Abstract
Background/Aim: Natural ingredient-containing complexes are used in dermo-cosmetics to ameliorate facial characteristics, yet human experimental approaches and trials demonstrating their efficacy remain limited. This study assessed the effects of a Greek yoghurt-based complex on the human facial skin microbiome.
Materials and Methods: Thirty-one volunteers (27 females, 4 males; 20-65 years) with stressed facial skin and 15 volunteers (10 females, 5 males) with balanced skin were enrolled in a 28-day clinical study. At baseline, skin swabs were collected from both groups. Participants with stressed skin were treated in a split-face design with a yoghurt-based complex applied to the right cheek and placebo to the left cheek for 28 days, with follow-up sampling on days 7, 14, and 28. Bacterial DNA was analyzed by 16S rRNA gene amplicon sequencing. Bioinformatic analyses assessed microbial composition, diversity, and relative abundance. Baseline differences between skin types and treatment effects versus placebo were evaluated over time.
Results: A lowered microbiota diversity was observed in “stressed” facial skin, compared to the “balanced” group, on day 0. The four most abundant microbiota genera were Corynebacterium, Propionibacterium, Staphylococcus and Actinomyces spp. A significant difference in the relative abundance of these species was noted in the two groups (stressed vs. balanced) at baseline, as well as a consistent increase in mean bacterial diversity after intervention with the yogurt-based complex.
Conclusion: A gradual restoration of the skin’s microbial balance essential for a healthy skin function and appearance, was noted following the yoghurt-based complex application.
Introduction
The human skin is the largest organ of the body, extending across the whole outer surface of the body and separating it from the external environment. It has an important, multifunctional role, which includes physical and immunological protection, sensation, endocrine-exocrine activity, along with assisting in the regulation of the body temperature and water balance (1). The protective barrier it forms against the environment serves as the organism’s first line of defence against physical injury and external aggressors, such as ultraviolet radiation, pollutants, pathogens and other toxins (2).
At the same time, skin provides a natural habitat for a myriad of microbes that comprise the “skin microbiome” (3, 4). The skin microbiome is defined as the sum of the genetic material of all the microorganisms present on the human skin (5). Resident microorganisms that colonize the skin, also known as skin microbiota, play an important role in maintaining the physiochemical balance of the skin (6). A balanced and diverse skin microbiota population, which includes viruses, fungi and bacteria, interacts synergistically with the skin to protect the host from pathogens and boost the immune system’s response by continuously training it (4, 7).
Human skin and in particular skin on the face contains a higher density of sebaceous glands, which help lubricate and protect the skin, but, also, make it more susceptible to oiliness, clogged pores and acne (5, 8). Healthy facial skin ecosystems contain mostly microorganisms that can tolerate these lipophilic, anaerobic conditions, such as Propionibacterium spp. (also known as Cutibacterium), Corynebacterium spp., Staphylococci spp., Demodex mites, Malassezia fungi and Propionibacterium phages (4, 6, 9-11). The microbiota communities that colonize the human face determine the skin’s physiochemical and physiological balance, ensuring its health and homeostasis, host immune defence, and its appearance (12). A heathy or “balanced” facial microbiome contributes to homeostatic mechanisms that fortify the skin’s barrier function, promoting hydration and overall wellbeing (13).
Disruption of this delicate microbial balance results in dysbiosis, a precursor of skin disease (14, 15). A plethora of factors may lead to dysbiosis, thereby potentially causing the facial skin to appear “stressed” and unhealthy. Internal factors include age, sex, hormones and genetics, whereas external causes comprise stress-inducing factors to the skin like poor hygiene, harsh cosmetic products, physical and psychological stress, diet, chemical exposure and medications (16, 17).
Recent dermatological research has increasingly focused on products with ingredients of natural origin to ameliorate characteristics of the human face with minimally invasive techniques (18-20). The development of microbiome-based formulations, such as bioactive compounds, pro- and prebiotics, has become of central focus in efforts to preserve skin homeostatic mechanisms. (21-24). These products have emerged as innovative options for cosmetic applications (22, 25, 26). For instance, Le et al. demonstrated the clinical anti-aging potential of caviar oil-derived products, highlighting the skin’s responsiveness to bioactive lipids and proteins (21). However, controlled experimental trials demonstrating their efficacy, mechanism of action and safety in human facial skin remain limited (22-24, 27, 28).
Advancements in the fields of genomics and bioinformatics and, more specifically, the consolidation of next generation sequencing (NGS) as the go-to tool in the study of microbiota have allowed the characterization of the skin’s microbiome in great detail (29, 30). Nowadays, bacteria are mostly analysed either by marker gene sequencing, e.g., 16S amplicon sequencing, or by whole-genome sequencing (shotgun sequencing). 16S RNA sequencing is widely used to determine the composition, abundance and diversity of the bacterial communities present in a sample without the need for further culturing (31, 32).
The aim of the study was to assess the effects of a Greek yoghurt-based complex on the human facial skin microbiome by plotting and characterizing bacterial flora using NGS of 16S bacterial ribosomal rRNA.
Materials and Methods
Yoghurt-based complex composition.
The yoghurt-based complex consisted of the following ingredients: (i) Greek Yoghurt: a cultured dairy product made by bacterial fermentation of milk, followed by a straining process to remove acid whey, resulting in a thicker texture and high protein count. This procedure concentrates its natural components, making it rich in proteins, minerals, lactic acid, vitamins, bioactive metabolites and active acid bacteria; (ii) An ingredient mix based on pre- and pro-biotics containing Alpha glucan-oligosaccharide, Polymnia sonchifolia root juice, Maltodextrin and inactivated Lactobacilli (L. casei and L. acidophilus); (iii) An ingredient mix based on marine algae extracts containing glycerin, water, seawater, Laminaria digitata extract, Chlorella vulgaris extract, saccharide isomerate and phenethylalcohol; and (iv) An ingredient mix based on oat milk containing water, glycerin, Avena Sativa (Oat) kernel extract, glyceryl oleate citrate and caprylic/capric triglyceride.
Study design. This was a 28-day, prospective, split-face clinical and microbiome study evaluating the effect of a topical active-complex formulation on facial skin. Participants applied the placebo cream (identical in formulation to the active-complex cream with absence of active ingredients) to the left cheek and the active-complex cream to the right cheek once daily throughout the study period.
Microbiome assessment and study groups. The effects of the yoghurt-based complex on the facial skin microbiome were evaluated by profiling bacterial communities in volunteers with “balanced” and “stressed” skin using next-generation sequencing of the 16S rRNA gene. The balanced-skin group served as a microbiome reference cohort and was sampled once at baseline (D0). Volunteers with stressed skin were sampled at baseline (D0) and subsequently followed longitudinally at D7, D14 and D28. After baseline sampling, the stressed-skin cohort was assigned in a split-face design to receive either the yoghurt-based complex or a placebo formulation, allowing comparison of microbiome changes over time between treatment conditions.
Participants and recruitment. A total of 31 adults with clinically assessed sensitive skin (27 females, four males; age range=20-65 years) were enrolled (Table I). In addition, 15 normal-skin subjects (10 females, 5 males) were included at baseline as a microbiome reference cohort (Table II) (Figure 1). From this group n=5 volunteers were subjected to emulsion check (sensory evaluation via 24-h patch test).
Demographic and skin characteristics of volunteers in the stressed skin group.
Demographic and skin characteristics of the microbiome reference group with normal skin.
Study flowchart. Study design and sampling scheme of the 28-day split-face clinical and microbiome study. A cohort of volunteers with stressed facial skin was sampled at baseline (D0) and subsequently assigned in a split-face design to receive the yoghurt-based complex on the right cheek and a placebo formulation on the left cheek, applied once daily for 28 days. Facial skin swab samples were collected from both cheeks at D0, D7, D14, and D28. A separate cohort of volunteers with balanced (normal) skin was included solely as a microbiome reference group and was sampled only once at baseline (D0), without receiving any topical treatment. IND: Individuals.
Patient selection, recruitment and enrollment as well as the clinical part of the study took place in the QACS Laboratory, Athens, Greece. Stressed skin status was confirmed by a board-certified dermatologist based on medical history, self-reported cutaneous hyperreactivity (stinging, burning, itching, tightness), and absence of visible inflammatory dermatosis at baseline. Stressed skin was defined as heightened neurosensory and barrier-related reactivity to environmental and cosmetic stimuli.
Participants were recruited from the laboratory’s volunteer registry. Eligibility was deemed based on predefined criteria, including no active dermatologic disease, no systemic therapy affecting skin function, and no antibiotic use within 30 days prior to enrollment. All participants provided written informed consent prior to study participation.
Skin sampling. Facial microbiome samples were collected at designated timepoints using a standardized, controlled swabbing protocol. Volunteers refrained from facial cleansing and topical product application for at least 12 hours prior to sampling. Sterile flocked swabs (4N6FLOQSwab®, Copan, Brescia, Italy) pre-moistened with 500 μl DNA-free buffer (0.15 M NaCl, 0.1% Tween-20) were used to sample a defined 4 cm2 area on each cheek.
To ensure sampling consistency, each site was swabbed with 40 strokes total: 20 horizontal (left/right) and 20 vertical (up/down), applied at ~75 g force (acceptable 50-100 g) and ~2 cm/s. The swab was rotated 90° every 5 strokes to expose fresh fibers. Pressure and motion parameters were standardized via operator training using a digital scale and a marked 2-cm guide path prior to study initiation. Total contact time per site was approximately 40 sec. Swabs were immediately placed into sterile tubes containing lysis buffer, transported on ice, and stored at −80°C until DNA extraction. Field blanks were collected during each sampling session to monitor environmental and reagent contamination.
Bacterial DNA extraction. Total DNA extraction from frozen swab samples was carried out with the co-application of enzymatic and mechanical lysis using the DNeasy Blood & Tissue kit (Qiagen, Milan, Italy), according to the manufacturer’s instructions. Approximately 180 μl of tissue lysis buffer were added directly to the frozen samples and vortexed for 60s to maximize microbial transfer from the swabs into the solution. To ensure thorough cell lysis, swabs were removed, and samples were bead-beaten at 30 Hz for 1 min using the Tissue Lyzer II (Qiagen, Germantown, MD, USA). Subsequently, samples were incubated with 20 μl proteinase K for 3 h at 56°C. The remaining bacterial DNA isolation steps were performed according to instructions from the DNeasy Blood & Tissue kit.
16S rRNA sequencing. Standard Illumina MiSeq® protocols were used for 16S metagenomic sequencing library preparation. For the amplification of the 16S rRNA gene variable V3-V4 regions, a total of 125 ng isolated microbial genomic DNA per sample was dispensed into a 96-well plate. PCR protocols were followed as recommended by the manufacturer using the suggested V3-V4 primer pairs. Initially, microbial DNA was used for a 25 μl PCR reaction containing 2× KAPA High Fidelity HotStart Ready Mix (Roche, Mannheim, Germany) and PCR primers specific to the studied hypervariable region. Thermal cycling conditions were 95°C for 3 min for the initial denaturation, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. A further extension step was carried out for 5 min at 72°C, followed by a cooling step to 4°C. A purification step of the V3, V4 hypervariable region amplicons was then carried out and Illumina sequencing adapters were attached by a 50 μl PCR reaction using the Nextera XT Index Kit (Illumina, San Diego, CA, USA). A purification step was repeated prior to library validation and quantification. Quality control of the libraries was carried out using the Agilent bioanalyzer DNA1000 kit and DNA quantitation with the PicoGreen fluorometric assay (Agilent, Santa Clara, CA, USA). Approximately 250 Million 2×300 bp Paired-End reads were generated using the Illumina MiSeq Sequencer.
Data analysis. Sequencing quality was assessed and corrected using the Trim Galore software package (33). To investigate the microbial community identified by the 16S gene sequencing, demultiplexed paired end FASTQ files were analysed with the Mothur software package (version 1.48.0), following the standardised MiSeq SOP (34). Paired-end raw sequence forward and reverse reads were merged using standard settings and sequences containing ambiguous data were removed in a quality control step. Sequences were then filtered towards removing duplicates prior to alignment to the reference database. Taxonomy of gene sequences was assigned using the SILVA bacterial reference database (version 102).
Bacterial community diversity was assessed using the Simpson diversity index. The mean community diversity for each group was calculated as the average Simpson index across samples.
Statistical analysis. All statistical analyses were performed using the ggplot2 software package, a graphical user interface for R version 3.2.2 (The R Foundation for Statistical Computing, Vienna, Austria). Relative abundance of major taxonomic groups was estimated as the ratio of sequence abundance to the total number of sequences identified in each sample and expressed as mean±standard deviation (SD) for each group. Comparisons of mean community diversity and mean relative abundance across all three study groups (“stressed”, “balanced”, “placebo”) and statistical significance was defined as p<0.05 (35).
Ethics statement. This study was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice (GCP) guidelines. Personal data were handled in compliance with GDPR (EU 2016/679) regulations, and all samples were pseudonymized prior to analysis to ensure participant confidentiality.
Results
Effects on skin microbiome. Analysis of 16S rRNA sequencing data revealed clear differences in bacterial community structure between balanced and stressed facial skin at baseline. Stressed skin exhibited significantly reduced microbial diversity compared to balanced skin, a pattern that progressively changed following application of the yoghurt-based complex. Longitudinal analysis across the four study time points (D0, D7, D14, and D28) demonstrated a statistically significant increase in bacterial diversity in the complex-treated skin, whereas no comparable changes were observed in the placebo-treated sites.
Taxonomic profile of facial skin bacteria. The microbiota genera with high representation were detected via 16s rRNA sequencing and the four most abundant included Corynebacterium, Propionibacterium, Staphylococcus, and Actinomyces spp., consistent with the findings reported in current literature (10, 36). There was a significant difference in the relative frequency of Corynebacterium, Propionibacterium, Staphylococcus, and Actinomyces among the “balanced” and “stressed” groups (p<0.05). In order to better compare these differences, the mean relative abundance was calculated. Deregulation of facial skin microbiome was evident, as reflected by the increase in mean relative abundance of Corynebacterium spp. and Staphylococcus spp. in “stressed” skin swabs. On the contrary, a significant decrease in the mean relative abundance of Propionibacterium spp. and Actinomyces was displayed in “stressed” skin swabs compared to those of the “balanced” group. Continued application of the complex resulted in the relative abundance of all four microbiota genera to gradually approach normal levels, accomplishing the best results on D28 (28 days post complex application), where their abundance resembled the levels of “balanced” skin (Figure 2).
Differences in the relative abundance of four major skin bacteria genera. Box plots showing the relative abundance of (A) Corynebacterium spp., (B) Propionibacterium spp., (C) Staphylococcus spp. and (D) Actinomyces spp. over the course of the study associated with yoghurt-based Complex and Placebo application on volunteer’s facial skin. Statistically significant at *p<0.05.
More specifically, following the application of the complex, the proportion of Corynebacterium bacteria compared to the total bacterial population in “stressed” skin swabs decreased from 5% on D0 to 3% on D28, in comparison with the 3% displayed on the “balanced” group (Figure 2A). Meanwhile, Propionibacterium spp. showed a remarkable progression increasing from 3% in “stressed” facial skin samples on D0 to 18% in the “complex” samples on D28, compared to the 19% present in the “balanced” group (Figure 2B). Staphylococcus bacteria in the “stressed” group decreased from 15% on D0 to 8% on D28, compared to the 9% of the “balanced” group (Figure 2C). Finally, Actinomyces spp. increased from 0.09% in the “stressed” group on D0 to 0.25% on D28 after the complex’s application, in comparison with 0.3% of the “balanced” samples (Figure 2D). It becomes evident that the application of the yogurt-based complex contributed to the bacterial population reaching “balanced” benchmarks over time. Such results were not evident in the “placebo” group where either the species’ percentages remained virtually the same as the “stressed” samples (Propionibacterium spp., Actinomyces spp.) or their populations became even more excessive (Corynebacterium spp. and Staphylococcus spp.), further disrupting the microbial ecosystem.
Bacterial community diversity before and after application. Bacterial community diversity was assessed using the Simpson Diversity Index. This showed distinctive differences between the volunteer groups. “Balanced” skin volunteers on D0 had a mean diversity index of 0.57±0.05, while the “Stressed” skin group exhibited a significantly lower mean index of 0.29±0.02. The “Stressed” group was then split into the “Complex” and “Placebo” groups, based on whether the complex or a placebo was applied on D7, D14 and D28. The mean index of the “Complex” group steadily increased after the application reaching 0.53±0.04 on the final day, while the “Placebo” group index remained unchanged at 0.25±0.03. The difference between all groups studied was statistically significant (p<0.05). Bar plot analysis demonstrates an upward shift in the median diversity of the “Complex” group compared to the “Placebo” group over time (Figure 3A). This shift is better depicted in a line chart displaying the mean community diversity over time, comparing the effects of Complex and Placebo application on volunteers’ facial skin (Figure 3B). Intervention with the yogurt-based complex could be associated with a consistent increase in mean bacterial community diversity, gradually restoring the facial skin’s microbiome balance. On the contrary, placebo application shows no improvements in biodiversity, with the mean diversity index remaining constantly on the same level as the “stressed” group.
Characterization of bacterial community diversity in facial skin swab samples. (A) Bar plot of community diversity across all study time points and study conditions (Balanced, Stressed, yoghurt-based Complex, Placebo) as estimated using the Simpson Diversity Index. (B) Alterations in mean community diversity over time as an effect of yoghurt-based Complex and Placebo application on volunteer’s facial skin. Statistically significant at *p<0.05.
Discussion
The facial microbiome plays a central role in maintaining the physiochemical balance of the skin, which is easily disturbed by lifestyle-related stimuli (6). When the skin microbiota balance is disrupted, this can lead to appearance alteration and high risk for abnormal skin conditions (16, 17, 37-39). Among the stressful factors for the skin are modern day skincare products that often rely on chemical-based compounds and even though are considered safe and meet regulatory standards, frequent use may change the skin’s metabolome and microbiome (40). Other factors include genetics, poor hygiene, stress or toxins exposure.
In order to study the effects of yogurt-based complex on an imbalanced human facial microbiome, the bacterial diversity on healthy and stressed skin was determined using NGS of the 16S bacterial ribosomal rRNA gene. This study showcases that the major genera detected in balanced skin were Propionibacterium, Staphylococcus, Corynebacterium, and Actinomyces. The most significant differences regarding the relative frequency of bacteria in balanced skin compared to the “stressed” samples were, also, mostly displayed in these four major genera. Corynebacterium spp. and Staphylococcus spp. concentrations displayed a statistically significant increase in “stressed” skin swabs, while a significant decrease was evident in the populations of Propionibacterium spp. and Actinomyces compared to those of the “balanced” group. Furthermore, in “stressed” facial skin a lowered microbiota diversity was observed underlining the fact that the remarkable decline of the microbiota diversity of a stressed condition may cause less abundant bacteria to disappear, resulting in a disturbance of the “balanced” condition’s microbiome. Afterwards, the yogurt-based formulation was applied four times over a 28-day period on imbalanced skin volunteers (D0, D7, D14, D28) and its ability to induce re-balancing effects on a “stressed” human face microbiome exhibiting dysbiosis was evaluated.
It is worth noting, that according to Howard et al., Lactobacillus and Cutibacterium (Propionibacterium) representation on the face’s microbiome demonstrated a statistically significant decrease with increasing age, compared to an increase in abundance of Staphylococcus and Corynebacterium among Caucasian women (41). Juge et al. reported that older aged Western European women displayed a significant decrease in Propionibacterium and Actinobacteria, with a simultaneous “striking increase” in Corynebacterium species compared to their younger counterparts (42). Several independent studies have reported comparable age-associated microbiome shifts across diverse cohorts in Western European, East Asian and North American populations. The similar patterns include decreases in Cutibacterium populations and an increase in Corynebacterium and, variably, Staphylococcus abundance (43-47). This could help explain the “stressed” facial appearance of the volunteers that participated in our study, since comparable results were reached.
Yogurt is a natural product that is produced with the fermentation of pasteurized milk with cultures of the bacteria Lactobacillus spp. and S. thermophilus and is considered a great probiotic (48, 49). Although studies on the benefits of topically applied whole yogurt formulations on the skin’s microbiome are limited, there is an increasing interest on how prebiotics and probiotics affect human health (50, 51). Streptococcus thermophilus can produce enzymes that, when applied topically, contribute to skin hydration, reducing skin dryness and loss of water and, consequently, decelerating the process of aging (52, 53). In vitro studies have, also, shown that S. thermophilus lysate has antifibrotic and antioxidant properties (54, 55). Likewise, in vitro and in vivo studies suggest Lactobacilli strains help improve skin hydration, elasticity and firmness, promote collagen synthesis and wound healing, reduce trans-epidermal water loss and inflammation and improve symptoms of photo-aging and skin diseases, such as acne and atopic dermatitis (28, 56-60). Both Lactobacilli and S. thermophilus bacteria have, also, shown to have antibacterial properties. This is possibly due to immunomodulation, and production of lactic acid, H2O2 and bacteriocin by those probiotics. This antibacterial activity has been shown to be effective against several pathogenic bacteria, including Staphylococcus aureus, Corynebacterium spp. and Escherichia Coli (61-70).
In accordance with these observations, our study concluded that following application of the yoghurt-based complex the overabundance of the opportunistic pathogens Staphylococci and Corynebacteria was reduced, while other beneficial bacteria, such as Propionibacterium, that were being suppressed started colonizing the skin again. Moreover, the lowered microbiota diversity in “stressed” facial samples, which was caused by the overgrowth of opportunistic microorganisms and leads to a deregulation of key functions, was gradually and steadily alleviated. This helped to stabilize the ecosystem and restore the homeostatic mechanisms of the skin.
Limitations. First, the small sample size of 31 volunteers limits the generalizability of the findings to broader populations. Additionally, investigation accrual of only four male volunteers prevents sex-based comparisons. Furthermore, the study did not account for environmental and lifestyle factors, such as the climate, diet, previous skincare routines and stress levels of the participants that may affect the final outcome. Further prospective studies including a larger cohort of participants will further amplify our results.
Conclusion
The study showcases the effects of application of a yogurt-based formula on “stressed” human facial skin. The yogurt-based complex seems to add to the restoration of the microbial equilibrium by reducing overrepresentation of certain bacteria species. This approach potentially counteracts the microbial disruption present in stressed skin, aiding a return to microbiota eubiosis and promotion of a “healthy- appearance” skin.
Footnotes
Authors’ Contributions
Design and conception: L.A and A.K.A.; data collection: I.V., G.V., D.V.; data analysis and interpretation: all authors; drafting of the article: all authors. Overall supervision: L.A and A.K.A.. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
G.S., A.L., C.S. and L.A. are employees of Korres SA. The company develops and markets skincare products including formulations related to the investigational product evaluated in the study. This study was sponsored by Korres SA. The sponsor had no role in data analysis, interpretation or the decision to publish.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received December 24, 2025.
- Revision received January 20, 2026.
- Accepted February 17, 2026.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
References
Jump to section
Related Articles
Cited By...
- No citing articles found.