentrations, PMC48 culture supernatant showed no significant inhibition on tyrosinase rate.However, at 25%-100% concentrations, it significantly inhibited tyrosinase.At 25% and 50% concentrations, inhibition rates of PMC48 supernatant for tyrosinase were moderate but significant (p < 0.05, 29% and 32%, respectively).At 100% concentration, it showed excellent inhibition activity with an inhibition rate of 40% (p < 0.01), similar to the inhibition activity of arbutin, the positive control.The antityrosinase activity of PMC48 culture supernatant indicates that it can be used as a natural inhibitor of melanin synthesis.

| Safety characteristics of the prepared formulation
Cytotoxicity of the prepared formulation containing PMC48 culture supernatant to RAW 264.7 macrophage cell line was evaluated.Cells were seeded, treated with the formulated product, and analyzed using an Ez-cytox reagent.Results (Figure 2) showed that viabilities of RAW 264.7 cells grown in the presence of different concentrations (1.5-100 μg/mL) of the formulated product were maintained above 60%, indicating that the test product containing PMC48 culture supernatant was not toxic to RAW 264.7 macrophage cell line.The presence of toxic components was also analyzed, and they were not detected in the prepared PMC48 formulation (Table S2).

| Skin patch test
A skin irritation test of the prepared formulation containing PMC48 culture supernatant was conducted.The formulation did not cause skin irritation or allergic reactions at 30 min or 24 h after patch removal.The mean skin reactivity scores of the formulation containing 2.5% and 100% PMC48 supernatant were 0.00 and 0.78, respectively.Thus, they were classified as having a low irritation potential (skin reactivity score range: 0.00-0.87)according to the classification criteria (Table 2).These results ensured the safety profile of PMC48 culture supernatant as a topical agent.

| Evaluation of prepared formulation using artificially induced tanned skin
The effect of the formulation containing PMC48 culture supernatant was evaluated using artificially induced tanned skin in an 8week clinical trial (Figure 3).Visual inspection was performed before and after the application of the test product.Results revealed that the formulation containing PMC48 culture supernatant significantly (p < 0.001) improved the tanned skin (Figure 3B, Table S3).After 2, 4, 6, and 8 weeks of treatment, color intensities of pigmented spots in subjects treated with F I G U R E 1 Inhibitory effect of PMC48 extract on DOPA oxidation.DOPA oxidation inhibition was measured using tyrosinase with a mixture solution of phosphate buffer and a wide range of concentrations of PMC48 extract in a 96-well plate.After incubation, oxidation inhibition was checked by measuring the optical density with a microplate reader.In the above assay, arbutin was used as a positive control.Statistically significant differences in comparison with the control were analyzed using an unpaired Student's t-test (**p < 0.01; *p < 0.05).Checking of skin abnormal skin reactions the prepared formulation were significantly decreased by 12.4%, 22.4%, 35.3%, and 47.6%, respectively, compared with the intensity at 0 week.In the control group (distilled water), color intensities at 2, 4, 6, and 8 weeks were decreased by 8.2%, 13.5%, 25.3%, and 37.0%, respectively.Significant differences existed between the PMC48 treated group and the control group (at 2 weeks, p < 0.01; at 4-, 6-, and 8-week, p < 0.001).
Skin brightness during the study period was also evaluated.
Results are shown in Figure 3C and Table S4.The L* value representing skin brightness was measured and analyzed.L* values of pigmented spots after 2, 4, 6, and 8 weeks of treatment with PMC48 culture supernatant were significantly increased by 3.5%, 5.1%, 6.1%, and 8.1%, respectively, compared with the initial value.In the control group, skin brightness values at 2, 4, 6, and 8 weeks were increased by 1.6%, 2.4%, 2.9%, and 4.5%, respectively.There were significant (p < 0.001) differences in skin brightness between the PMC48-treated and control groups.
Melanin indices of pigmented spots in subjects treated with PMC48 were also measured and analyzed (Figure 3D, Table S5).
Skin moisture contents of pigmented sites in participants treated with PMC48 were also measured and analyzed (Figure 3E, Table S6).

| NGS-based 16S rRNA profiling
To determine distributions of bacterial community at different taxonomic levels after PMC48 extract treatment, a 16S rRNA amplicon sequencing was performed, and resulting data were analyzed at different taxa levels (Figure 4, Table S7).Actinobacteria accounted for the highest percentage at the phylum level in both groups, followed by Firmicutes, Proteobacteria, and Bacteroidetes (Figure 4A).However, data indicated no significant difference in phylum level between cohorts.At the class level, Actinobacteria dominated over other taxa in both groups.Only Flavobacteria showed a distinctively increased population in the control group (Figure 4B).At the order level, only Flavobacteriales was abundant at 8 weeks in the control group (Figure 4C).Furthermore, the family Lactobacillaceae was the only prominent taxa in the PMC48 treatment group at the family level (Figure 4D).Interestingly, our data showed no change in taxa composition at the genus or species level.
Alpha diversity indices calculated by ACE (Species richness) and Simpson (Species diversity) methods indicated no significant differences between before and after treatment with either the extract or the control (Figure 4E).Furthermore, principal coordinate analysis (PCoA) based on the UniFrac divergence method presented that controls had similar distribution areas before and after 8 weeks of treatment.In contrast, the PMC48 treatment group showed different distribution patterns before and after the PMC48 treatment (Figure 4F).Beta-set-significance analysis based on Jensen-Shannon divergence showed a significant difference after treatment with the PMC48 extract (Table 3).However, there was no significant difference between samples treated with PMC48 extract and control samples before or after treatment.

| DISCUSS ION
In this study, we evaluated the clinical effect of P. acidilactici (PMC 48) on hyperpigmented skin by recruiting 22 participants.We observed that PMC 48 treatment showed significant skin-whitening effects by enhancing melanin degradation and inhibiting tyrosinase.
Skin hyperpigmentation is a significant concern.Most available treatment options are intense and associated with multiple side effects. 34Thus, there is a need to develop a treatment option with minimal or no adverse effects.Some studies have indicated that probiotics could be used as skincare products against hyperpigmentation and collagen degradation. 35Previously, our team has demonstrated the role of a kimchi-derived PMC48 in melanin degradation and hyperpigmentation. 9Here, we evaluated the effects of PMC48 as a potential skincare drug.
We performed different in vitro assays, such as microbial safety, drug stability, and cell toxicity assays.Our data showed that PMC48 was safe for industrial-scale production and stable when stored from 4°C to 45°C for 28 days.
Generally, keratinocyte or fibroblast cell lines are used for skin irritation and cytotoxicity test.However, we used the RAW 264.7 macrophage cell line because this cell line is known for studying overall toxicity and cellular responses to microbes. 36The data showed no cytotoxicity of PMC48 formulation up to 100 μg/mL.Moreover, none of the 32 volunteers complained about skin problems such as irritation or allergic reactions.Furthermore, the tyrosinase inhibition test based on L-DOPA showed that PMC 48 had tyrosine inhibitory effects like arbutin (a positive control used in this study), a known drug for skin depigmentation. 37However, the exact mechanism by which PMC48 performs DOPA oxidation inhibition is unknown.A detailed study is needed to discover the mechanism involved.Next, skin characteristics such as whitening, brightness, and melanin index levels were examined after 8 weeks of PMC48 treatment.Our data Cutibacterium acnes, and Candida albicans. 38Furthermore, PMC48 treatment for 8 weeks resulted in no significant change in alpha diversity, although prominent differences in beta diversities among groups were observed at genus and species levels.
Pediococcus acidilactici has been reported to have many applications as a probiotic. 39It also possesses anticancer, 40 antiobesity, 41 and antipathogen 42 effects.In addition, it can be used as a natural product for food preservation. 43Furthermore, some studies have demonstrated the use of P. acidilactici as a drug and a skincare product. 44In this study, we recruited 22 human participants to evaluate the application of P. acidilactici as a skincare microbe against hyperpigmentation, melanin deposition, and skin darkening.The results of this study are expected to benefit the masses in the skincare field and people suffering from skin pigmentationrelated problems.
Our study has certain areas for improvement.Firstly, the number of recruited participants needed to be increased.Secondly, this research needs in-depth analysis to determine the mechanisms involved in melanin degradation by PMC48.Thus, further detailed studies are needed to further elaborate the role of PMC48 as a probiotic against melanin degradation.Despite these shortcomings, our data suggest that PMC48 can be a potential drug candidate against hyperpigmented skin in the cosmetics and pharmaceutical industries.

| CON CLUS IONS
In summary, the Pediococcus acidilactici PMC48 strain has skinwhitening, hyperpigmentation-inhibiting, and melanin-degrading effects.Eight weeks of PMC48 treatment significantly improved pigmented skin by decreasing the melanin index through its tyrosinase inhibition capacity.In addition, 16S ribosomal RNA-based amplicon sequencing analysis showed a distinct increase in the family Lactobacillaceae without changing other skin microbiota.Furthermore, PMC48 improved skin moisture content and skin elasticity.These results demonstrate that P. acidilactici PMC48 can be a potential probiotic for the cosmetic industry against different skin disorders.
24 h, the patch chambers were taken out.Experts evaluated the reaction site at 30 min and 24 h after patch removal according to the International Contact Dermatitis Research Group's (ICDRG) and Frosch and Kligman's recommendations (Frosch and Kligman 1979; Lachapelle and Maibach 2020).

2. 8 |
Selection of test subjects for evaluating the whitening effect of PMC48 in a clinical trial To evaluate the whitening effect of PMC48 culture supernatant on artificially induced pigmented skin, we arranged an 8-week clinical trial.This study enrolled 22 Korean females according to inclusion and exclusion criteria.Inclusion criteria were as follows: (1) persons who voluntarily filled out and signed the consent form after receiving sufficient explanations about the purpose, contents, and possible adverse reactions of the study; (2) adults 19 years of age or older up to 60 years old without acute or chronic physical disease including skin disease; (3) those who fell under types II, III, and IV categories according to the Fitzpatrick skin type classification table; and (4) those who could follow up the visit schedule during the test period.Exclusion criteria were as follows: (1) pregnant or lactating women and women who were planning to become pregnant during the trial period; (2) those who had a history of photoallergy or photosensitivity; (3) those who had used external skin preparations containing steroids for more than 1 month for the treatment of skin diseases; (4) those who consumed a drug or food with a skin-whitening effect; and versal) primers (forward primer: T CGT CG G C AG CGT CA G A TG TGT AT A A GA GAC AG G T GC CAGCAGCCGCGGTAA and reverse primer: GTCTC GTG GGC TCG GAG ATG TGT ATA AGA GACAGGACT ACCAGGGTATCTAAT), 2 × KAPA HiFi HotStart ReadyMix enzyme (Roche, USA), 23 μL of DNA, and PCR grade water (Invitrogen, USA) in a final volume of 50 μL for each sample separately.PCR cycles conditions were as follows: initial denaturation at 94°C for 3 min; 35 cycles of denaturation at 94°C for 45 s, annealing at 50°C for 1 min, and extension at 72°C for 1 min and 30 s; and a final extension step at 72°C for 10 min.AMPure beads (Beckman Coulter, USA) were used for PCR product purification following the manufacturer's protocol.
Figure 3A depicts a schematic diagram of the clinical trial.Photographs of skin areas clinically tested are presented in the Appendix S1 (Figure S1).

F I G U R E 2
Cell viability assay results.RAW 264.7 cells were treated with a prepared formulation containing different concentrations of PMC48 culture supernatant for 24 h.Cytotoxicity was evaluated using an Ez-cytox reagent.Results are expressed as percentages of cell viability relative to the control.Statistically significant differences in comparison with the control were analyzed using an unpaired Student's t-test (**p < 0.01; *p < 0.05).

F I G U R E 4
16S rRNA gene-based skin metagenomics profiling.16S rRNA profiling was conducted using 0-week and 8-week samples to know bacterial compositions throughout the treatment.The resulting data were analyzed at (A) phylum, (B) class, (C) order, and (D) family levels.Wilcoxon rank-sum test was used to analyze the significance between samples.(E) Alpha diversity was also analyzed.Species richness was analyzed with Ace (left).Species diversity was analyzed with Simpson (right).The horizontal thick black band represents the median value.Boxplot margins indicate the first and third quartiles.(F) Beta diversity was evaluated through Jenson-Shannon-based principal coordinate analysis (PCoA).In the above assay, A and B indicate PMC48 treated and control samples, respectively.) showed that all factors above were significantly improved by PMC48 treatment.The decreased melanin index by PMC48 might have increased skin whitening and brightness.In addition, a significant change in skin hydration level was observed after 2, 4, and 8 weeks of PMC48 treatment, indicating its moisturizing capacity.Our 16S rRNA gene-based metagenomics results indicated a distinct increase in the family Lactobacillaceae in the subjects' skin after 8 weeks of PMC48 formulation treatment.In addition, some studies have shown that members of the Lactobacillaceae family have probiotic and skincare properties, especially the genus Lactobacillus (Lactobacillus salivarius and Limosilactobacillus fermentum) could compete directly with pathogens such as Staphylococcus aureus,