Heat‐treated Pediococcus acidilactici LM1013‐mediated inhibition of biofilm formation by Cutibacterium acnes and its application in acne vulgaris: A single‐arm clinical trial

Acne vulgaris is a common skin disease accompanied by chronic inflammation in the pilosebaceous follicles, resulting from excessive Cutibacterium acnes. This study aimed to investigate the inhibition of biofilm formation by C. acnes ATCC 6919 using heat‐treated Pediococcus acidilactici LM1013 (HT‐LM1013), previously isolated from the Korean traditional fermented alcoholic beverage—makgeolli, and its application as a leave‐on‐type product for patients with acne vulgaris.


| INTRODUC TI ON
2][3] Although it is not a deadly disease, it can cause depression and lower self-esteem. 4Acne vulgaris accompanied by multifactorial chronic inflammation in pilosebaceous follicle results in excessive sebum production and follicular keratinization associated with Cutibacterium acnes. 1,4,5Overpopulation of C. acnes in pustules aggravates acne vulgaris symptoms, and therefore antibiotics have been prescribed in patients with acnes vulgaris. 2Clindamycin, a lincosamide antibiotic, is effective in the treatment of acne vulgaris. 2,3indamycin blocks protein synthesis and inhibits free fatty acid production in C. acnes. 3However, clindamycin therapy can induce antibiotics resistance on C. acnes. 2,3tibacterium acnes thrives in microcomedones developed by excessive sebum production.Microcomedones provide a lipid-rich and anaerobic environment for the organism. 5C. acnes releases lipase for hydrolyzing triglycerides in the sebum to free fatty acids and glycerol. 5,6Free fatty acids play a role as inflammatory, chemotaxis, and comedogenic agents 4 and induce damage to sebaceous follicle cells. 4,5ycerol is a nutrient source for C. acnes. 4,5Biofilm formation is another property of C. acnes associated with pathogenicity and antibiotics resistance. 6,7Biofilm is initiated by adhering planktonic bacterial cells to environmental surfaces and developing extracellular matrix consisting of glycosyl residues, 7 extracellular DNA, proteins, 7,8 phospholipids, and exopolysaccharides 8 for the bacterial community. 6diococcus acidilactici LM1013 (previously reported as P. acidilactici K3) was isolated from nuruk, a starter of makgeolli, which is a traditional Korean fermented alcoholic beverage. 9,10P. acidilactci LM1013 showed high alcohol tolerance given its intrinsic genomic properties, such as genes for alcohol dehydrogenase, aldehyde dehydrogenase, and malate dehydrogenase. 9Probiotics are wellknown natural antimicrobial agents, 11 although live microorganisms are restricted as cosmetic ingredients because of spoilage.However, postbiotics, which are inactivated probiotics, including lysates and metabolites, 12 show remarkable stability during storage and can be applied to various cosmetics.Therefore, heat-treated P. acidilactici LM1013 (HT-LM1013) was investigated for the inhibition of C. acnes biofilm formation and applied as a leave-on-type product for patients with acne vulgaris.Lyophilized HT-LM1013 received the product number (S-210426-2)

| Chemicals and microorganism
and was stored at −80°C until further use.
To investigate the inhibition of biofilm formation by C. acnes ATCC 6919, HT-LM1013 lysate was prepared using a high-pressure homogenizer (Nano DeBEE High-Pressure Homogenizer, BEE International).Briefly, a 1% (w/v) HT-LM1013 suspension was homogenized using a high-pressure homogenizer at 2500 bar.The homogenized suspension was collected in a sterile bottle and filtered through a 0.20μm PES filter under vacuum conditions.The filtered lysate was stored at −20°C until further use.

| Analysis of HT-LM1013 fatty acids
Cellular fatty acid composition was analyzed using a gas chromatography/mass selective detector (GC/MSD) according to our previous study. 12GC/MSD system was composed of an Agilent 8890 gas chromatography system with a 5977 B MSD and a 7693A automated liquid sampler (Agilent).Fatty acid methyl esters (FAME) were extracted using the Bligh and Dyer method with modifications.FAME were separated by Agilent J&W DB-FastFAME capillary column packed with cyanopropyl (30 m × 0.25 mm, 0.25 μm).Ultrapure helium was used as the carrier gas at a flow rate of 1 mL/min.The injection port temperature was 250°C under constant flow, and 1 μL of the sample was injected using the split mode of 20:1.The oven temperature was initiated at 50°C for 1 min, raised from 50 to 165°C at a rate of 60°C/min, maintained at 165°C for 1 min, raised from 165 to 230°C at a rate of 2.5°C/min, and maintained for 3 min.
The temperatures of the ion source and transfer line were 230 and 250°C, respectively.Mass spectra were obtained using electron ionization at 70 eV and recorded m/z 40-550 of mass range.Methyl undecanoate was used as the internal standard.

| Antimicrobial activity of P. acidilactici LM1013 and its lysate
The antimicrobial ability of P. acidilactici LM1013 was assessed using the agar spot method. 11Five microliters of P. acidilactici LM1013 culture was spotted on MRS agar and incubated at 37°C for 24 h.After incubation, 5 mL of RCM agar was overlaid onto P. acidilactici LM1013 cultured on MRS agar.The overlaid RCM agar was solidified for 30 min.Approximately 8 Log CFU/mL of was spread on overlaid RCM agar and further incubated for 72 h at 37°C under anaerobic condition.The inhibition zones indicated the antimicrobial effect of P. acidilactici LM1013.
The minimum inhibitory concentration (MIC) value was determined by microdilution assays. 13The lysate was diluted twofold using RCM broth in a 96-well microplate.After dilution, approximately 6 Log CFU/mL of C. acnes was added to each well and further incubated at 37°C for 72 h under anaerobic conditions.The lowest concentration in which C. acnes was not visually detected was set as the MIC.The number of viable C. acnes ATCC 6919 cells was counted to determine the bacteriostatic effect of the lysate.

| Tricarboxylic acid cycle and lipase inhibition
Tricarboxylic acid (TCA) cycle activity was measured using INT solution, according to a previous study. 13Briefly, approximately 7 Log CFU of C. acnes ATCC 6919 was incubated with the lysate (1/4 × MIC, 1/2 × MIC, and 1 × MIC, respectively) for 24 h.After incubation, C. acnes ATCC 6919 was harvested by centrifugation at 13 572 g and 4°C for 10 min.The harvested cells were washed by phosphate-buffered saline (pH 7.2).Washed cells were diluted to an OD 600 of 0.1 and INT solution was added to 1 mM.After 30 min, the amount of soluble formazan was measured using a microplate reader (SpectraMax iD3; Molecular Devices) at 630 nm.
To evaluate lipase inhibition of the lysate, supernatants were separated from C. acnes ATCC 6919 cultures treated with the lysate of HT-LM1013 for measuring TCA cycle inhibition.Lipase substrates (4-MU palmitate, 4-MU stearate, and 4-MU oleate) were dissolved in a 13 mM Tris-HCl solution containing 150 mM sodium chloride and 1.3 mM calcium chloride (pH 8.0).Fifty microliters of each supernatant was mixed with the same volume of lipase substrate (0.2 mg/mL).The mixtures were then incubated at 25°C for 30 min under illumination.The enzyme activity was terminated using 100 μL of 100 mM sodium citrate solution (pH 4.2).Enzyme activities were measured by measuring the fluorescence of 4-MU using a microplate reader.The excitation and emission wavelengths were 360 and 449 nm, respectively. 1

| Inhibition and degradation of biofilm
The inhibitory effect on biofilm formation was measured as described in a previous study [6][7][8] with modifications.To investigate the effect, C. acnes ATCC 6919 (3 × 10 8 CFU/mL) was added to a 96-well microplate, after which each well was treated with the lysate ranging from 1/256 × MIC to 1 × MIC. C. acnes ATCC 6919 without lysate treatment was confirmed by the formation of mature biofilms as a control.The biofilms were incubated for 72 h at 37°C under anaerobic conditions.Biofilm degradation was measured in mature biofilms treated with the lysates.Briefly, C. acnes ATCC 6919 (1.5 × 10 8 CFU/mL) was incubated in a 96-well microplate to establish mature biofilms.After incubation, the supernatants were removed and mature biofilms were gently washed with phosphate-buffered saline (PBS).Lysates (1/256 × MIC to 1 × MIC) were added to mature biofilms and incubated for 24 h at 37°C under anaerobic conditions.The biofilms were gently washed with PBS and dried at 37°C for 30 min.Dried biofilms were stained with 0.1% crystal violet solution for 30 min and excess dye was gently removed.Crystal violet was extracted from the biofilm using 100% ethanol, and the absorbance was measured using a microplate reader at 570 nm.

| Study design
This study was a single-arm clinical trial that compared acne vulgaris conditions in patients for 4 weeks.The products for the clinical trial were prepared by Celltrion Skincure.The products, such as alpha-hydroxy acids and salicylic acid, did not contain any ingredients for acne vulgaris treatment.

| Irritation test
An irritation test was conducted using an IQ Ultimate chamber (Chemotechnique MB Diagnostics AB).Briefly, 20 μL of the leaveon type product was added to the chamber and applied to the skin.
After 24 h, skin irritation was assessed by experts.

| Experimental procedure
All participants applied the test product to the face before sleeping for 4 weeks.Briefly, a white transparent leave-on-type product containing HT-LM1013 was evenly applied once a day, taking an appropriate amount on the face before bedtime for 4 weeks.During the clinical trial, the participants were instructed not to use skin moisturizers or drugs, except test products, and were checked daily for adverse reactions.

| Efficacy assessment
The efficacy assessment was compared to the initially evaluated value at week 0, which established the baseline.Participants were evaluated for comedones (blackhead and whitehead), sebum content, and clinical photographs during their follow-up visits at weeks 0, 2, and 4. Before the evaluation for efficacy, all participants washed their face and the temperature and relative humidity were maintained at 20-24°C and 45%-55%, respectively, for 30 min.
For this clinical trial, the attended expert which has been trained and performed in clinical trials more than 5 years.In addition, Professor Ju Hee Lee (MD & PhD, Department of Dermatology, Yonsei University Hospital) supervised all the experts in clinical trial for determining comedones.The sebum content between the eyebrows was measured using a sebumeter (Sebumeter® SM 815; Courage+Khazaka electronic GmbH).Clinical photographs were obtained using a facial skin analyzer (Mark-Vu®).

| Statistical analysis
Statistical analyses were performed using the SPSS Statistics version 25 software (IBM).Mean values were analyzed using one-way analysis of variance (ANOVA) followed by Duncan's multiple range, Tukey's range, Games Howell, and Friedman tests were followed by the Wilcoxon signed-rank test at p < 0.05.

| Fatty acid composition of HT-LM1013
The fatty acid composition of HT-LM1013 is shown in Table 1, and a total of 15 types of fatty acids were detected in its cellular materials.cis-11-Vaccenate (32.48%), palmitate (29.07%), and cis-10nonadecylate (25.13%) were identified as the major fatty acids in HT-LM1013.The total saturated fatty acid (SFA), unsaturated fatty acid (USFA), and cyclic fatty acid (CFA) contents were 33.97%, 65.13%, and 0.89%, respectively.The ratio of USFA/SFA was evaluated as 1.92, and USFA and CFA/SFA were measured as 1.94.

| TCA cycle and lipase inhibition
The inhibition rates of the TCA cycle and lipase activity of

| Biofilm inhibition and degradation
The    3).No adverse reactions including skin irritation were observed during the clinical trial.

| DISCUSS ION
Interest on the human microbiome niche annually increases in the cosmetic and personal care markets. 15Skin is the largest microbiome ecosystem in the body, consisting of bacteria, fungi, yeast, and other microorganisms. 16,17Skin microbiota play an important role in the maintenance of a heathy cutaneous barrier. 17Skin and commensal microbiota defend from pathogens, chemicals, and physical aggressions by modulating the skin innate and adaptive immune systems. 16,17These important roles of skin microbiota result in probiotics-related cosmetic products such as balm, cleanser, cream, deodorant, foundation, gel, mask, exfoliant, serum, and soap bar.However, safety concerns impose limitations on probiotic ingredients, and postbiotics such as lysates, derivates, and ferments are recommended. 15Moreover, postbiotics have the advantage of formulating cosmetics with favorable physicochemical properties, in contrast to probiotics. 18The HT-LM1013 is inactivated by heat stress and has been applied as a cosmetic ingredient.
In the human skin microbiota, C. acnes shows opportunistic pathogenicity in a disrupted skin barrier.The symptoms of disrupted skin barrier such as increased pH, water loss, skin flaking, keratinocyte apoptosis, and inflammation afford pathogenicity to C. acnes. 17In inflammatory acne lesions, the relative abundance of C. acnes phylotype IA strain increases, whereas that of C. acnes phylotypes IB and II strains decrease. 17,19C. acnes phylotype IA strain releases more virulence factors such as triacylglycerol lipase, porphyrins, hyaluronate lyase, and Christie-Atkins-Munch-Petersen factor than the other phylotype strains. 17,20These virulence factors generate oxidative stress in keratinocytes and perifollicular inflammation in acne. 17,19 addition, C. acnes phylotype IA strain are more prone to establish biofilm formation and colonization. 20In this study, HT-LM1013 lysate reduced the release of lipase and biofilm formation against C. acnes phylotype IA strain (C.acnes ATCC 6919).These results show that HT-LM1013 contributes to the regulation of the skin microbiota.
Biofilms, another pathological property of C. acnes, play a critical role in antibiotic resistance. 6,7,20Like C. acnes ATCC 6919, phylotype I strains have linear plasmids that are essential for biofilm formation and colonization of the skin.Biofilms act as diffusion barriers that disturb the transport of antimicrobial agents.Biofilms also reduce the metabolism of antimicrobial agents in the host. 20For these reasons, non-antibiotic therapies are constantly required in acne vulgaris treatment.Photodynamic therapy (PDT) is widely used in acne vulgaris treatment, accompanied by photosensitizing (PS) agents, to produce reactive oxygen species.The efficacy of PDT, such as bacteriostatic effect and inhibition of biofilm formation against C.
acnes, without PS is decreased. 21Huo et al 22  (Table 1).Additionally, palmitate, other abundant fatty acid in HT-LM1013, was reported as inhibiting biofilm formation by C. acnes 7 and Staphylococcus aureus. 23Similar to HT-LM1013, Tsai et al 24   HT-LM1013 has the potential to be a novel cosmetic ingredient.

2 . 2 |
Iodonitrotetrazolium chloride (INT) and crystal violet solutions were purchased from Sigma-Aldrich.4-Methylumbelliferyl palmitate (4-MU palmitate), 4-methylumbelliferyl stearate (4-MU stearate), and 4-methylumbelliferyl oleate (4-MU oleate) were obtained from Santa Cruz Biotechnology.Pediococcus acidilactici LM1013 was cultured in de Man-Rogosa-Sharpe (MRS) at 37°C.P. acidilactici LM1013 was, sub-cultured three times at 12-h intervals, and stored in MRS medium containing 20% glycerol at −80°C until further use.C. acnes ATCC 6919 was purchased from the American Type Culture Collection, cultured in reinforced clostridial Media (RCM) at 37°C for 72 h in anaerobic conditions using a 20% CO 2 gas pack (Mitsubishi Gas Chemical Company), and stored in RCM medium containing 20% glycerol at −80°C until further use.Preparation of HT-LM1013 and its lysate HT-LM1013 was prepared by the Department of Production of Lactomason.The number of heat-treated cells (without viable cells), Escherichia coli contamination, and heavy metal contents were authorized by the Quality Management Team in Lactomason.

2. 8
.2 | Patients This study included 23 patients (11 males and 12 females) aged 14-40 years (average 27.83) old with clinically diagnosed acne vulgaris (acne severity score: 1 or 2).Exclusion criteria were pregnancy, breastfeeding, lesions in the face, infectious skin diseases, allergy and anaphylaxis, skin rash caused by drugs, cosmetics and light exposure, steroid and phototherapy within 1 month, skin treatment including botox, filer injection, tattoo within 3 months, and mental disorders.All the participants clearly understood the clinical study and provided signed consent before participation.

Figure 3
Figure 3 shows a microscopic image of lysates treated C. acnes ATCC 6919.Compared to the control (non-treated C. acnes), 1 × MIC of lysate-treated C. acnes showed concave and harshness shapes.In addition, 1 × MIC of lysate-treated C. acnes was elongated by the inhibition of cell division.

Figures 4 and 5
Figures 4 and 5 show a comparison of closed comedones (whitehead), open comedones (blackhead), and facial sebum content in volunteers during the clinical trial.HT-LM1013 decreased the number of closed comedones from 14.04 to 12.70 (at week 2) and 10.22 (at week 4) (p < 0.01).During the same period, open comedones decreased from 7.22 to 5.87 (at week 2) and 4.39 (at week 4) (p < 0.01).The sebum content also decreased to 83.03% and 76.23% at weeks

E 2
Inhibition of biofilm formation and degradation of mature biofilm using heat-treated Pediococcus acidilactici LM1013 lysate.(A) Inhibition of biofilm.(B) degradation of mature biofilm.p < 0.05, p < 0.01, and p < 0.001, compared to the control.F I G U R E 3 Field-emission scanning electron microscopy of heat-treated Pediococcus acidilactici LM1013 lysate-treated Cutibacterium acnes ATCC 6919.Observations were performed at a magnification of 50 000×.

5 | 7 Note:
reported heat-treated Lactiplantibacillus plantarum-GMNL6 inhibited biofilm formation against S. aureus and improved facial conditions.This study clearly showed that HT-LM1013 contributed the positive effect for acne vulgaris.The HT-LM1013 containing leave-on-type product for the clinical trial was designed not to contain any ingredient for acne treatment like salicylic acid.The detailed information of formulas was not provided for the confidential point of view.The clinical F I G U R E 4 Change in comedones and sebum contents in clinical trial participants.(A) Change in closed comedones.(B) Change in open comedones.(C) Change in sebum content.p < 0.01 and p < 0.001, compared to the control.trial was performed in single-arm and absence of placebo control with a few limitations, but positive effect of leave-on-type product was provided.In addition, all participants showed low level of acne severity score; therefore, more limitations are required for better clinical result.CON CLUS ION HT-LM1013, a postbiotic ingredient, can regulate C. acnes growth, lipid metabolism, and biofilm formation.It can be applied as a cream TA B L E 3 Satisfaction score of cream containing heat-treated Pediococcus acidilactici LM1013.Data are shown as mean ± standard deviation.

F I G U R E 5
Representative clinical photograph of clinical trial participants.Clinical photographs were obtained using facial skin analyzer.forpatients with acne vulgaris and can attenuate comedones and sebum content on the face.Most of the participants were satisfied with the HT-LM1013-containing cream.These results suggest that

Table 2
shows the antimicrobial effect of P. acidilactici LM1013 and its lysate.In agar spot assay, 17.17-mm inhibition zone was TA B L E 1 Fatty acid composition of heat-treated Pediococcus acidolactici LM1013.