Repurposing INCI-registered compounds as skin prebiotics for probiotic Staphylococcus epidermidis against UV-B

Abstract

Repurposing existing compounds for new indications may facilitate the discovery of skin prebiotics which have not been well defined. Four compounds that have been registered by the International Nomenclature of Cosmetic Ingredients (INCI) were included to study their abilities to induce the fermentation of Staphylococcusepidermidis (S. epidermidis), a bacterial species abundant in the human skin. Liquid coco-caprylate/caprate (LCC), originally used as an emollient, effectively initiated the fermentation of S. epidermidis ATCC 12228, produced short-chain fatty acids (SCFAs), and provoked robust electricity. Application of LCC plus electrogenic S. epidermidis ATCC 12228 on mouse skin significantly reduced ultraviolet B (UV-B)-induced injuries which were evaluated by the formation of 4-hydroxynonenal (4-HNE), cyclobutane pyrimidine dimers (CPD), and skin lesions. A S. epidermidis S2 isolate with low expressions of genes encoding pyruvate dehydrogenase (pdh), and phosphate acetyltransferase (pta) was found to be poorly electrogenic. The protective action of electrogenic S. epidermidis against UV-B-induced skin injuries was considerably suppressed when mouse skin was applied with LCC in combination with a poorly electrogenic S. epidermidis S2 isolate. Exploring new indication of LCC for promoting S. epidermidis against UV-B provided an example of repurposing INCI-registered compounds as skin prebiotics.

Full text



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Repurposing INCI‑registered
compounds as skin prebiotics
for probiotic Staphylococcus
epidermidis against UV‑B
Arun Balasubramaniam1, Prakoso Adi1, Do Thi Tra My1, Sunita Keshari2, Raman Sankar3,
Chien‑Lung Chen4 & Chun‑Ming Huang1*
Repurposing existing compounds for new indications may facilitate the discovery of skin prebiotics
which have not been well dened. Four compounds that have been registered by the International
Nomenclature of Cosmetic Ingredients (INCI) were included to study their abilities to induce the
fermentation of Staphylococcus epidermidis (S. epidermidis), a bacterial species abundant in the
human skin. Liquid coco‑caprylate/caprate (LCC), originally used as an emollient, eectively initiated
the fermentation of S. epidermidis ATCC 12228, produced short‑chain fatty acids (SCFAs), and
provoked robust electricity. Application of LCC plus electrogenic S. epidermidis ATCC 12228 on mouse
skin signicantly reduced ultraviolet B (UV‑B)‑induced injuries which were evaluated by the formation
of 4‑hydroxynonenal (4‑HNE), cyclobutane pyrimidine dimers (CPD), and skin lesions. A S. epidermidis
S2 isolate with low expressions of genes encoding pyruvate dehydrogenase (pdh), and phosphate
acetyltransferase (pta) was found to be poorly electrogenic. The protective action of electrogenic S.
epidermidis against UV‑B‑induced skin injuries was considerably suppressed when mouse skin was
applied with LCC in combination with a poorly electrogenic S. epidermidis S2 isolate. Exploring new
indication of LCC for promoting S. epidermidis against UV‑B provided an example of repurposing INCI‑
registered compounds as skin prebiotics.
Drug repurposing or repositioning is an eective approach to rapidly identify novel indications from knowncom-
pounds1,2. ere have been numerous successful cases of repurposed drugs, including sildenal citrate (Viagra)
as a medicine for erectile dysfunction and pulmonary arterial hypertension and raloxifene hydrochloride (Evista)
as a treatment for osteoporosis in postmenopausal women3. Repurposing drugs, including Remdesivir4 and
Dexamethasone5, to discover potential forms of treatment for severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) is actively ongoing. Skin diseasesaect half of the world’s population and novel drugs for treat-
ments of skin diseases are in high demand. eskin isthe largest organ in humans and continuously protects
the humans from the harmful environment6,7. Over 100 distinct species, which contribute to making up a total
of 1 million microbes in the skin microbiome, conquer every square centimeter of human skin6,8. Our previous
studies have demonstrated that fermentation of skin probiotic bacteria generated benecial metabolites, such
as short-chain fatty acids (SCFAs), that can attenuate skin disorders9. For example, Staphylococcus epidermidis
(S. epidermidis), a common member in the human skin microbiome, can fermentatively metabolize carbon-rich
molecules as prebiotics to yield SCFAs against pathogenic Staphylococcus aureus (S. aureus)10.
Prebiotics were originally dened by Gibson and Roberfroid in 1995 as nondigestible food ingredients11. is
denition was later revised in12 and13 as “Glucose-based dietary bers and non-carbohydrate substances including
polyunsaturated fatty acid (PUFA) have been used as prebiotics for gut bacteria”. Prebiotics for bacteria in the
skin and other human organs are not yet dened. Prebiotics can provide probiotic bacteria as carbon sources
to initiate the fermentation and produce SCFAs as acetate and butyrate14. It has been reported that oxidation of
acetate or butyrate served as an electron donor to discharge electron to electron acceptors15. e gene-encoding
proteins in the extracellular electron transfer (EET) family of homologs are believed to be present in both Gram-
negative and Gram-positive bacteria16. Unlike Gram-negative bacteria, Gram-positive bacteria, not having an
OPEN
Department of Biomedical Sciences and Engineering, National Central University, Taoyuan, Taiwan. Department
of Life Sciences, National Central University, Taoyuan, Taiwan. Institute of Physics, Academia Sinica, Nankang,
Taipei, Taiwan. Division of Nephrology, Landseed International Hospital, Taoyuan, Taiwan. *email: chunming@
ncu.edu.tw
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outer membrane, carry a cell envelope with a textured peptidoglycan layer and teichoic acids that are thought
to be poorly electrogenic17. However, a avin-based EET process has been recently identied in Gram-positive
bacteria to produce electricity16 by activation of type II NADH hydrogenase, which can catalyze electron exchange
from cytosolic NADH to a quinone derivative such as quinone demethylmenaquinone (DMK)16. Our recent
studies demonstrated that S. epidermidis can mediate glycerol as a source to generate electricity and enhanced
bacterial resistance to UV-B18.
In this study, we selected four carbon-rich molecules that have been listed on the International Nomencla-
ture of Cosmetic Ingredients (INCI) to examine their prebiotic activities. e liquid coco-caprylate/caprate
(LCC) with C8-10 fatty acid connected to C12-C18 fatty alcohols is currently used as an emollient on ultraviolet
(UV) lter absorbance19. Isononyl isononanoate (ININ) (C18H36O2) is an ingredient in cosmetics and personal
care products as an emollient, texture enhancer, and plasticizer20. Polyethylene glycol (PEG)-150 distearate
(PDS) [(C2H4O)n.C36H70O3] is a thickening agent for shampoo products21. PEG-150 pentaerythrityl tetrastearate
(PETIS) (C77H148O8) has been used for increasing viscosity of an aqueous agent in cosmetics22. To screen these
four carbon-rich molecules for repurposing, we examined their prebiotic activities for induction of fermenta-
tion of S. epidermidis. It has been documented that S. epidermidis can mediate fermentation to down-regulate
UV-B-induced inammation in mouse skin9. We thus further explored the mechanism by which repurposing
carbon-rich molecules as skin prebiotics inuence the skin damage induced by UV-B. UV radiation which
provokes free radical formation, making it a primary risk factor for skincancer23. It has been reported that UV,
in particular UV-B, radiation can up-regulate the local neuroendocrine axes, induce the release of hormones to
circulation, activate the central hypothalamic–pituitary–adrenal axis, and reset body hemostasis against skin
disorders including cancers, aging and autoimmune diseases24. Repurposing may facilitate the discovery of new
mechanisms of action for INCI-registered ingredients as skin prebiotics against UV injuries.
Methods
Ethics statement. All animal protocols and experiments have been approved by the National Central
University (NCU), Taiwan. Experiments were conducted in accordance with the protocols (NCU-106-016, 19
December 2017) of the Institutional Animal Care and Use Committee (IACUC) of National Central University
(NCU). Female ICR mice (8–9weeks old) were purchased from the National Laboratory Animal Center Tai-
pei, Taiwan. CO2 sedation was used to sacrice mice in an encased chamber. All human study protocols were
approved by Institutional Review Board (IRB) (No. 19-013-B1, 22 May 2019) and Ethics Committee of Land-
seed International Hospital, Taiwan. e methods followed for skin swab sampling procedure were carried out
in accordance with relevant guidelines and regulations of IRB which was approved by Landseed International
Hospital, Taiwan. Skin swabs were collected from three healthy subjects and informed consent was obtained
from all study participants.
Bacterial fermentation. Staphylococcus epidermidis ATCC 12228 in tryptic soy broth (TSB) (Sigma, St.
Louis, MO, USA) was cultured overnight at 37°C. Bacterial growth was determined at 600nm wavelength
(OD600). e bacterial pellet was collected aer centrifugation at 5,000 × g for 10min, resuspended with 1 × PBS
and diluted to 107CFU/ml before further incubation in rich media [1.5g/l KH2PO4, 10g/l yeast extract (Biokar
Diagnostics, Beauvais, France), 2.5g/l K2HPO4, 3g/l TSB, and 0.002% (w/v) phenol red (Merck, Darmstadt,
Germany)] at 37°C. For fermentation, bacteria in rich media in the presence of 2% LCC, ININ, PDS or PETIS
(TNJC corporation, Chiayi, Taiwan) were incubated for 12h. e color change of phenol red from red to yellow
indicated bacterial fermentation, which was quantied by measurement of OD562. Bacteria alone or rich media
with or without LCC, ININ, PDS or PETIS served as controls. To examine the eect of LCC on the bacterial
growth, S. epidermidis ATCC 12228 [107 colony-forming unit (CFU)/ml)] was incubated with 2% LCC or phos-
phate buer saline (PBS) for 12h at 37°C. Aer incubation, bacteria were serially diluted 1: 100–1:105 in a 96 well
plate. 10μl of serially diluted bacteria were dropped on a TSB agar plate for CFU counts.
Electricity detection. Electricity produced by S. epidermidis was detected in vitro using a chamber
equipped with cathode and anode. A carbon felt (2.5cm × 10cm) and a carbon cloth (10cm × 10cm) (Homy
Tech, Taoyuan, Taiwan) were utilized to fabricate anode and cathode., respectively. e cathode was wrapped up
to a Naon membrane N117 (6cm × 6cm) (Homy Tech), which served as a proton exchange membrane (PEM).
Copper wires were used to connect anode and cathode with external resistance (1,000 Ω)18. S. epidermidis in
the presence or absence of LCC, ININ, PDS or PETIS was pipetted on the surface of the anode. Electricity was
recorded by the changes in voltage (mV) against time (min) using a digital multimeter (Lutron, DM-9962SD,
Sydney, Australia). e recorded voltages in every 10s were used for plotting a graph.
Cyclic voltammetry. e three-electrode autolab potentiostat (PGSTAT 128N, Metrohm Autolab, Utrecht,
Netherland) was used for conducting cyclic voltammetry. e screen-printed carbon electrode (SPCE) (SE-100,
Zensor R&D, Taichung, Taiwan) served as a working electrode with a working area of 5mm. e Ag/AgCl elec-
trode and platinum electrode acted as the reference against applied potential and counter electrode, respectively.
All electrodes were purchased from Metrohm Autolab. S. epidermidis (107CFU/μl) in the presence or absence of
2% of LCC, ININ, PDS or PETIS was drop-coated on the surface of a working electrode. e potential windows
were inspected between −0.8 and 0.2V at 0.005V/s. PBS at 7.4 pH was used as an electrolyte. e potentiostat
was operated using Autolab Nova 2.0 soware (https ://metro hm-autol ab.com/Produ cts/Echem /Sow are/Nova.
html/; Metrohm Autolab, Utrecht, Netherland).
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Extraction of bacterial RNA. RNA was extracted from overnight cultured S. epidermidis ATCC 12228
or a S2 isolate (107CFU/ml). e cultured bacteria were centrifuged at 5,000 × g for 10min and the pellet was
collected. RNA in bacterial pellet was extracted using a total RNA mini purication kit (Biokit, Miaoli, Taiwan)
and quantied by UV spectrophotometry in a Synergy HT multi-mode microplate reader (BioTek Instruments
Inc., Highland Park, Winooski, Vermont, USA).
Real‑time qPCR (RT‑qPCR). RT q-PCR was used to analyze the expression of genes encoding pyruvate
dehydrogenase (pdh), phosphate acetyltransferase (pta) and intracellular adhesion A (ica A) in S. epidermidis
ATCC 12228 and S2 isolate. RNA (1ng) was converted to cDNA using an iScript cDNA Synthesis Kit (Bio-Rad,
Hercules, CA, USA). e cDNA was served as a template in StepOnePlus RT PCR System (ermo Fisher Scien-
tic, Waltham, MA, USA), which was executed using Power SYBR Green and PCR Master Mix (ermo Fisher
Scientic). e primer-Blast tool (https ://blast .ncbi.nlm.nih.gov/Blast .cgi/; Rockville Pike, Bethesda MD, USA)
from the National Center for Biotechnology Information (NCBI) was used for designing all primers. Total one
step RT-PCR reaction condition was xed for 40 cycles as follows: 95°C for 10min followed by 95°C for 15s,
60°C for 60s, and 72°C for 30s. Gene expression was normalized with the 16S rRNA gene. e cycle threshold
(2−ΔΔCt) was implemented to analyze the relative expression of genes. e designed primers for all genes were
shown in TableS2.
Gas chromatography‑mass spectrometry (GC–MS) analysis. Staphylococcus epidermidis ATCC
12228 (107CFU/ml) with 2% LCC in rich media was cultured for 24h and then centrifuged at 5,000 × g for
10min. Supernatants of bacterial culture were collected and ltered through 0.22µm lters. e GC–MS proto-
col for SCFAs detection were obtained following the method25.
UV‑B exposure. Mouse hair in the dorsal skin was removed using Nair cream (Church and Dwight, Ewing
Township, NJ, USA) one day before experiments started. e dorsal skin of each mouse was exposed to 100mJ/
cm2 UV-B irradiation using a UV lamp (Model EB-280C, Spectronics Corp., Westbury, NY, USA) twice a week,
followed by subsequent application of 107CFU/ml S. epidermidis with or without 2% LCC three times per week
for two weeks. e images of mouse skin were captured on day 0, 7 and 14. Skin lysates and sections from dorsal
skin (1cm2) was prepared. Skin sections were stained with hematoxylin and eosin (H&E) and visualized using
the Olympus BX63 microscope (Olympus, Tokyo, Japan).
Western blotting. Tissue Protein Extraction Reagent (T-PER) (ermo Fisher Scientic) was used for pre-
paring the skin lysates. Protein concentrations of skin lysates were measured by a bicinchoninic acid (BCA)
assay (Bio-Rad). Skin lysates (30μg) were loaded to a 10% sodium dodecyl sulphate–polyacrylamide gel elec-
trophoresis (SDS-PAGE) gel and transferred to a PVDF membrane (Millipore Sigma, Burlington, MA, USA).
e membrane was blocked with 5% (w/v) non-fat milk, and incubated with primary antibodies to cyclobutane
pyrimidine dimer (CPD) (1:1,000; Cosmo Bio, Tokyo, Japan), 4-hydroxynonenal (4-HNE) (1:2,000; Abcam,
Cambridge, MA, USA), or β-actin (1:5,000; ACE Biolabs, Taoyuan, Taiwan) overnight at 4°C. e membrane
was subsequently incubated with secondary antibodies goat anti-rabbit or anti-mouse IgG (H + L) horseradish
peroxidase (HRP) (1:5000; ACE Biolabs) for 1h. Protein bands in membranes were developed using chemilu-
minescent detection reagent (ermo Fisher Scientic) and visualized by an Omega Lum C Imaging System
(Gel Co., San Francisco, CA, USA). ImageJ soware (https ://image j.nih.gov/ij/index .html/; National Institutes
of Health, Bethesda, MD, USA) was employed to quantify the intensities of protein bands.
Statistical analysis. GraphPad Prism 8 (https ://www.graph pad.com/; GraphPad Soware, La Jolla, CA,
USA) soware was employed for data analysis by unpaired t-test. e signicant dierence was considered by
P-values observation as follows: P-values of < 0.05 (*), < 0.01 (**), and < 0.001 (***). e mean ± standard devia-
tion (SD) was obtained from at least three separate experiments.
Results
INCI‑registered compounds function as skin probiotics for induction of SCFA production and
bacterial electricity. Our previous studies have demonstrated the fermentation and electrogenic activities
of S. epidermidis in the presence of glycerol as a carbon source18. To examine if INCI-registered compounds can
act as skin prebiotics that provides carbon sources to induce fermentation of skin bacteria, we cultured S. epider-
midis ATCC 12228 (107CFU/ml), a non-biolm forming skin bacterium, in rich media containing phenol red
with 2% each individual INCI-registered compound including LCC, ININ, PDS, and PETIS for 12h. Media with
bacteria alone served as controls. A change in color of phenol red from red to yellow and a signicant reduction
of the optical density of 562nm (OD562) due to low pH values26 in the culture media of the S. epidermidis in
the presence of 2% each individual INCI-registered compound served as indications of bacterial fermentation.
As shown in Fig.1A, 2% LCC and ININ, compared to PDS and PETIS, induced signicant fermentation of S.
epidermidis by detection of yellowish media and OD562 reduction. e reduction of OD562 induced by LCC was
greater than that by ININ. To validate the occurrence of fermentation, six SCFAs including acetate, butyrate,
hexanoate, isobutyrate, isovalerate, and propionate in the media of LCC fermentation were detected by GC–MS
analysis (Fig.1B). A high amount of acetate (> 15mM) was produced by LCC fermentation of S. epidermidis.
SCFAs, especially acetate and butyrate, have been proved as potent electron donors during the EET process in
bacteria27–29. We thus investigated the electrogenic activity of S. epidermidis in the presence of INCI-registered
compounds. Changes in voltages in an invitro chamber and current values detected by cyclic voltammetry30
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were used to monitor bacterial electricity. Compared to media with S. epidermidis alone, media with S. epider-
midis plus ININ, PDS or PETIS did not elicit high voltage changes and currents. By contrast, a robust increase
in voltage changes with a peak voltage of approximately 6mV and currents was detected in the media with S.
epidermidis plus LCC (Fig.2). e result provided evidence for repurposing INCI-registered LCC as a prebiotic
to provoke the fermentation and electricity production of skin S. epidermidis bacteria.
Figure1. LCC fermentation of S. epidermidis. (A) S. epidermidis ATCC 12228 (107CFU/ml; B) was incubated
for 12h in rich media in the presence or absence of 2% LCC, ININ, PDS or PETIS. e prevalence of
fermentation was indicated by colour change of phenol red from red to yellow and quantied by OD562. (B) S.
epidermidis ATCC 12228 (107CFU/ml) in the presence of 2% LCC in rich media was cultured for 24h. e
levels (mM) of six SCFAs (acetate, butyrate, hexanoate, isobutyrate, isovalerate and propionate) in fermentation
media were quantied. Data are the mean ± SD from three separate experiments. ***P < 0.001 (two-tailed t-test).
Figure2. Electricity production by LCC fermentation of S. epidermidis. (A) e electricity measured by voltage
changes (mV) was recorded for 20min aer pipetting S. epidermidis alone or with LCC (B/LCC), ININ (B/
ININ), PDS (B/PDS) or PETIS (B/PETIS) on the surface of an anode. (B) Cyclic voltammetry was employed to
measure the current (µA) generated by various experimental conditions above. Data presented the mean ± SD
from three separate experiments. ***P < 0.001 (two-tailed t-test).
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Topical application of S. epidermidis plus LCC reduced UV‑B‑induced the formation of 4‑HNE
and CPD. It has been documented that UV-B-induced free radicals can cause skin hyperplasia31, lipid
peroxidation32 and the CPD formation33. We next assessed the inuence of fermentation and electricity pro-
duced by S. epidermidis plus LCC on the UV-B-induced skin injuries. e recurrent exposure of UV-B signi-
cantly fostered the formation of 4-HNE and CPD on mouse skin topically applied with LCC or S. epidermidis
alone. In addition, UV-B-induced epidermal hyperplasia, as characterized by an increase in epidermal thickness
(Figure S1) and lesions (Fig.3C), can be detected on mouse skin topically applied with LCC or S. epidermidis
alone. However, the UV-B-induced the formation of 4-HNE, CPD, epidermal hyperplasia and lesions were con-
siderably attenuated when mouse skin was topically applied with S. epidermidis plus LCC (Fig.3, Figure S1).
Topical application of PBS or LCC alone without S. epidermidis on mouse skin before UV-B irradiation exhibited
the same levels of 4-HNE and CPD as well as skin lesions (Figure S2), indicating that LCC itself was insucient
to impede the UV-B-induced skin injuries. Data above dened a novel function for the repurposed LCC in con-
junction with S. epidermidis for suppressing UV-B-induced skin injuries.
The S. epidermidis S2 isolate with low expression of pdh and pta genes was poorly electro‑
genic. Since LCC eectively induced S. epidermidis to undergo fermentation and yield electricity, we next
determined the expression of genes related to fermentation, acetate production, and biolm formation in S.
epidermidis ATCC 12228 and a S. epidermidis S2 strain isolated from human skin. e pdh gene encodes for
pyruvate dehydrogenase, which catalyzes pyruvate to acetyl coenzyme A (acetyl-CoA) at the upstream site of the
fermentation pathway34. e pta gene encoding for phosphate acetyltransferase is involved in the conversion of
acetyl-CoA to acetate35. e icaA gene encoding for intracellular adhesion A has been well characterized in the
engagement of the biolm formation in S. epidermidis36,37. e 16s rRNA sequence of S. epidermidis S2 isolate
shared 99.8% identity to that of S. epidermidis ATCC 12228 (TableS1). However, the expressions of pdh and pta
genes in S. epidermidis S2 isolate were much lower than those in S. epidermidis ATCC 12228 (Fig.4A). Further-
more, the activity of LCC fermentation monitored by OD562 reduction of phenol red-containing rich media for
S. epidermidis S2 isolate was relatively low compared to that in S. epidermidis ATCC 12228 (Fig.4B). e high
expression of icaA gene (Fig.4A) and obvious biolms (Fig.4D) were detected in S. epidermidis S2 isolate, but
not in the non-biolm forming bacterial strain, S. epidermidis ATCC 12228. Interestingly, unlike S. epidermidis
ATCC 12228, the S. epidermidis S2 isolate that expressed low levels of pdh and pta genes was poorly electrogenic.
As shown in Fig.4C, consistent with Fig.2, a peak voltage of approximately 6mV was detected in media with S.
epidermidis ATCC 12228 plus LCC whereas little or no voltage change was measured in S. epidermidis S2 isolate
Figure3. Eect of S. epidermidis in the presence of LCC on the UV-B-induced formation of 4-HNE, CPD and
lesions. e dorsal skin of ICR mice topically applied with LCC alone, S. epidermidis ATCC 12228 alone (B), or
S. epidermidis plus LCC (B/LCC) was irradiated with (+ UV) or without (− UV) UV-B. e images of protein
bands of (A) 4-HNE or (B) CPD analyzed by western blot were displayed. e ratio of intensities of protein
bands of 4-HNE or CPD normalized to β-actin was shown. (C) Morphologies of mouse skin irradiated with
(+ UV) or without (− UV) 100mJ/cm2 UV-B 0, 7, and 14days aer irradiation were shown. Skin lesions were
indicated by white arrows. Data are the mean ± SD from three independent experiments. ***P < 0.001 (two-tailed
t-test). ns = non-signicant.
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plus LCC. Collectively, the pdh and pta genes may participate in fermentation and electricity production of S.
epidermidis triggered by LCC.
LCC plus S. epidermidis S2 isolate did not confer protection against UV‑B‑induced skin inju‑
ries. To conrm the critical roles of pdh and pta genes in LCC fermentation of S. epidermidis against UV-B
invivo, dorsal skin of ICR mice was topically applied with S. epidermidis ATCC 12228 or S2 isolate in the pres-
ence of LCC before UV-B irradiation. e levels of 4-HNE and CPD in mouse skin were measured by western
blot. In agreement with data in Fig.3, aer UV-B irradiation, the formation of 4-HNE, CPD and skin lesions
were detected at low levels and moderate on mouse skin topically applied with S. epidermidis plus LCC. How-
ever, when mouse skin was topically applied with S. epidermidis S2 isolate plus LCC (Fig.5A–C), UV-B induced
signicantly increasing levels of 4-HNE and CPD as well as skin lesions. Since the expressions of pdh and pta
genes in S. epidermidis S2 isolate were much lower than those in S. epidermidis ATCC 12228 (Fig.4), the expres-
sions of pdh and pta genes may mediate the LCC-triggered promotion of S. epidermidis against skin injuries
caused by UV-B.
Discussion
LCC is an ester obtained from the reaction of the coconut alcohol-derived fatty acids with a mixture of caprylic
acid and capric acid38. Both caprylic acid and capric acid, as medium-chain fatty acids, can be extracted from
coconut oil and have been used as ingredients for skincare formulation to protect against UV radiation39,40. It
has been reported that addition of caprylic acid into the fermentation process enhanced acetate production41.
Coconut oil has been used as a fuel for electricity production42. Although many medium-chain fatty acids exhib-
ited potent bactericidal activities43, our result in Figure S3 demonstrated that LCC did not change the growth of
S. epidermidis. Compared to other INCI-registered compounds (ININ, PDS and PETIS), LCC displayed higher
activity in promoting fermentation and electricity production of S. epidermidis (Fig.1). Biolms are electroactive
and can promote the electricity production of bacteria44. By using S. epidermidis ATCC 12228, a non-biolm
forming strain, we demonstrated that skin bacteria can produce electricity without biolm formation on elec-
trodes. Data in our recent publication has revealed that addition of glycerol into the S. epidermidis culture can
induce fermentation and instantly produce detectable electricity, highlighting a possible mechanism that skin
bacteria underwent fermentation to accumulate SCFAs as electron donors to intensify electricity18.
Results in Fig.1 demonstrated that the electricity measured by changes in voltage and currents was con-
siderably produced when the anode was pipetted with S. epidermidis ATCC 12228 plus 2% LCC. However, the
electricity produced by bacteria plus LCC was largely reduced in the S. epidermidis S2 isolate which expressed
Figure4. e gene (pdh, pta, and icaA) expression, electricity and biolm formation in S. epidermidis ATCC
12228 and S2 isolate. (A) Relative expression of pdh, pta, and icaA genes normalized to 16S rRNA was analyzed
by the RT-qPCR. (B) LCC fermentation for 12h in rich media was quantied by measurement of OD562. (C)
Electricity production of bacteria in the presence of 2% LCC was analyzed by voltage changes (mV) in an
invitro chamber. (D) Bacterial biolms were stained by crystal violet aer culture of bacteria in TSB on a
24-well plate for 48h. B/LCC: S. epidermidis ATCC 12228 plus 2% LCC; S2/LCC: S. epidermidis S2 isolate plus
2% LCC. Scale bars = 1cm. Data are the mean ± SD from three separate experiments. **P < 0.01; ***P < 0.001
(two-tailed t-test).
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lower levels of pdh and pta genes (Fig.3). e data indicated that proteins corresponding to pdh and pta genes
may play a role in electricity production of S. epidermidis in the presence of LCC. Electrons are derived by the
reduction reaction of NAD+ to NADH in the metabolic pathway of bacterial fermentation45. e conversion of
NAD+ to NADH is involved in pyruvate dehydrogenase (pdh), which initiates the process of electron transport
chain45,46. Previous studies have shown that electricity production of Shewanella oneidensis MR-1, a representa-
tive electrochemically active bacterium (EAB) extensively studied in the laboratory, was mediated by activation
of NAD+-linked PDH47. Phosphate acetyltransferase (pta) can catalyze the conversion of acetyl-CoA to acetate,
a known electron donor. A pta knockout strain of Shewanella oneidensis strain has been used to study the eect
of electricity on bacterial fermentation48. e protective eect of bacterial fermentation on the suppression of
the UV-B-induced formation of 4-HNE and CPD was remarkably diminished when mouse skin was topically
applied with LCC and S. epidermidis S2 isolate which yielded low electricity and expressed lower levels of pdh and
pta genes (Fig.5). e data suggested that SCFAs and electricity induced by LCC fermentation of S. epidermidis
may synergistically provide mice protection from UV-B injuries. Future studies will include the construction
of a pdh knockout S. epidermidis strain and investigation of the essential role of pdh gene in S. epidermidis for
production of electricity against UV-B injuries.
4-HNE is known to be genotoxic and damages DNA by producing bulky 1,N2-propano-2′-deoxyguanosine
adducts49. Here, we showed that 4-HNE and CPD were formed when mouse skin was constantly bombarded
with UV-B. Results in our previous studies have demonstrated that butyrate produced by glycerol fermentation
of S. epidermidis can down-regulate UV-B-induced pro-inammatory interleukin (IL)-6 cytokines through SCFA
receptor 2 (FFAR2)50. us, skin bacteria may take advantage of endogenous glycerol as a carbon source to pro-
voke fermentation and simultaneously produced SCFAs and electrons. Besides being electron donors, SCFAs may
regulate FFAR2 and/or histone deacetylases (HDAC)51 to mitigate the inammation induced by UV irradiation.
Electrons may play a key role in neutralizing free radicals generated by UV irradiation. It has been shown that UV
light can mediate nitrate, a constituent of sweat in skin, to generate free radicals52, which can subsequently induce
lipid peroxidation to produce 4-HNE53. However, commensal bacteria can utilize nitrate as an electron acceptor
Figure5. e UV-B-induced formation of 4-HNE, CPD and lesions in mouse skin applied with LCC in
combination with S. epidermidis S2 isolate. e dorsal skin of ICR mice was topically applied with S. epidermidis
ATCC 12228 (B/LCC) or S. epidermidis S2 (S2/LCC) in the presence of 2% LCC. e levels of (A) 4-HNE, (B)
CPD related to β-actin in western blot analysis and (C) lesions (at 0, 7, and 14days post-irradiation) on mouse
skin irradiated with 100mJ/cm2 UV-B (+ UV) were shown. Skin lesions were indicated by white arrows. Data
are the mean ± SD from three separate experiments. **P < 0.01; ***P < 0.001 (two-tailed t-test).
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for energy transduction54. For example, under anoxic or oxygen-depleted conduction, Pseudomonas species can
mediate denitrication by using nitrate as an electron acceptor to convert nitrate to nitrogenous gases55. us,
a possible mechanism behind the protective eect of S. epidermidis plus LCC on UV-B skin injuries is that LCC
triggers bacteria to produce electrons, activating bacterial denitrication to reduce UV-B-induced free radicals.
In summary, prebiotics for skin probiotic bacteria have been not dened by the Food and Drug Administra-
tion (FAD) even though they may become novel therapeutics for treatments of skin disorders via induction of
fermentation of skin bacteria. Our study here demonstrated an approach by repurposing INCI-registered com-
pounds as skin prebiotics and revealed their capabilities of generating electricity from skin bacteria to combat
UV-B-induced skin damages.
Received: 6 October 2020; Accepted: 12 November 2020
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Acknowledgements
e study was supported by the Ministry of Science and Technology (MOST) Grants (108-2622-B-008-001-CC1;
108-2314-B-008-003-MY3, and 107-2923-B-008-001-MY3) and 106/107/108-Landseed Hospital-NCU joint
Grants. We thank Supitchaya Traisaeng at National Central University for assistance at biolm staining.
Author contributions
A.B.: Methodology; A.B., and P.A.: Soware; A.B., P.A., D.T.T.M., S.K., and R.S.: Data curation; A.B.: Writing-
Original dra preparation; C.-M.H. and C.-L.C.: Ethical evaluation; C.-M.H.: Conceptualization, Visualization,
Investigation, Methodology, Supervision, Writing-Reviewing and Editing.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-78132 -5.
Correspondence and requests for materials should be addressed to C.-M.H.
Reprints and permissions information is available at www.nature.com/reprints.
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