Investigational Product and Application

The synbiotic supplement used in this study is commercially available from Codex Labs (San Jose, CA, USA) under the trade name Shaant Clear Skin and contains the following ingredients: Bifidobacterium lactis, Lactobacillus acidophilus, Bacillus coagulans, guggul extract (Commiphora mukul), epigallocatechin gallate (EGCG), hypromellose (capsule), rice extract blend, gum fiber blend, organic rice fiber, fiber blend (rice fiber, oat fiber, sunflower lecithin), and silicon dioxide. Subjects who were allocated to this group were instructed to take two capsules (77 mg) daily with water.

The myoinositol-based herbal supplement used in this study is commercially available from Codex Labs (San Jose, CA, USA) under the trade name Shaant Skin De-Stress and contains the following ingredients: myoinositol, folate, pantothenic acid, sodium, methylsulfonylmethane, organic holy basil extract (Ocimum sanctum leaves), turmeric root extract (Curcuma longa), (50 mg curcuminoids), barberry root extract (Berberis vulgaris), l-theanine, milk thistle extract (Silybum marianmum, 140 mg Silymarin), natural flavors, citric acid, organic rice fiber, sea salt, stevia leaf extract. Subjects who were allocated to this group were instructed to mix one scoop (5.6 g) into a single serving of water (8 oz. or 240 mL) once daily.

Inclusion and Exclusion Criteria

Inclusion criteria: male and female patients between the ages of 12 and 45 years. The presence of non-cystic, mild to moderate acne based on investigator global assessment (grade 2 or 3) and presence of at least 10 inflammatory lesions and at least 5 non-inflammatory lesions.

Exclusion criteria: the presence of severe acne as noted by the investigator global assessment (grade 4) or the presence of cystic acne. Those who are unwilling to discontinue oral probiotic-based supplementation, or supplement containing ingredients found in the study’s oral product 1 month prior to enrollment. Those who are unwilling to discontinue topical antibiotics and topical benzoyl peroxide for 2 weeks prior to enrollment. Those who are unwilling to keep their non-prescription facial regimen the same throughout the study. Individuals who have been on an oral antibiotic for acne within a month prior to enrollment. Individuals who are pregnant or breastfeeding. Individuals who have changed any of their hormonal based contraception or therapies within 3 months prior to enrollment. Individuals with use of isotretinoin within 3 months prior to enrollment. Individuals on finasteride or dutasteride. Current tobacco smoker or a tobacco smoking history.

Study Design and Recruitment

This study was an 8-week, randomized, head-to-head clinical trial conducted between July 2023 and July 2024. The study was conducted according to the guidelines of the Declaration of Helsinki, approved by the Allendale Institutional Review Board (CB_Acne_Supp, June 26, 2023), and registered on www.clinicaltrials.gov (NCT05919810). All participants or guardians (in the case of minor) provided signed informed consent prior to participation and all minors provided signed informed assent. All participants or guardians (in the case of minor) provided written consent for their photographs to be used in publication. Male and female patients, ages 12 to 45 years, in the greater Sacramento area were recruited and screened for eligibility. All study procedures were completed at Integrative Skin Science and Research in Sacramento, CA. A priori randomization was performed with a computer-based randomization program and eligible subjects were assigned to groups through the use of blinded sealed envelopes by the study coordinator. Study visits occurred at baseline, week 4, and week 8.

Stool Sample Collection, DNA Extraction, and 16S rRNA Sequencing

All subjects were provided with pre-assembled stool sample collection kits and were instructed to bring back samples collected within 72 h of their baseline, week 4, and week 8 visits.

Full Length 16S rRNA Sequencing

Total DNA extraction was performed using the MagMax Microbiome Ultra Nucleic Acid Isolation Kit (Applied Biosystems) and the KingFisher Flex Purification System (Thermo Fisher Scientific) according to the manufacturer’s instruction. Cells were processed using the homogenizer MP FastPre-24 5G (MP Biomedical). The total DNA recovered was quantified using the Qubit 4 Fluorometer (Thermo Fisher Scientific) and the dsDNA high-sensitivity (HS) kit.

The full-length 16S rRNA gene (approximately 1500 bp in length) was amplified with some modifications to the previously described method [29]. The polymerase chain reaction (PCR) was carried out in 25 µl total volume containing 12.5 µl LongAmp Taq 2× Master Mix (New England Biolabs), 400 nM primers concentration, and 8.5 µl template DNA. Primers 27f (5′-TTTCTGTTGGTGCTGATATTGC-AGRGTTYGATYMTGGCTCAG-3′) and 1492r (5′-ACTTGCCTGTCGCTCTATCTTC-CGGTTACCTTGTTACGACTT-3′) were used to amplify the full-length 16S gene. The PCR amplification program was set as follows: initial denaturation at 95 °C for 4 min, followed by 30 cycles at 95 °C for 20 s, 51 °C for 30 s, and 65 °C for 4 min and a final extension 65 °C (5 min). Successfully amplified samples were cleaned with 0.6× Agencourt AMPure XP beads (Beckman Coulter) and resuspended in 15 µl of nuclease-free water.

Each sample was assigned a unique molecular barcode and added in a second PCR reaction using a modified version of the “PCR Barcoding Expansion 1–96 kit” (Oxford Nanopore Technologies). This barcoding PCR was carried out with 12.5 µl LongAmp Taq 2× Master Mix (New England Biolabs), 0.5 µl barcoding primer mix (10 µM), and 70 fmol 16S amplicons from the previous reaction. The PCR thermal profile included an initial denaturation at 95 °C for 3 min; 12 cycles of 95 °C for 15 s, 62 °C for 15 s, and 65 °C for 4 min; and a final extension at 65 °C for 15 min. The barcoded samples were pooled, and sequencing adapters were added according to ligation sequencing kit SQK-LSK114 instructions (Oxford Nanopore Technologies). Briefly, 12.5 µl Ligation Buffer, 5 µl Quick T4 Ligase, and 2.5 µl Ligation Adapter were added to the pool, and the reaction was incubated at room temperature for 10 min. Lastly, 10 fmol of DNA library was gently loaded onto an R10.4.1 flow cell (Oxford Nanopore Technologies). Sequencing was performed using a GridION Mk1b device (Oxford Nanopore Technologies) with live basecalling in super accurate mode (quality threshold Q12).

Data Analysis Workflow

FastQ reads were pre-processed with cutadapt v. 3.5 to remove sequencing adapters and filtered with the dada2 v. 1.28.0 R package [30]. Reads ranging from 1200 and 1800 nucleotides in length were retained. Chimeras were detected and filtered out using Minimap2 v. 2.16 and yacrd v. 1.0.0 tools. Microbial taxonomy was determined using emu v. 3.4.4 [31] mapping filtered fastQ reads against the reference Genome Taxonomy Database (GTDB) database [32].

SCFA Measurement: Stool and Plasma

All subjects were provided with pre-assembled stool sample collection kits and were instructed to bring back samples collected within 72 h of their baseline, week 4, and week 8 visits. Subjects underwent venipuncture at baseline, week 4, and week 8. The collection tubes were centrifuged, and the supernatant plasma was selectively collected and stored at − 80 °C until they were shipped for analysis.

SCFAs in stool and plasma were quantified by Metabolon (Morrisville, NC, USA). Stool or plasma samples were analyzed for the following SCFAs: acetic acid (C2), propionic acid (C3), isobutyric acid (C4), butyric acid (C4), 2-methylbutyric acid (C5), isovaleric acid (C5), valeric acid (C5), and caproic acid (hexanoic acid, C6). SCFAs were analyzed by the Metabolon Method TAM148. Prior to analysis, individual analytes were preset to target standard concentrations and calibrated. Then, all samples were spiked with stable labelled internal standards and subsequently induced to protein precipitation with an organic solvent. The sample was centrifuged, and an aliquot of the supernatant was derivatized and injected onto an Agilent 1290/SCIEX QTrap 5500 LC MS/MS system (with a C18 reverse-phase UHPLC column). The settings for the mass spectrometer were set to negative mode with electrospray ionization. Briefly, the peak area of each individual analyte product’s ions is measured and compared against the peak area of corresponding internal standards. SCFAs were then quantified by performing a weighted linear squares regression analysis. LC–MS/MS raw data was collected using SCIEX software Analyst 1.7.3 and processed with SCIEX OS-MQ software v3.1.6. Microsoft Excel for Microsoft 365 MSO was utilized to perform data reduction.

Hormone Quantification

This study quantified hormones from saliva rather than serum because previous studies that compared salivary to serum-based collections of hormones determined that both collection methodologies were similar [33]. Subjects were given saliva collection kits (ZRT Laboratory, Beaverton, OR, USA) at screening, baseline, and week 4 to bring back at baseline, week 4, and week 8, respectively. Each kit contained specific instructions on how to collect four samples of saliva, label tubes, and complete paperwork. The first sample was collected upon waking up for the day, the second sample before lunch, third before dinner, and fourth right before bedtime. Subjects were instructed to complete their collections within 24 h prior to their next visit. Upon retrieval, samples were kept frozen at − 80 °C prior to shipping and quantification by ZRT Laboratory (Beaverton, OR, USA). Salivary hormones analyzed included estradiol, estrone, DHEA, cortisol, cortisone, progesterone, 17-hydroxyprogesterone (17-OHP), androstenedione, aldosterone, and testosterone.

Investigator’s Global Assessment and Lesion Counting

A board-certified dermatologist performed acne lesion counts for inflammatory and non-inflammatory lesions at screening, baseline, week 4, and week 8. The Investigator’s Global Assessment (IGA) was utilized to calculate and grade acne severity at these same visits.

Facial Photography

Facial photographs were obtained by using high-resolution digital photographic tools such as the BTBP 3D imaging Photography System (BrighTex Bio-Photonics, LLC, San Jose, CA, USA) at screening, baseline, week 4, and week 8.

Statistical Analysis

Parametric statistical analyses were performed using Student’s t test to assess within-group (two-tailed, paired) and between-group (two-tailed, unpaired) differences. Any values of p < 0.05 were considered statistically significant and p < 0.10 was considered a trend.

Statistical analysis of microbiome abundance was performed using RStudio v. 524 (R v. 4.3.2). Community analysis was performed using phyloseq R package v. 1.46 evaluating alpha diversity indexes. The Wilcoxon rank sum test was implemented to evaluate significative differences. Beta diversity was evaluated by running a principal coordinate analysis (PCoA) based on Bray–Curtis dissimilarity index. Significant differences in beta diversity were tested using permutational multivariate analysis of variance (PERMANOVA) test (adonis2 function, vegan package).