1. Introduction
Skin is the first line of defense against external intruders and serves as a haven for various bacteria. Skin bacteria’s degree of richness and diversity is varied and related to distinct skin locations and their physiological properties [
1]. Some skin parts have higher microbial diversity than the gut and dental cavities. Within the community investigated, individual variation differed widely, while the temporal stability of the assessed skin microbial populations was reasonably consistent [
2,
3]. They were considered commensals (non-pathogenic permanent inhabitants) or transients (temporary residents) [
4,
5]. Most skin pathogens are opportunistic, i.e., living on the skin as commensals; in their favorable conditions, including microbial dysbiosis or microbial imbalance, antibiotic use, and host immune status, they become pathogens [
6].
Skin functionality was severely affected by reduced skin moisture, age-related dermal and epidermal junction modification, degeneration of vital skin structural protein, microbial alteration, and fragility resulting in fine lines and wrinkles, uneven skin tone, and dryness [
7]. Consequently, maintaining the balance of the skin microbiome with the dermal and epidermal is essential for skin health [
8].
In recent years, skin disorders have received much attention. People have been concerned about skin aging, wrinkles, pigmentation, and dryness [
9]. Researchers are focusing on developing skin care products such as an anti-aging facial moisturizer with a neutral skin pH (4.5–5.5) and other active ingredients, such as
Astragalus membranaceus root extract, ursolic acid, and tetrahexyldecyl ascorbate, rhamnose, silanetriol, and other natural extracts, to prevent the skin from UV-A radiation and improve skin thickness [
10,
11].
Cosmetics with chemicals and herbal extracts may be toxic or cause side effects and are less effective for skin health [
12]. Nowadays, micro-ecological skincare is preferable, and using probiotics to improve skin health is a choice of several studies [
9,
13]. Parameters such as moisture retention, transepidermal water loss (TEWL), elasticity, and wrinkles are the determinants of the efficacy of orally ingested probiotics in maintaining healthy skin by reducing the roughness and dryness on the skin [
14,
15,
16].
Probiotics are reported for their role in preventing, treating, and managing skin aging and diseases [
17,
18,
19].
Lactobacillus spp. and
Bifidobacterium spp. are the most used probiotics to treat skin diseases [
17,
20]. Furthermore, research on topical probiotics in skincare and dermatological therapy is still in its infancy. Further investigation is warranted to establish their security, effectiveness, and mode of action in various skin conditions [
13].
The paraprobiotics (non-viable cells; intact or broken) and postbiotics (non-viable bacterial products or metabolic byproducts of probiotics) have attracted attention from the food and pharmacological industries recently [
21]. Several questions, including the bioactive components of postbiotics, the processes through which they confer health benefits [
22,
23], and the immunological aspects of postbiotics, still need to be answered completely [
21]. However, many researchers have accelerated their research, implementing postbiotics to identify its benefits in areas including the food industry, pharmaceutics, and cosmetics.
Recently, Kim et al. reported that the
L. plantarum K8 strain isolated from kimchi could improve skin moisture by regulating the expression of hyaluronic acid synthase-2 and aquaporin-3 in HaCaT cells. The results showed that paraprobiotics of
L. plantarum K8 might be used in moisturizing cosmetic products [
24].
The studies on paraprobiotics are very limited, and the proficiency of paraprobiotics in skin care products has not been elucidated in detail. Studies on human subjects are necessary to confirm the activity of the paraprobiotics-containing moisturizing product. Thus, the study’s objective was to analyze the impact of paraprobiotics (Bifidobacterium lactis (B. lactis) and Lactobacillus plantarum (L. plantarum)-containing moisturizer on the skin hydration and skin microbiome of healthy subjects.
2. Materials and Methods
2.1. Preparation of Moisturizer
L. plantarum and B. lactis were purchased from LactoMason Co., Ltd., Gyeongsangnam-do, Republic of Korea, as a freeze-dried powder. The probiotic powders were autoclaved (121 °C for 15 min) and used to prepare the moisturizer. The formulations comprised autoclaved L. plantarum and B. lactis cells (1 × 1010 CFU each), xanthan gum (1% w/w), and water. First, xanthan gum was dispersed into water and stirred until the dispersion was complete. Then, the autoclaved probiotics were combined into the mixture (APM). The placebo formulation was prepared without autoclaved probiotics (PM).
2.2. Study Design and Analysis
Fifty healthy subjects, either men or women, were included in the study. The inclusion and exclusion criteria of the subjects are as follows. Inclusion criteria: (1) Aged between 20–60 years old; (2) No history of skin diseases, tattoos, or scars; (3) No history of cosmetics allergy; (4) No psychiatric illness; (5) No hospitalization during the past 6 months before the study; (6) No history of immune-related diseases; (7) No antibiotic use during the past 14 days before the study. Exclusion criteria: (1) Pregnancy or breastfeeding mother. The subjects were asked to stop using other moisturizing cosmetics for 3 days before the study. The subjects were randomly allocated into two groups (Placebo (P); n = 25 and treatment (T); n = 25). Each group received either APM or PM.
Skin hydration was measured using MoistureMeterSC (Delfin Technologies Ltd., Kuopio, Finland) and VapoMeter® (Delfin Technologies Ltd., Kuopio, Finland). There were two courses of study, namely short-term and long-term studies. For the short-term study, subjects’ skin moisture was measured and recorded at 0 h (baseline) and then measured again every hour after the moisturizer application for 4 h. For the long-term study, the subjects were asked to apply their respective moisturizers twice a day on their skin for 4 weeks. The changes in their skin were assessed after 4 weeks.
The study protocol was approved by the ethical committee of the Faculty Pharmacy, Chiang Mai University (Approval no. 11/2563 dated 28 February 2020).
2.3. Next-Generation Sequencing
Due to the insufficient quality and quantity of skin metagenomes, we sequenced only 10 samples (Control; n = 5; treatment; n = 5) to determine the microbiome in the present study. The baseline and after four weeks of treatment samples were denoted as pre- and post-samples of control (Cpre and Cpost) and treatments (Tpre and Tpost). The samples codes are denoted as SK11-1-Cpre/post, SK12-1-Cpre/post, SK16-1-Cpre/post, SK19-1-Cpre/post, SK9-1-Cpre/post for the control group, and SK10-1-Tpre/post, SK14-1-Tpre/post, SK2-1-Tpre/post, SK6-1-Tpre/post, SK8-1-Tpre/post for the treatment group.
The sequencing of metagenomics isolated from the skin swab was performed as detailed in our previous study. In short, the 16S rRNA gene was amplified using 16S V3-V4 primers and sparQ HiFi PCR master mix (Quantabio, Beverly, MA, USA). Subsequently, the 16S amplicon was purified using sparQ PureMag Beads (Quantabio, Beverly, MA, USA). The amplicon was indexed using Nextera XT index primer and amplified. Then, the amplicon was cleaned, pooled, and diluted to the final loading concentration (4 pM). Cluster generation and 250-bp paired-end read sequencing were performed on an Illumina MiSeq at Omics Sciences and Bioinformatics Center (Chulalongkorn University, Bangkok, Thailand).
2.4. Microbiome Analyses
The paired-end data for each sample were obtained as forward reads and reverse reads in two separate fastq files, which were paired using QIIME2.0™ using 16S rRNA workflow. Phylogenetic diversity (PD) was annotated from the rarefaction curve. The Shannon diversity index was calculated for the control and treatment samples. A separate comparison between post-samples of control and treatment groups was performed to estimate species diversity.
The Shannon diversity index was used to calculate the significant differences between the groups by implementing the Kruskal–Wallis (pairwise) test. The relation between samples was identified using PCoA and visualized in QIIME 2 View. Herein, the first three main coordinates were used to create PCoA plots and labeled according to their variance. The taxonomy (phylum to species) was assigned to the samples. The intra (Cpro vs. Cpost; Tpro vs. Tpost) and inter-group comparisons (Cpro vs. Tpro; Cpost vs. Tpost) were also performed.
2.5. Statistical Analysis
All the statistical analysis was performed using STATA/IC 14.0 (StataCorp L.L.C., College Station, TX, USA). The demographic parameters such as gender, age, and BMI were assessed by implementing the exact Fisher test, rank-sum, and t-test with a statistical significance of p ≤ 0.05.
The differences in TEWL and SCM were calculated by paired t-test and Wilcoxon’s sign rank test and expressed as the mean differences. Additionally, the linear regression analysis was performed to obtain the coefficients for the TEWL and SCM at the 4th h and 4th weeks (p ≤ 0.05). Significant changes in the microbiome at the phylum, genus, and species in the control and treatment groups were analyzed using Wilcoxon’s sign rank and rank-sum tests (p ≤ 0.05). Power analysis was performed to confirm the validity of the statistical analysis.
5. Conclusions
Four weeks of APM treatment significantly improved the TEWL and SCM in healthy subjects compared to the control and respective baseline values. APM treatment increased the abundance of commensal microbes belonging to the phylum Planctomycetes, Chloroflexi, Verrucomicrobia, Gemmatimonadetes, Planctomycetes, and Nitrospirae. The abundance of Proteobacteria and Firmicutes and Cyanobacterial growth was reduced after APM treatment. The beneficial bacterial genera were increased after APM treatment. Meanwhile, some of the pathogenic and opportunistic pathogenic bacteria were detected in the treatment group.
In conclusion, APM treatment improved the skin microbiota associated with skin aging, UV-induced skin damage, acne, skin lesions, and skin diseases. The current study primarily reported the effect of paraprobiotics (B. lactis and L. plantarum)-containing moisturizer on the skin microbiome in healthy Thai subjects. However, as detailed previously, the study has some limitations, so further detailed clinical studies are needed to confirm the beneficial effect of APM on skin hydration and skin microbiome, which could aid in the development of paraprobiotics-based skin care formulations to manage skin diseases.