Characteristics of Malassezia furfur at various pH and effects of Malassezia lipids on skin cells

Abstract

Abstract

Malassezia species are commensal and opportunistic fungi found in human skin. All Malassezia species lack fatty acid synthesis genes and survive by utilizing several lipases to degrade and absorb fatty acids from external lipid sources, but little research has been done on their optimal active pH and temperature. Our skin protects itself from external stimuli and maintains homeostasis, involving bacteria and fungi such as Malassezia species that inhabit our skin. Hence, dysbiosis in the skin microbiome can lead to various skin diseases. The skin’s pH is slightly acidic compared to neutral, and changes in pH can affect the metabolism of Malassezia species. We used keratinocyte cell lines to determine the effect of lipids bio-converted by Malassezia furfur, Malassezia japonica, and Malassezia yamatoensis under pH conditions similar to those of healthy skin. Lipids bio-converted from Malassezia species were associated with the regulation of transcripts related to inflammation, moisturizing, and promoting elasticity. Therefore, to determine the effect of pH on lipid metabolism in M. furfur, which is associated with seborrheic dermatitis, changes in biomass, lipid content, and fatty acid composition were determined. The results showed that pH 7 resulted in low growth and reduced lipid content, which had a negative impact on skin health. Given that bio-converted Malassezia-derived lipids show positive effects at the slightly acidic pH typical of healthy skin, it is important to study their effects on skin cells under various pH conditions.

Key points

• pH 6, Malassezia spp. bio-converted lipid have a positive effect on skin cells

• Malassezia spp. have different lipid, fatty acid, and growth depending on pH

• Malassezia spp. can play a beneficial role by secreting lipids to the outside

Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-024-13292-2.

Introduction

The skin, the largest organ in the human body, consisting of the epidermis, dermis, hair follicles, sweat glands, sebaceous glands, and fat layer, performs important physical barrier dynamics that protect the interior from various external environmental factors (de Szalay and Wertz 2023; Lai-Cheong and McGrath 2013). Most notably, when exposed to ultraviolet (UV) rays, the skin naturally produces a pigment called melanin to protect itself from the harmful rays (Brenner and Hearing 2008). It also plays an essential role in preventing harmful chemicals from entering the body through mechanical impacts or scratches, which can lead to skin diseases (Hänel et al. 2013). Interestingly, our skin, especially the epidermis, is home to various bacteria, fungi, and viruses that live in symbiosis and form a biological barrier called the skin microbiome (Grice and Segre 2011). Their role is to protect the skin by preventing the colonization of harmful pathogens that may invade from the outside and participating in the differentiation and regeneration of skin cells. They also positively impact skin health and immunity by producing beneficial substances or inducing the host to produce antimicrobial substances. Sometimes, an imbalance between these microorganisms can occur due to various environmental factors, causing a breakdown of the skin barrier (Harris-Tryon and Grice 2022). This altered host response can lead to skin diseases such as dermatitis, psoriasis, seborrheic dermatitis, alopecia areata, acne, and in the worst cases skin cancer (De Pessemier et al. 2021; Gaitanis et al. 2008). Therefore, research is actively underway to identify the composition of the skin microbiome, the centerpiece of the skin barrier, to prevent and treat various skin diseases and to positively affect the skin. In particular, the development of next-generation sequencing (NGS) technology has led to significant advances in research by fundamentally revealing the composition of the human microbiome. The traditional method of identifying microorganisms is to take skin cells from a specific location and perform actual cultures. This method has a major limitation in identifying microorganisms when culture is not possible due to complex culture conditions or the inability to realize realistic growth conditions. However, NGS technology has overcome these limitations by using microbial DNA extracted from samples rather than cultures, and it also allows us to determine their relative distribution (Hodkinson and Grice 2015). The human microbiome project, which aims to characterize human microorganisms based on NGS, has found that the microbiome of the body can be broadly divided into dry, moist, toenail, and sebaceous areas. In addition, it was revealed that the relative abundance of microorganisms coexisting in each area differs depending on the environment of the body part. As a result, in contrast to the rich bacterial diversity present in each skin region, the fungi present in the sebaceous areas of most skin, except two web space including toenails and plantar heels, were identified as belonging to the Malassezia genus, which have lipophilic properties (Byrd et al. 2018; Grice and Segre 2011). Although the Malassezia genus is a resident flora in the skin, it is also known as an opportunistic infectious agent due to its prevalence in dermatologic lesions such as seborrheic dermatitis, dandruff, alopecia, atopic dermatitis, and pityriasis versicolor (Huang et al. 2019; Park et al. 2021).

The Malassezia genus is known to lack fatty acid synthase genes, and due to this characteristic, Malassezia genus must obtain fatty acids from outside for cell metabolism, growth, and proliferation (Park et al. 2017; Wu et al. 2015). Sebum from human sebaceous glands is composed of wax esters, sterol esters, cholesterol, cholesterol esters, fatty acids, triglycerides, and squalene, making it an excellent lipid source for Malassezia genus (Lovászi et al. 2017). Since the Malassezia genus cannot directly utilize the lipids in sebum, it secretes enzymes such as lipase, esterase, lipoxygenase, and protease to decompose and oxidize sebum and use it as a lipid nutrient source (Ramirez et al. 2017). In particular, the Malassezia genus is known to ingest saturated fatty acids from the scalp surface and produce oleic acid (C18:1 ω-9), a type of unsaturated fatty acid, causing fatty acid imbalance, destroying the skin barrier, and possibly causing seborrheic dermatitis and dandruff (Ro and Dawson 2005). The activity of these enzymes is known to vary depending on factors such as substrate concentration, metal ions, pH, and temperature, which in turn affects the types of fatty acids produced. It is worth noting that the genus Malassezia has not been well studied regarding which fatty acids are produced and utilized under different pH conditions and how they affect the skin. While the skin of normal people is slightly acidic with a pH of about 5.5 ± 0.6, the skin of patients suffering from skin diseases such as seborrheic dermatitis or atopic eczema has a high pH (Eberlein-Konig et al. 2000; Jung et al. 2013). Therefore, in this study, we investigated how changes in pH, an external environmental factor, affect the growth potential, lipid content, and fatty acid composition of three representative Malassezia species. Furthermore, we examined the soothing, moisturizing, anti-aging, and elasticity effects of the extracts under each condition on skin cells.

Materials and methods

Preparation of buffer medium for Malassezia species culture according to pH

A modified Leeming & Notman broth solution (mLNB) was used to prepare culture medium for culturing Malassezia species (Suzuki et al. 2022). In addition, citrate–phosphate buffer (CPB) was added to the prepared media to keep the pH constant (Prins and Billerbeck 2021). First, 0.22 µm filtered distilled water (D.W.) was sterilized at 121 °C for 15 min to make CPB. The sterilized D.W. was used to prepare 0.1 M citric acid solution (citric acid, DAEJUNG, Siheung, Korea) and 0.2 M sodium phosphate solution (dibasic anhydrous sodium phosphate, DAEJUNG, Siheung, Korea), respectively. Finally, sterilized D.W, 0.1 M citrate solution, 0.2 M sodium phosphate solution, and mLNB broth solution were mixed to prepare pH 6.0 which is similar to the pH of normal human skin (detailed CPB-mLNB broth solution composition is described in Supplemental Table S1). The pH range of people with normal skin environments is 5.5 ± 0.6, but two representative points within this range were selected, and pH 5 and pH 6 were selected as experimental conditions to clearly analyze the change in skin response according to pH change.

Malassezia species culture according to pH conditions and primary human sebocytes culture

We conducted cultures to determine the effects of lipids derived from Malassezia species and lipids derived from human sebocytes on human skin cells. Malassezia species (Malassezia furfur KCTC 7546, Malassezia japonica KCTC 17611, and Malassezia yamatoenesis KCTC 17656; the biological resources used in this research were distributed from KCTC) stocked at − 80 ℃ were spread on mLNB agar plates to make an actively growing culture stage and cultured at 32 ℃ for 7 days. Afterward, single colonies were collected and inoculated into 14-mL test tubes containing pH 6.0, 5 mL mLNB broth solution and cultured for 3 days in a shaking incubator at 120 revolutions per minute (rpm), 32 °C. To increase the amount for injection, each Malassezia species from a 14-mL test tube was inoculated into a 250-mL baffled shake flask containing pH 6.0, 100 mL mLNB. After culturing for 3 days under the same conditions, it was used for inoculation in this experiment. The Malassezia species culture medium cultured for 3 days in a 250-mL baffled shake flask was transferred to a 50-mL conical tube and centrifuged at 4000 rpm for 10 min. Because of the oil present in the culture medium, it was difficult to measure the correct absorbance, so the supernatant was removed, 10 mL of phosphate-buffered saline (PBS) was added, and the optical density was measured at 600 nm using a BioPhotometer D30 (Eppendorf, Hamburg, Germany). Subsequently, a new 10-day culture was conducted, adjusting to an optical density (O.D.) of 0.1 at 600 nm. Primary human sebocytes were obtained from Celprogen (Torrance, CA, USA) and cultured in a 37 °C, CO₂ 5% incubator using Human Sebocyte Complete Growth Media (Celprogen, Torrance, CA, USA). Following the cells’ sub-confluency, they were harvested and subcultured using 0.25% trypsin–EDTA (Gibco BRL, NY, New York, USA). Cells were subcultured on a 150 × 25 mm plate to culture a large quantity of cells.

Malassezia species and human sebocytes lipid extraction for skin cell treatments

To determine the effect of bio-converted lipids within Malassezia species on skin cells, lipids were extracted from three species of Malassezia. Considering the point at which biomass and lipid content stabilize, we proceeded with lipid extraction on the 10th day. To harvest the cells after completion of the culture, cells were centrifuged at 4000 rpm for 10 min using a 5840R centrifuge (Eppendorf, Hamburg, Germany). After removing the supernatant, the collected cells were transferred to cellulose extraction thimble filter (Whatman, Kent, UK) and extracted using a soxhlet extraction apparatus (SciLab, Seoul, Korea). Hexane was used as the solvent, and the heating temperature was set at 55 °C to extract the lipids overnight (de Jesus and Filho 2020). Subsequently, a reduced-pressure extraction was performed using an efficient rotary evaporator, Laborota 4000 (Heidolph, Schwabachm, Germany), to remove all the liquid and obtain only the lipids. Afterwards, to treat the cells, lipids were re-dissolved using DMSO (Sigma, St. Louis, MI, USA). Sebocyte lipids were extracted using the same method for extracting lipids from Malassezia species.

Skin cell culture and Malassezia bio-converted lipid treatment

In vitro efficacy analysis was performed with the human keratinocyte cell line, HaCaT (ATCC, Manassas, VA, USA) and human fibroblast cell line, Hs68 (ATCC, Manassas, VA, USA). Cells were cultured in high glucose DMEM media (HyClone, Logan, UT, USA) supplemented with 10% of fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 1% of antibiotics/antimycotics solution (Welgene, Daegu, Korea). Sebocyte lipid (SL), artificial sebum (AS, Biochemazone, Leduc, AB, Canada), and Malassezia species bio-converted lipid (M. furfur bio-converted lipid: MF BCL, M. japonica bio-converted lipid: MJ BCL, M. yamatoensis bio-converted lipid: MY BCL) were used as samples for cell treatment. HaCaT cells were stabilized for 24 h with a total of 2 mL dispensed to inoculate 4 × 10⁵ cells per well and passage 8 was at the time of inoculation. In addition, culture media was replaced with FBS-free media before sample treatment. To analyze the anti-inflammatory effect, FBS-free DMEM media containing 10 µg mL⁻¹ of poly (I:C) HMW (InvivoGen, San Diego, CA, USA) and 10 ng mL⁻¹ of recombinant human IL-4 protein (R&D Systems, Minneapolis, MN, USA) were used as an inflammation-inducing agent. Simultaneously, 1 µM of the anti-inflammatory drug dexamethasone (Sigma, St. Louis, MI, USA) was treated as a positive control. To evaluate the anti-inflammatory efficacy of the samples, all sample treatment groups were treated with poly (I:C) + IL-4 to induce inflammation. Additionally, the samples were treated with 10 ppm and 100 ppm, respectively. After 4 h of treatment, the supernatant was removed to extract RNA, and cells were harvested using TRIzol reagent (Invitrogen, Waltham, MA, USA). For the moisturizing effect evaluation group, 1 µM of retinoic acid (Sigma, St. Louis, MI, USA) was used as a positive control group. Samples were treated at 10 and 100 ppm. After 24 h of culture, the supernatant was removed, and cells were harvested using TRizol reagent. Finally, Hs68 cells were used and prepared under the same conditions as HaCaT cells to evaluate elasticity. As a positive control, recombinant human TGF-beta 1 protein (TGF-β1; R&D Systems, Minneapolis, MN, USA) was treated at 10 ng mL⁻¹, and samples were treated at 10 and 100 ppm. After 24 h of culture, the supernatant was removed and cells were harvested using the TRIzol reagent.

Relative gene expression for evaluating inflammation, moisture, and elasticity effects

RNA concentration was measured using Qubit flex fluorometer (Invitrogen, Waltham, MA, USA) and Qubit RNA high sensitivity assay kits (Invitrogen, Waltham, MA, USA). For cDNA synthesis, RNA was quantified to 1 µg and then synthesized into cDNA using MiniAmp Thermal Cycler (Thermofisher, Waltham, MA, USA) equipment using ReverTra Ace™ qPCR RT Master Mix (TOYOBO, Osaka, Japan). For real-time quantitative PCR (RT-qPCR), PowerSYBR® Green PCR Master Mix (Applied biosystems, Warrington, UK) reagent was used and was performed using StepOne Plus™ real-time PCR equipment. Analysis conditions were 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 10 s, and 60 °C for 1 min. Ct (Cycle threshold) values ​​through RT-qPCR were obtained through StepOne Plus™ software (Applied Biosystems, Warrington, UK), and relative gene expression was confirmed using the delta delta Ct method. The graph was created using prism software version 9.5.1, and the p-value was calculated using a one-way ANOVA test within Prism software (Wu et al. 2023). The expression of interleukin-6 (IL-6) and thymic stromal lymphopoietin (TSLP) was compared to evaluate inflammation, aquaporin-3 (AQP3) and hyaluronan synthase-3 (HAS3) to assess moisturization, and fibrillin-1 (FBN1) and elastin (ELN) to evaluate elasticity. Primers used are listed in the Supplemental Table S2.

Gas chromatography–mass spectrometry conditions for fatty acid analysis

The analysis of the fatty acid information was conducted by gas chromatography–mass spectrometry (GC–MS; Agilent 7010C QQQ GC–MS; Agilent, Santa Clara, CA, USA) with an HP-5MS UI column (30 m × 0.25 mm × 0.25 µm; Agilent, Santa Clara, CA, USA). Helium gas was used as the mobile phase, and the flow was 1.3 mL min⁻¹, and the pressure was 10.785 psi. Inlet temperature was set at 250 ℃, pressure at 10.785 psi, total flow at 17.3 mL min⁻¹, and septum purge at 3 mL min⁻¹. The injector used a split-splitless inlet, and analysis was performed in split mode at a 10:1 ratio. The oven temperature increased by 10 °C per min from 150 to 300 °C and was held at 300 °C for 10 min. Ion source temperature was 290 °C and mass range was measured at 45 to 600 amu (atomic mass unit).

Fluorescence microscopy of bio-converted lipid release from Malassezia species

To determine whether Malassezia species release lipids containing fatty acids outside the cells, M. furfur, M. japonica, and M. yamatoensis were cultured for 2 weeks on mLNB agar using the same amount of AS instead of olive oil. To stop the supply of AS from the agar plates, the colonies were carefully transferred to mLNB agar that did not contain olive oil or AS. After 0, 3, and 6 h, the lipids of each Malassezia species were stained using NileRed, and the cell walls were stained using Calcofluor-White. Visualization was performed using an inverted fluorescence microscope (Olympus IX83, Olympus Co., Tokyo, Japan) was used. We used VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Inc., Newark, CA, USA). The emission and detection wavelengths were confirmed to be 568 nm and 570–620 nm for NileRed, and 461 nm and 430–470 nm for Calcofluor-White.

Results

Effects of bio-converted lipids from Malassezia species on various human skin cell lines via real-time quantitative PCR

Cell culture, processing, and gene expression analysis were performed to determine the effect of lipids extracted from three species of Malassezia cultured for 10 days under pH 6, a slightly acidic condition of normal skin group, on human skin cell lines. (The Ct values for the housekeeping gene and target gene are presented in Supplemental Table S3.) To confirm changes in inflammatory factors, we compared the expression of interleukin-6 (IL-6), a cytokine known to be upregulated by inflammatory response in HaCaT cells (Das et al. 2022; Swaroop et al. 2024). As expected, higher expression of IL-6 was confirmed in the inflammation-inducing group compared to the control group (increased 93.08 ± 4.54-fold). Additionally, a 60.02% decrease in IL-6 expression was observed in the inflammation-induced group treated with dexamethasone, which is commonly used for acute and chronic inflammatory diseases (Bronicki et al. 2000). IL-6 expression in the inflammation-induced group treated with SL 10 ppm was similar to that in the inflammation-induced group, and the expression value was lower in the SL 100 ppm–, AS 10 ppm–, and AS 100 ppm–treated groups (SL 10 ppm: 91.16 ± 4.78-fold, SL 100 ppm: 75.42 ± 2.46-fold, AS 10 ppm: 71.26 ± 1.87-fold, AS 100 ppm: 42.33 ± 3.32-fold). In the groups treated with 10 ppm of lipids derived from M. furfur and M. japonica, IL-6 levels were similar to or higher than those of the inflammation-induced group (MF BCL 10 ppm: 99.42 ± 1.39-fold, MJ BCL 10 ppm: 113.27 ± 5.04-fold). In contrast, IL-6 expression was lowered when treated with 100 ppm of both MF BCL and MJ BCL (MF BCL 100 ppm: 56.07 ± 0.69-fold, MJ BCL 100 ppm: 60.8 ± 2.03-fold). M. yamatoensis showed low expression values ​​at both 10 and 100 ppm (MY BCL 10 ppm: 71.66 ± 6.92-fold, MY BCL 100 ppm: 50.09 ± 3.22-fold). This trend of decreased IL-6 expression with increasing treatment dose was similar to the results observed with SL and AS treatments (Fig. 1a).

Fig. 1.

Relative gene expression of Malassezia spp. bio-converted lipid treatments on cells. a, b Inflammatory response: changes in inflammation-related factor genes (IL-6, TSLP) were measured in HaCaT cells induced with 10 µg mL⁻¹ poly (I:C) and 10 ng mL⁻¹ IL-4. HaCaT cells, at passage 8, were seeded at 4 × 10⁵ cells per well and allowed to stabilize for 24 h. All treatment groups, except the control group, were induced with poly (I:C) + IL-4. Dexamethasone was used as a positive control. After 4 h of treatment, RNA was extracted for RT-qPCR analysis. c, d Moisturizing effect: retinoic acid was used as a positive control, and AQP3 and HAS gene expression were measured in HaCaT cells. HaCaT cells, at passage 8, were seeded at 4 × 10⁵ cells per well and allowed to stabilize for 24 h. After 24 h of treatment, RNA was extracted for RT-qPCR analysis. e, f Elasticity-related factors: Hs68 cells were used to measure changes in the expression of elasticity-related factors (FN1, ELN), with human recombinant TGF-β1 serving as a positive control. Hs68 cells, at passage 8, were seeded at 4 × 10.⁵ cells per well and allowed to stabilize for 24 h. After 24 h of treatment, RNA was extracted for RT-qPCR analysis. The data shown represent one of three independent experiments, which yielded similar results across all replicates. Values are expressed as mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA with multiple comparisons. SL, sebocyte lipid; AS, artificial sebum; MF BCL, Malassezia furfur bio-converted lipid; MJ BCL, Malassezia japonica bio-converted lipid; MY BCL, Malassezia yamatoensis bio-converted lipid. Each bar represents the mean relative gene expression normalized to β-actin. The p-values were annotated on the graphs, with significance levels indicated as follows: ns (not significant), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

We confirmed the expression of thymic stromal lymphopoietin (TSLP), which is secreted in response to inflammatory cytokines or Type 2 T-helper cell (Th2)–inducing cytokines, and is known to induce the activation of dendritic cells in the skin lesions of atopic dermatitis patients (Fig. 1b) (Das et al. 2022). Like the IL-6 expression results, TSLP expression increased in the inflammation-inducing group compared to the control group, and TSLP expression decreased in the dexamethasone-treated group (inflammation-inducing group: 5.85 ± 0.72-fold, dexamethasone-treated group: 4.46 ± 0.5-fold). The SL 10 and 100 ppm and AS 10 ppm treatment groups showed higher or similar expression values ​​compared to the inflammation-inducing, while the AS 100 ppm treatment group showed lower TSLP expression values ​​than dexamethasone-treated (SL 10 ppm: 6.67 ± 0.18-fold, SL 100 ppm: 7.21 ± 1.24-fold, AS 10 ppm: 5.87 ± 1.01-fold, AS 100 ppm: 2.68 ± 0.39-fold). The group treated with lipids derived from Malassezia species showed higher or similar TSLP expression values ​​than the inflammation-induced group, but a decrease in TSLP was confirmed in the group treated with MY BCL 100 ppm (MF BCL 10 ppm: 9.23 ± 1.15-fold, MF BCL 100 ppm: 5.17 ± 0.68-fold, MJ BCL 10 ppm: 9.9 ± 0.9-fold, MJ BCL 100 ppm: 5.31 ± 0.62-fold, MY BCL 10 ppm: 5.53 ± 0.45-fold, MY BCL 100 ppm: 4.05 ± 0.54-fold). Based on the observed trends in the groups treated with 10 and 100 ppm of lipids derived from Malassezia species, it appears that both AS and Malassezia species–derived lipids decrease TSLP expression as the inoculum concentration increases.

The stratum corneum is important for skin barrier function, and it is known that when this barrier is damaged, skin moisture is lost (Jeon et al. 2015). Aquaporin genes, found in various body parts, are passive transporters of water and are important for intracellular and extracellular water homeostasis (Kitchen et al. 2015). The aquaporin-3 (AQP3) gene is known to be more highly expressed in keratinocytes than other AQP genes, and the AQP3 protein is known to transport not only water but also glycerol. To determine the effect of Malassezia-derived lipids on HaCaT cell moisturization, we checked the expression of the AQP3 gene, which is primarily known to be associated with skin moisturization (Fig. 1c). As expected, high AQP3 expression was confirmed in the retinoic acid–treated group, which is known to upregulate AQP3 expression (retinoic acid–treated group: 2.03 ± 0.08-fold). The expression of AQP3 in the SL 10 ppm, SL 100 ppm, and Malassezia-derived lipid 10 ppm–treated groups, and MY BCL 100 ppm was similar to or slightly higher than that in the control group (SL 10 ppm: 1.04 ± 0.02-fold, SL 100 ppm: 1.13 ± 0.11-fold, MF BCL 10 ppm: 1.04 ± 0.06-fold, MJ BCL 10 ppm: 1.29 ± 0.23-fold, MY BCL 10 ppm: 0.99 ± 0.03-fold, MY BCL 100 ppm: 1.1 ± 0.08-fold). The expression of AQP3 was reduced in the AS 10 ppm, AS 100 ppm, MF BCL 100 ppm, and MJ BCL 100 ppm treatment groups, showing a tendency to decrease with increasing doses (AS 10 ppm: 0.85 ± 0.15-fold, AS 100 ppm: 0.62 ± 0.04-fold, MF BCL 100 ppm: 0.44 ± 0.02-fold, MJ BCL 100 ppm: 0.4 ± 0.03-fold).

As a result of confirming the expression of hyaluronan synthase 3 (HAS3), a gene that synthesizes hyaluronic acid, known for its moisturizing effect on the skin due to its hydrophilic nature with many hydroxyl groups, HAS3 expression was increased as expected (retinoic acid–treated group: 4.02 ± 0.38-fold) (Choi et al. 2022). The expression of HAS3 was the same or decreased in the SL 10 ppm, SL 100 ppm, AS 100 ppm, and MY BCL 10 ppm and 100 ppm treatment groups (SL 10 ppm: 0.92 ± 0.07-fold, SL 100 ppm: 0.7 ± 0.02-fold, AS 100 ppm: 1.07 ± 0.07-fold, MY BCL 10 ppm: 0.87 ± 0.02-fold, MY BCL 100 ppm: 0.74 ± 0.07-fold). Following the retinoic acid treatment group, the highest HAS3 expression was confirmed in the AS 10 ppm treatment group, and the expression of MF BCL 10 ppm, MF BCL 100 ppm, MJ BCL 10 ppm, and MJ BCL 100 ppm was all higher compared to the control group (AS 10 ppm: 1.97 ± 0.31-fold, MF BCL 10 ppm: 1.22 ± 0.04-fold, MF BCL 10 ppm: 1.42 ± 0.04-fold, MJ BCL 10 ppm: 1.34 ± 0.21-fold, MJ BCL 100 ppm: 1.18 ± 0.09-fold) (Fig. 1d).

To determine the effect on skin elasticity, the expression of fibrillin-1 (FN1), which is involved in microfibril homeostasis, elastic fiber assembly, and the formation of the extracellular matrix (ECM), was examined in the fibroblast cell line Hs 68 (Fig. 1e) (Baldwin et al. 2013). Consistent with previous research results showing that FN1 synthesis is increased by TGF-β1, a growth factor that regulates cell proliferation, migration, differentiation, and survival, FN1 expression was increased in the TGF-β1 treatment group (TGF-β1-treated group: 3.23 ± 0.96-fold) (Jiao et al. 2016). The expression of FN1 in the SL 10 ppm–treated group was similar to the control group, but the remaining groups showed higher expression, with the highest expression in the MF BCL–treated group (SL 10 ppm: 0.96 ± 0.18-fold, SL 10 ppm: 1.31 ± 0.35-fold, AS 10 ppm: 2.12 ± 0.12-fold, AS 100 ppm: 1.74 ± 0.24-fold, MF BCL 10 ppm: 2.52 ± 0.45-fold, MJ BCL 10 ppm: 2.14 ± 0.75-fold, MY BCL 10 ppm: 1.74 ± 0.31-fold).

The expression of elastin (ELN), another component of the ECM that provides elasticity and flexibility to the skin and plays an important role in wound healing by regulating cell migration, proliferation, and differentiation, was confirmed through the fibroblast cell line Hs68 (Fig. 1f) (Carney et al. 2017; Diller and Tabor 2022; Mbundi et al. 2021). Consistent with previous research findings, our study confirmed that ELN expression is upregulated in the TGF-β1 treatment group (TGF-β1-treated group: 283.65 ± 17.33-fold) (Katsuta et al. 2008; Roodnat et al. 2022). The MF BCL 10 ppm treatment group exhibited higher ELS expression compared to M. japonica and M. yamatoenesis and showed the highest ELS expression (SL 10 ppm: 6.59 ± 0.54-fold, SL 100 ppm: 6.57 ± 1.04-fold, AS 10 ppm: 29.54 ± 3.51-fold, AS 100 ppm: 4.96 ± 0.63-fold, MF BCL 10 ppm: 69.8 ± 5.67-fold, MJ BLC 10 ppm: 28.34 ± 4.24-fold, MY BCL: 25.74 ± 1.01-fold). These results indicate that treatment with 10 ppm of lipids derived from M. furfur positively influences skin elasticity. All relative gene expression values and p-value are shown in Supplemental Tables S4 and S5, and cell experiments under pH 5 conditions, which are slightly acidic conditions of normal skin environments, showed almost the same trend as pH 6, and the data are not shown separately.

M. furfur culture medium pH changes during incubation period

M. furfur culture and pH measurement methods are detailed in Supplemental Method S1. Measurements of pH over the incubation period show a common decrease in pH over the cultivation period across all conditions (Fig. 2a and Supplemental Table S6). The pH 7 condition decreased to pH 6.76 ± 0.04, the pH 6.6 condition decreased to pH 6.35 ± 0.05, the pH 6 condition decreased to pH 5.08 ± 0.11, and finally, the pH 5 condition decreased to pH 4.28 ± 0.04. It is important to note that the pH 7 and 6.6 conditions did not change significantly, with decreases of 3.43% and 3.64%, respectively, but a more considerable decrease was observed in the pH 6 and 5 conditions, with reductions of 15.33% and 14.4%, respectively. Although the pH change in all conditions was minimized using a citrate–phosphate buffer, these pH changes can be indirectly explained by various factors such as organic acid production from microorganisms, CO₂ accumulation, nitrogen compound metabolism, cell growth, and weakening of the buffer solution (Lund et al. 2020; Sánchez-Clemente et al. 2018; Sanil et al. 2014). For the changes identified under pH 5 and 6 conditions, we focused on fatty acid breakdown, the release of absorbed fatty acids, and the growth of Malassezia species. Since Malassezia species does not have genes encoding fatty acid synthesis enzyme (FAS1), fatty acids supplied in the medium are brought into the cells and used for metabolic activities (Triana et al. 2017; Wu et al. 2015). One might question whether synthesizing fatty acids within a cell can significantly impact pH changes, but most microorganisms actually utilize fatty acid–binding proteins present in the cell membrane or create endosomes containing fatty acids and secrete them outside the cell (de Carvalho and Caramujo 2018; Nolan et al. 2006; Weisiger 2007). Additionally, Malassezia species have a large number of extracellular lipases that can decompose fatty acids from external lipids and lower the pH of the medium by releasing protons from the carboxylic acid groups of fatty acids (Park et al. 2021; Ramirez et al. 2017).

Fig. 2.

Malassezia furfur was cultured at an initial optical density (O.D.) of 0.1 at 600 nm in 1-L flasks containing 300 mL of citrate–phosphate buffer and mLNB broth solution, with shaking at 120 rpm and 32 °C. Sampling was conducted daily at 2 PM, with 11 mL of culture taken each time. From this sample, 1 mL was used for pH measurement. The remaining 10 mL was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and 10 mL of PBS was added. Of this PBS mixture, 3 mL was used for biomass measurement. a Culture medium pH change according to pH conditions during the culture period: changes in pH levels during the incubation period for M. furfur cultured under different initial pH conditions (pH 7, 6.6, 6, and 5) using citrate–phosphate buffer and mLNB broth solution. The pH levels were measured at various time points over a 10-day period. The final pH levels on day 10 were pH 7 decreased to 6.76 ± 0.04, pH 6.6 decreased to 6.35 ± 0.05, pH 6 decreased to 5.08 ± 0.11, and pH 5 decreased to 4.28 ± 0.04. Detailed buffer information is provided in Supplemental Table S1. Detailed pH values measured during the culture period are provided in Supplemental Table S6. b Comparison of biomass concentration changes over time at different pH conditions: Changes in biomass concentration (g L⁻¹) over a 10-day period at different initial pH conditions (pH 7, 6.6, 6, and 5). Biomass concentration was determined by measuring the dry weight of cells at various time points. The final biomass concentrations on day 10 were 5.11 ± 0.2 g L⁻¹ at pH 7, 8.22 ± 0.04 g L⁻¹ at pH 6.6, 12.38 ± 1.88 g L⁻¹ at pH 6, and 9.71 ± 1.01 g L⁻.¹ at pH 5. Detailed biomass values are provided in Supplemental Table S7. Values are expressed as mean ± standard error of the mean (SEM). Three independent experiments were conducted, each with three replicates for each pH condition, using 12 flasks in total (4 pH conditions × 3 replicates). pH and biomass measurements were made once per sample. Paired t-tests were conducted to compare the pH values and biomass concentrations between day 0 and day 10. The p-values were annotated on the graphs, with significance levels indicated as follows: ns (not significant), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Comparison of changes in biomass concentration of M. furfur according to pH

After 10 days of incubation in a shaking incubator, the final biomass concentration was highest at pH 6, followed by pH 5, pH 6.6, and pH 7 (Fig. 2b and Supplemental Table S7). At pH 6, the biomass concentration was 12.38 ± 1.88 g L⁻¹, which is an improvement of 27.5%, 50.61%, and 142.27% compared to pH 5 (9.71 ± 1.01 g L⁻¹), pH 6.6 (8.22 ± 0.04 g L⁻¹), and pH 7 (5.11 ± 0.2 g L⁻¹), respectively. The pH 5 and pH 6 conditions, similar to the slightly acidic skin pH of 4.5 to 6, were found to be favorable for M. furfur growth. In particular, a rapid increase was observed between days 2 and 4 in all conditions, indicating a period of active cell proliferation using fatty acids. M. furfur, which cannot synthesize C14 and C16 fatty acids due to the absence of FAS1, is known to secrete lipase to break down external lipids into fatty acids and use them for growth (Harada et al. 2015). Malassezia species, including M. furfur, carry multiple lipase genes, and M. furfur is well known for the activity of MfLIP1, an extracellular lipase. The optimal activity of MfLIP1 is known to be at pH 5.8, and compared to that, it decreases by 35% at pH 7 and decreases more than threefold around pH 4 (Brunke and Hube 2006). In our results, pH 7 condition had the lowest biomass concentration. These results show that even if extracellular lipase such as MfLIP1 decomposes more fatty acids with high activity, it may not lead to an increase in biomass. Malassezia species have several intracellular lipases in addition to extracellular lipases. The reason biomass did not increase at pH 7 might be that pH 7 conditions are not suitable for the intracellular lipase enzyme to function. In other words, even if external lipids are broken down into fatty acids by extracellular lipase and enter the cell, an environment is needed where they can be converted into usable fatty acids by intracellular lipase. Therefore, it can be confirmed that a pH around 6 can properly decompose lipids outside the cell and enable efficient utilization of fatty acids inside the cell. The method used to measure the biomass of M. furfur is described in Supplemental Method S1.

Changes in M. furfur total lipid contents under different pH

Lipid content changes were measured throughout the incubation period, except for the adaptation period on days 1 and 2 when intracellular lipid concentrations were deficient and difficult to quantify (Fig. 3 and Supplemental Table S8). The method used to measure lipid content is detailed in Supplemental Method S2. The lipid content included fatty acids, phospholipids, waxes, sphingolipids, glycolipids, steroids, and other substances. As shown in Fig. 3 on day 3, the lipid content was highest at pH 6 (62.97 ± 9.2%), followed by pH 5 (51.58 ± 3.33%), pH 6.6 (39.53 ± 4.14%), and pH 7 (25.96 ± 2.16%). pH 7 had at least 58% lower lipid content than pH 6, indicating that the activity of extracellular lipase does not correlate with intracellular lipid content. A decreasing trend in lipid content was observed between days 3 and 5 for all pH conditions, as inferred from the growth rates in the biomass concentration graphs. The growth rates for each condition from day 3 to day 5 were 2.21 g L⁻¹ day⁻¹ at pH 6, 1.55 g L⁻¹ day⁻¹ at pH 5, 1.76 g L⁻¹ day⁻¹ at pH 6.6, and 0.93 g L⁻¹ day⁻¹ at pH 7. In addition to being utilized for growth during the exponential phase, fatty acids are also used for adenosine triphosphate (ATP) synthesis, necessary for survival, resulting in a significant decrease in fatty acids during periods of metabolic activity (Jia et al. 2023; Merritt et al. 2020; Zhao and Li 2021). For pH 7, with the lowest lipid content, the lipid content continued to decrease until day 10, unlike the other conditions. This indicates that fatty acids are being consumed continuously because the maximum biomass concentration has not yet been reached. On day 10, the lipid content was highest in the following order: pH 6.6 (45.64 ± 2.14%), pH 5 (39.38 ± 2.41%), pH 6 (38.35 ± 1.19%), and pH 7 (5.2 ± 0.42%), with pH 7 having a final lipid content at least 8.78, 7.38, and 7.6 times lower than the respective conditions.

Fig. 3.

Lipid contents under different pH conditions. Malassezia furfur was cultured at an initial optical density (O.D.) of 0.1 at 600 nm in 1-L flasks containing 300 mL of citrate–phosphate buffer and mLNB broth solution, with shaking at 120 rpm and 32 °C. Sampling was conducted daily at 2 PM, where 11 mL of culture was taken. From this sample, 10 mL was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and 10 mL of PBS was added. Of this mixture, 2 mL was used for lipid extraction. Lipid accumulation in M. furfur cells was measured during the culture period under different initial pH conditions (pH 7, 6.6, 6, and 5) using a modified Bligh and Dyer method (Bligh and Dyer 1959; Yu et al. 2022). Lipid extraction involved cell lysis with chloroform and methanol, followed by separation and drying of the chloroform layer (Yu et al. 2021). Lipid content was not measured on days 1 and 2 due to difficulty in accurate weight determination. Detailed lipid content values are provided in Supplemental Table S8. Values are expressed as mean ± standard error of the mean (SEM). Experiments were conducted in three independent sets, each with three replicates per pH condition, totaling 12 flasks. To assess the statistical significance of changes from day 3 to subsequent time points up to day 10, a one-way ANOVA with multiple comparisons was performed. The resulting p-values were calculated and annotated on the graph. The p-values were annotated on the graphs, with significance levels indicated as follows: ns (not significant), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Comparison of M. furfur cell fatty acid composition by pH conditions and culture period

Through fatty acid analysis using GC–MS, retention times and area values of peaks were confirmed for each sample. The method for extracting fatty acids from M. furfur for GC–MS analysis is described in Supplemental Method S2. Using the National Institute of Standards and Technology (NIST) library, 7 fatty acids were identified: palmitoleic acid (C₁₆H₃₀O₂), palmitic acid (C₁₆H₃₂O₂), linoleic acid (C₁₈H₃₂O₂), oleic acid (C₁₈H₃₄O₂), octadecanoic acid (C₁₈H₃₆O₂), 11-eicosenoic acid (C₂₀H₃₈O₂), and arachidic acid (C₂₀H₄₀O₂). By comparing the area values of each fatty acid, different ratios were observed within the samples depending on pH conditions and culture period (Fig. 4 and Table 1). Detection times were as follows: palmitoleic acid: 7.47, 7.51; palmitic acid: 7.69; linoleic acid: 9.29; oleic acid: 9.33, 9.38; octadecanoic acid: 9.55; 11-eicosenoic acid: 11.09; arachidic acid: 11.29. The fatty acid composition of olive oil used in this study is palmitic acid (C16:0): 11.5 ± 4%; linoleic acid (C18:2): 7.0 ± 3.3%; oleic acid (C18:1): 78.4 ± 4.3%; octadecanoic acid (C18:0): 2 ± 0.5%; arachidic acid (C20:0): 0.22 ± 0.12%. Analysis of the fatty acid composition of M. furfur indicates that it decomposed lipids from olive oil, absorbed and accumulated all fatty acids, and used them for metabolism. 11-eicosenoic acid and arachidic acid began to be detected on day 3. These results suggest that the number of cells used to extract fatty acids was too low, that M. furfur absorbed and used arachidic acid and 11-eicosenoic acid immediately, or that suitable conditions for metabolism were not present. Oleic acid had the highest composition and was consistent across all pH conditions. Palmitic acid and linoleic acid showed high variability at pH 5, pH 6, and pH 6.6. Octadecanoic acid, 11-eicosenoic acid, and arachidic acid were present at relatively constant concentrations and did not show significant fluctuations compared to other fatty acids.

Fig. 4.

Fatty acid composition of Malassezia furfur under different pH conditions over time. M. furfur was cultured at an initial optical density (O.D.) of 0.1 at 600 nm in 1-L flasks containing 300 mL of citrate–phosphate buffer and mLNB broth solution, with shaking at 120 rpm and 32 °C. Sampling was conducted daily at 2 PM. From each sample, 10 mL was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and 10 mL of PBS was added. Of this mixture, 3 mL was used for fatty acid extraction. The extracted fatty acids were analyzed using gas chromatography–mass spectrometry (GC–MS), with identification based on the National Institute of Standards and Technology (NIST) library. The composition of palmitoleic acid, palmitic acid, linoleic acid, oleic acid, octadecanoic acid, 11-eicosenoic acid, and arachidic acid within M. furfur cells is shown. Results are displayed as pie charts representing the area value of each fatty acid relative to the total area of all peaks. All analyses were conducted in triplicate. Variations in fatty acid composition according to pH and cultivation period are provided in Table 1

Table 1.

Variations in fatty acid composition according to pH and cultivation period

pH value	Day	Palmitoleic acid	Palmitic acid	Linoleic acid	Oleic acid	Octadecanoic acid	11-Eicosenoic acid	Arachidic acid
7	1	0 ± 0	8.6 ± 7.45	0 ± 0	87.14 ± 6.9	4.25 ± 0.58	0 ± 0	0 ± 0
	2	0.94 ± 0.24	7.4 ± 0.22	4.2 ± 0.28	85.18 ± 1.14	2.28 ± 0.55	0 ± 0	0 ± 0
	3	0.55 ± 0.48	7.56 ± 1.7	2.85 ± 0.95	86.87 ± 1.39	2.18 ± 1.05	0 ± 0	0 ± 0
	4	0.95 ± 0.18	8.69 ± 5.76	6.25 ± 4.83	81.9 ± 12.55	2.14 ± 2.03	0.03 ± 0.05	0.04 ± 0.07
	5	0.98 ± 0.14	7.75 ± 4.63	6.55 ± 4.48	82.41 ± 11.12	2.24 ± 2.02	0.03 ± 0.05	0.05 ± 0.08
	6	0.98 ± 0.24	5.48 ± 1.26	4.85 ± 1.53	86.92 ± 3.35	1.71 ± 0.69	0.02 ± 0.04	0.04 ± 0.06
	7	0.7 ± 0.18	4.42 ± 0.14	2.79 ± 0.1	90.79 ± 0.03	1.27 ± 0.14	0 ± 0	0.04 ± 0.07
	8	0.49 ± 0.17	5.45 ± 0.42	6 ± 5.19	86.11 ± 5.82	1.83 ± 0.81	0.06 ± 0.07	0.05 ± 0.05
	9	0.76 ± 0.1	4.89 ± 0.09	2.74 ± 0.23	90.02 ± 0.54	1.6 ± 0.19	0 ± 0	0 ± 0
	10	0.66 ± 0.14	4.71 ± 0.43	2.74 ± 0.11	90.34 ± 0.71	1.55 ± 0.05	0 ± 0	0 ± 0
6.6	1	0 ± 0	12.46 ± 1.54	0 ± 0	84.36 ± 2.46	3.19 ± 1.17	0 ± 0	0 ± 0
	2	0.8 ± 0.06	8.53 ± 0.45	4.58 ± 0.31	84.62 ± 0.73	1.47 ± 0.07	0 ± 0	0 ± 0
	3	0.54 ± 0.06	10.61 ± 1.02	6.04 ± 0.55	80.56 ± 1.78	2.13 ± 0.32	0.06 ± 0.01	0.05 ± 0
	4	0.61 ± 0.07	16.6 ± 0.58	11.23 ± 0.91	66.34 ± 1.72	5 ± 0.24	0.08 ± 0	0.14 ± 0.02
	5	0.61 ± 0.09	15.96 ± 0.9	13.11 ± 0.86	64.52 ± 2.27	5.56 ± 0.49	0.09 ± 0	0.15 ± 0.02
	6	0.47 ± 0.09	11.35 ± 3.72	10.75 ± 5.86	73 ± 12.19	4.22 ± 2.39	0.08 ± 0.04	0.13 ± 0.09
	7	0.35 ± 0.03	8.68 ± 1	7.1 ± 1.51	80.54 ± 3.07	3.14 ± 0.62	0.09 ± 0.03	0.1 ± 0.02
	8	0.31 ± 0.11	7.64 ± 2.2	4.96 ± 1.81	84.4 ± 4.68	2.5 ± 0.83	0.07 ± 0.02	0.13 ± 0.06
	9	0.29 ± 0.05	8.46 ± 0.67	6.94 ± 0.66	80.71 ± 1.58	3.43 ± 0.24	0.07 ± 0.01	0.11 ± 0.01
	10	0.28 ± 0.07	7.22 ± 0.52	7.46 ± 0.61	81.35 ± 1.12	3.48 ± 0.3	0.08 ± 0.01	0.12 ± 0.02
6	1	0 ± 0	10.94 ± 1.06	0 ± 0	86.25 ± 1.29	2.82 ± 0.51	0 ± 0	0 ± 0
	2	0.46 ± 0.17	9.25 ± 1.12	3.06 ± 0.29	85.48 ± 1.34	1.73 ± 0.04	0 ± 0	0 ± 0
	3	0.55 ± 0.1	16.92 ± 2.47	13.09 ± 4.48	64.32 ± 8.68	4.89 ± 1.58	0.07 ± 0.02	0.15 ± 0.06
	4	0.86 ± 0.12	18.28 ± 0.29	19.57 ± 1.32	53.65 ± 2.67	7.26 ± 0.87	0.13 ± 0.02	0.26 ± 0.04
	5	0.79 ± 0.05	16.48 ± 0.22	20.68 ± 0.55	54.54 ± 0.93	7.15 ± 0.43	0.13 ± 0.01	0.24 ± 0.02
	6	0.36 ± 0.01	8.25 ± 0.75	12 ± 1.21	76.01 ± 2.41	3.23 ± 0.47	0.06 ± 0	0.09 ± 0.01
	7	0.31 ± 0.03	6.74 ± 0.42	12.26 ± 1.39	77.38 ± 1.95	3.15 ± 0.24	0.07 ± 0.01	0.09 ± 0.02
	8	0.3 ± 0.02	6.09 ± 0.6	10.2 ± 5.4	80.5 ± 5.82	2.77 ± 0.69	0.06 ± 0.01	0.09 ± 0.01
	9	0.31 ± 0.01	4.47 ± 0.93	11.74 ± 2.89	80.57 ± 4.57	2.76 ± 0.72	0.07 ± 0.01	0.09 ± 0.02
	10	0.34 ± 0.03	3.39 ± 0.25	14 ± 0.96	79.64 ± 1.41	2.47 ± 0.21	0.08 ± 0.01	0.08 ± 0.01
5	1	0 ± 0	13.19 ± 1.65	0 ± 0	81.81 ± 1.55	5.01 ± 0.61	0 ± 0	0 ± 0
	2	0.42 ± 0.72	13.37 ± 5.29	1.86 ± 2.11	79.75 ± 5.75	4.6 ± 3.38	0 ± 0	0 ± 0
	3	0.28 ± 0.13	11.18 ± 2.42	8.07 ± 2.52	77.2 ± 5.86	3.12 ± 0.77	0.05 ± 0.02	0.1 ± 0.02
	4	0.53 ± 0.29	13.5 ± 2.41	13.94 ± 6.24	66.61 ± 11.34	5.2 ± 2.29	0.08 ± 0.04	0.15 ± 0.12
	5	0.51 ± 0.17	12.66 ± 0.63	16.8 ± 4.31	63.93 ± 6.18	5.83 ± 1.31	0.09 ± 0.03	0.17 ± 0.05
	6	0.22 ± 0	4.63 ± 0.73	8.03 ± 1.22	84.75 ± 1.36	2.25 ± 0.31	0.05 ± 0.02	0.07 ± 0.01
	7	0.26 ± 0.03	4.43 ± 0.86	10.25 ± 4.46	82.1 ± 6.45	2.81 ± 1.06	0.06 ± 0.01	0.08 ± 0.02
	8	0.26 ± 0.03	4.24 ± 1.03	10.81 ± 3.63	81.58 ± 5.35	2.96 ± 0.82	0.06 ± 0.01	0.09 ± 0.02
	9	0.3 ± 0.03	3.5 ± 1.08	12.47 ± 4.43	80.69 ± 6.42	2.89 ± 0.92	0.08 ± 0.01	0.08 ± 0.02
	10	0.31 ± 0	2.98 ± 0.23	14.64 ± 2	79.08 ± 2.35	2.83 ± 0.22	0.08 ± 0.01	0.09 ± 0.01

Values are expressed as percentages (%)

Comparative evaluation of fatty acid content in cultured cells under different pH conditions and culture period

The weight of fatty acids in M. furfur cultured in the flask was calculated using the composition ratio of each fatty acid and the weight of lipids (Fig. 5 and Table 2). Due to the characteristic of M. furfur, which must absorb C16 from the outside, the results suggested that pH 6 was the most suitable condition for fatty acid accumulation and synthesis, with pH 5 being the next highest. The contents of oleic acid, linoleic acid, and palmitic acid were generally highest at pH 6, followed by pH 5. The amount of oleic acid continued to increase as M. furfur’s growth. The weight of linoleic acid, which can be synthesized from oleic acid and absorbed from olive oil, was highest at pH 6. At pH 6 and 5, linoleic acid and palmitic acid had the highest weights and tended to increase rapidly and then decrease. The weights of linoleic acid and palmitic acid at pH 6.6 and pH 7 were low, but, like pH 6 and pH 5, they tended to increase somewhat and then decrease. Palmitoleic acid, octadecanoic acid, 11-eicosenoic acid, and arachidic acid showed the highest amounts at pH 6, and a tendency to increase and then decrease, like linoleic acid and palmitic acid, was confirmed at pH 6 and pH 5. These results suggest that fatty acid absorption and biosynthesis are most active at pH 6 and pH 5 and then stabilize or decrease to maintain a constant amount.

Fig. 5.

Fatty acid weight under different pH conditions over the Malassezia furfur cultivation period. The fatty acid weights shown were calculated based on the lipid content data presented in Fig. 3 and the fatty acid composition data shown in Fig. 4. The overall fatty acid weight at pH 7 (a), pH 6.6 (b), pH 6 (c), and pH 5 (d) is shown. The amounts of oleic acid, palmitoleic acid, palmitic acid, linoleic acid, octadecanoic acid, 11-eicosenoic acid, and arachidic acid were measured. Values are presented as mean ± standard error of the mean (SEM) from three independent replicates. Statistical significance of changes from day 3 to later time points up to day 10 was evaluated using a one-way ANOVA with multiple comparisons. p-values were calculated relative to day 3 and have been indicated below the x-axis. The corresponding p-values were determined and marked on the graph, with significance levels represented as follows: ns (not significant), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Table 2.

Fatty acid quantities of Malassezia furfur in the flask during cultivation

pH value	Day	Palmitoleic acid	Palmitic acid	Linoleic acid	Oleic acid	Octadecanoic acid	11-Eicosenoic acid	Arachidic acid
7	3	3.2 ± 2.78	46.28 ± 13.46	17.07 ± 4.87	526.67 ± 24.93	13.45 ± 7.36	0 ± 0	0 ± 0
	4	7.59 ± 1.03	82.58 ± 79.53	60.51 ± 63.99	661.2 ± 95.62	21.44 ± 25.61	0.36 ± 0.62	0.49 ± 0.85
	5	8.41 ± 3.33	86.03 ± 94.86	74.53 ± 86.82	712.6 ± 285.87	27.24 ± 35.66	0.45 ± 0.79	0.73 ± 1.26
	6	6.32 ± 1.15	40.57 ± 27.52	36.75 ± 27.86	595.85 ± 238.82	13.24 ± 11.12	0.24 ± 0.41	0.38 ± 0.66
	7	3.18 ± 0.9	21.58 ± 9.26	13.76 ± 6.09	442.56 ± 184.17	6.2 ± 2.66	0 ± 0	0.22 ± 0.39
	8	2.14 ± 0.98	22.97 ± 5.7	23 ± 16.2	362.91 ± 83.6	7.37 ± 2.19	0.29 ± 0.34	0.22 ± 0.2
	9	2.44 ± 1.27	15.84 ± 8.25	8.79 ± 4.46	292.65 ± 155.29	5.29 ± 2.94	0 ± 0	0 ± 0
	10	1.74 ± 0.3	12.45 ± 0.57	7.26 ± 0.12	239.62 ± 13.94	4.11 ± 0.07	0 ± 0	0 ± 0
6.6	3	6.71 ± 1.27	132.29 ± 35.35	75.5 ± 21.53	992.4 ± 168.65	26.68 ± 8.5	0.74 ± 0.19	0.68 ± 0.18
	4	14.99 ± 1.32	414.55 ± 55.36	280.87 ± 46.61	1650.68 ± 147.54	125.06 ± 18.26	2.09 ± 0.18	3.41 ± 0.76
	5	14.09 ± 2.66	371.04 ± 49.8	304.94 ± 45.3	1493.22 ± 74.36	129.4 ± 20.8	2.1 ± 0.28	3.54 ± 0.73
	6	13.62 ± 6.23	337.35 ± 201.19	330.88 ± 265.87	1998.01 ± 133.19	130.25 ± 107.04	2.52 ± 1.94	4.04 ± 3.74
	7	9.14 ± 0.5	231.28 ± 48.83	190.19 ± 57.82	2123.6 ± 144.93	84.15 ± 24.25	2.34 ± 0.61	2.64 ± 0.73
	8	10.54 ± 5.74	244.93 ± 56.25	158.3 ± 50.73	2815.09 ± 767.18	79.52 ± 21.21	2.15 ± 0.43	4.46 ± 2.85
	9	9.27 ± 1.44	278.53 ± 60.05	229.38 ± 58.03	2700.15 ± 812.67	113.47 ± 28.15	2.3 ± 0.46	3.57 ± 1.14
	10	10.6 ± 2.07	271.32 ± 28.72	280.81 ± 38.2	3051.27 ± 139.62	130.84 ± 18.17	3.03 ± 0.48	4.57 ± 1.06
6	3	15.46 ± 5.6	470.56 ± 158.01	371.61 ± 180.93	1727.24 ± 168.21	138.85 ± 67.21	2.02 ± 0.99	4.26 ± 2.26
	4	42.54 ± 9.82	895.73 ± 95.53	961.88 ± 149.98	2619.28 ± 131.87	358.17 ± 72.89	6.38 ± 1.69	12.67 ± 3.21
	5	32.87 ± 5.39	688 ± 117.72	865.47 ± 167.39	2279.46 ± 416.31	300.47 ± 68.71	5.3 ± 1.35	10.1 ± 2.52
	6	15.33 ± 1.39	355.41 ± 55.65	517.17 ± 87.94	3256.34 ± 108.7	139.45 ± 29.84	2.46 ± 0.2	3.85 ± 0.58
	7	13.97 ± 2.78	303.1 ± 44.25	553.86 ± 120.62	3465.18 ± 265.2	141.8 ± 23.17	3.18 ± 0.17	3.92 ± 0.81
	8	15.49 ± 0.38	322.38 ± 62.48	517.68 ± 244.16	4258.7 ± 764.37	143.16 ± 23.79	2.95 ± 0.15	4.64 ± 0.44
	9	15.02 ± 3.43	224.88 ± 95.1	594.22 ± 274.79	3914.84 ± 552.44	140.07 ± 66.73	3.32 ± 1.35	4.32 ± 1.74
	10	16.37 ± 5.17	162.48 ± 44.9	672.98 ± 194.38	3786.01 ± 769.5	119.29 ± 36.98	3.66 ± 0.86	4.07 ± 1.46
5	3	5.84 ± 4.97	221.19 ± 139.49	163.41 ± 113.57	1407.84 ± 494.3	62.17 ± 40.41	1.01 ± 0.76	1.87 ± 1.09
	4	17.33 ± 9.74	441.79 ± 87.61	458.89 ± 212.55	2173.02 ± 360.74	171.24 ± 78.04	2.6 ± 1.42	4.8 ± 3.9
	5	14.21 ± 9.06	331.8 ± 114.98	458.08 ± 261.6	1630.61 ± 333.21	158 ± 84.92	2.49 ± 1.63	4.81 ± 2.95
	6	5.34 ± 0.94	114.17 ± 22.52	201.32 ± 61.92	2089.87 ± 297.88	56.23 ± 16.51	1.28 ± 0.59	1.79 ± 0.62
	7	8.58 ± 3.83	149.38 ± 78.64	363.37 ± 269.32	2603.79 ± 670.19	98.48 ± 68.34	1.96 ± 0.93	2.76 ± 1.74
	8	9.58 ± 3.14	160.07 ± 64.61	420.16 ± 231.48	2974.11 ± 598.55	113.95 ± 56.15	2.18 ± 0.77	3.29 ± 1.38
	9	12 ± 4.35	146.68 ± 73.83	531.51 ± 291.2	3172.74 ± 715.11	122.37 ± 64.19	3.08 ± 1.27	3.53 ± 1.7
	10	11.71 ± 1.82	114.63 ± 25.68	565.13 ± 141.83	3016.91 ± 374.93	108.85 ± 22.28	3.12 ± 0.55	3.42 ± 0.93

All values in the table are expressed in mg L⁻¹

Secretion of intracellular lipids from Malassezia species cells to the outside

Staining the cell wall (chitin) and lipids of Malassezia species transferred to a medium without artificial sebum with Calcofluor-White and NileRed, respectively, it was confirmed that red-stained lipids were present within the blue-stained cells (Fig. 6). Intracellular lipids were observed to be secreted from inside the cell to the outside within just 3 h of culture. These results suggest the possible role of Malassezia species as a sebum depot or refinery, replenishing epidermal surface lipids when sebum secretion is insufficient. In our M. furfur fatty acid weight analysis, it was confirmed that the weight of some fatty acids decreased after a certain period. There appears to be a relationship between the loss of intracellular lipids and the decrease in fatty acid weight. However, additional experiments are needed to verify whether fatty acids are actually released to the outside and whether the lipid weight inside the cell decreases.

Fig. 6.

Intracellular lipid secretion observed through confocal microscopy in three Malassezia species. To observe the behavior of three types of Malassezia species when cultured on agar plates with a lipid source and then transferred to a medium without a lipid source, they were labeled with NileRed and Calcofluor-White. Lipids are stained red with NileRed, and cells are stained blue with Calcofluor-White. Images were taken at 0 h (T0), 3 h (T1), and 6 h (T2) after transfer to a lipid source-free medium. Panels show (a) Malassezia furfur, b Malassezia japonica, and (c) Malassezia yamatoensis. All images have the same scale. Data were collected from three independent experiments, and representative images are shown

Discussion

Among the various fungi that commonly cause skin diseases, Malassezia spp. is the most abundant and is also common in ordinary people. One environment factor that differs between normal people and patients with skin diseases is pH conditions. Therefore, in this study, we closely examined the effects of pH conditions on the growth, lipid content, and fatty acid composition of M. furfur, M. japonica, and M. yamatoensis, and the impact of these Malassezia-derived lipids on human skin cell lines. First, we analyzed the growth of M. furfur under different pH conditions and found that the biomass concentration of this microorganism was highest at pH 6, followed by pH 5, 6.6, and 7. The biomass concentration increased remarkably between the first and fourth day of culture, indicating a period of active cell proliferation utilizing fatty acids. Additionally, lipid content was highest at pH 6 and lowest at pH 7, indicating differences in extracellular lipase activity and metabolism in the uptake of lipids from outside to inside cells, and use or accumulation of absorbed lipids. Analysis of the fatty acid composition revealed that oleic acid accounted for the highest percentage, while the percentage of linoleic acid tended to increase under most pH conditions. Linoleic acid plays a vital role in cell growth and metabolism. As an essential fatty acid, it maintains the fluidity of cell membranes and is involved in cell signaling processes. The increased percentage of linoleic acid in pH 6 conditions suggests that M. furfur is more active in growth and metabolism under these conditions. On the other hand, the lower linoleic acid ratio in the pH 7 condition indicates that this condition is unfavorable for the metabolic activity of M. furfur. All of these results suggest that the optimal growth of M. furfur occurs under slightly acidic conditions, indicating that M. furfur may thrive better in an environment similar to the skin’s natural pH. Further study is needed to determine whether M. japonica and M. yamatoensis, which showed good efficacy in cell evaluation, exhibit similar trends to the results of M. furfur.

Under pH 6 conditions, Malassezia-derived lipids had specific effects on human skin cell lines. RT-qPCR analysis of the expression of pro-inflammatory cytokines IL-6 and TSLP revealed that the expression of both cytokines significantly increased in the inflammation-induced group. In contrast, the expression tended to decrease in a concentration-dependent manner in the M. furfur–derived lipid treatment group, suggesting that Malassezia-derived lipids may help modulate the inflammatory response. Stabilizing and reducing inflammatory responses caused by external infections can benefit skin health by maintaining the skin barrier. IL-6 is particularly highly expressed in inflammatory skin diseases such as psoriasis and is known to be involved in abnormal proliferation and differentiation of keratinocytes. The psoriasis patient’s skin pH is approximately 5.2, which is lower than the normal pH (5.6). Therefore, it is expected that the growth of M. furfur will be reduced, and the production of lipids and fatty acids will be lower. As a result, there may be difficulties in reducing the expression of inflammation, including IL-6 (Cannavo et al. 2017; Swaroop et al. 2024). Additional research is needed to determine whether bio-converted lipids derived from Malassezia at pH 6 can help improve skin health and reduce inflammation in psoriasis patients. TSLP released by keratinocytes is known to be highly expressed in atopic dermatitis patients. In particular, TSLP regulates Th2 immune responses, producing IL-4, IL-5, and IL-13, and increases the production of inflammatory cytokines such as IL-31 (Das et al. 2022). It is known that the skin pH of atopic dermatitis patients is higher than normal pH Based on the finding that M. furfur does not grow properly at pH 7, which is higher than the normal skin pH, additional research on the effects of Malassezia-derived biosynthetic lipids appears to be necessary (To confirm the effect of lipids biosynthesized from Malassezia on keratinocytes under conditions of pH 7 and pH 6.6, the same treatment was performed; however, a significant reduction in the induced inflammatory response was not observed).

The expression of AQP3 and HAS3 was also identified in relation to the moisturizing function of maintaining skin hydration. AQP3 expression was found to be reduced in the AS, MF BCL, and MJ BCL high-dose treatment groups. It is known that AQP3-deficient mice exhibit delayed skin wound healing and reduced keratinocyte proliferation. In addition, studies have shown that AQP3-deficient mice have reduced water content in the stratum corneum (Ma et al. 2002; Nakahigashi et al. 2011). These studies found that both deficient mouse models were restored by glycerol supplementation. The decreased expression of AQP3 in the high-dose treatment group could be due to the presence of lipid components from Malassezia biosynthesis that either directly deliver water to keratinocytes or act as substitutes for glycerol. Malassezia biosynthesis–derived lipids may play a role in preventing water from escaping the cells or in bringing water in from the outside. Therefore, further studies beyond fatty acids are needed to determine the role of Malassezia in skin moisturization.

HAS3 is an enzyme that synthesizes hyaluronic acid, which is important in skin moisturization. The results showed that the expression of HAS3 in the SL 10 ppm, SL 100 ppm, AS 100 ppm, and MY BCL 10 ppm and 100 ppm treatments was similar to or slightly lower than the control. These results indicate that SL and AS do not have a significant effect on HAS3 expression. On the other hand, HAS3 expression tended to increase in the AS 10 ppm, MF BCL, and MJ BCL treatments, suggesting that these lipids may contribute to enhanced moisturization with increasing concentration. FN1 primarily provides skin elasticity and flexibility, and its expression is known to be increased by TGF-β1. While FN1 expression in the SL 10 ppm treatment group was similar to the control, the rest of the groups showed higher expression, with the highest expression in the MF BCL 10 ppm treatment group, suggesting that Malassezia-derived lipids may positively affect skin elasticity.

For ELN, the expression was highest in the TGF-β1 treatment group, consistent with previous studies. The MF BCL 10 ppm treatment group showed higher ELN expression compared to other Malassezia species, suggesting that M. furfur–derived lipids may positively influence skin elasticity. In conclusion, this study identifies the effects of pH changes on the growth and lipid metabolism of Malassezia species, providing a new approach to maintaining and improving skin health. We found that Malassezia species exhibit optimal growth in a slightly acidic environment and that lipids produced under these conditions can positively impact the modulation of the inflammatory response in human skin cell lines and improve skin moisturization and elasticity.

Future studies should analyze the specific fatty acids Malassezia species produce at each pH condition to determine which ones have positive and negative effects on skin cells. Additionally, it is necessary to verify what types of lipids Malassezia species secrete to the outside and through what methods. We also need to examine how the ingredients change depending on pH. In addition to fatty acids, studies are needed to analyze the content of lipid components such as triacylglycerols, phospholipids, waxes, sphingolipids, and glycolipids using equipment such as liquid chromatography–mass spectroscopy–quadruple time of flight (LC–MS/Q-TOF) to determine what effect each composition has on skin cells. Furthermore, the effects of Malassezia-derived lipids need to be evaluated using a variety of skin cell lines, and their impact on inflammatory mediators and immune responses needs to be studied in greater depth. In particular, the impact of Malassezia-derived lipids on the immune system must be systematically elucidated by investigating the responses of inflammatory mediators and immune cells, including IL-1, TNF-α, and IFN-γ. To better understand skin lesions and pathological changes that may occur when Malassezia infection becomes chronic, it is necessary to study changes in skin cells exposed to Malassezia-derived lipids over prolonged and repeated periods. Finally, it is important to develop therapeutic strategies based on in vitro findings through clinical studies with actual patients to practically evaluate the impact of Malassezia-derived lipids on skin diseases. Through this, we have laid a new foundation for understanding why Malassezia species, known as an opportunistic pathogen, are a core microbiome not only in patients but also in normal skin, why their abundance is high, and how they coexist. This will allow us to systematically understand the different effects of Malassezia species on the skin, enabling us to develop effective methods for preventing and treating skin diseases.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

Data curation: Y.P, writing—original draft preparation: Y.P and D.K, writing—review and editing: K.H, conceptualization: B.Y, Y.H, and K.H, validation: B.Y, investigation: Y.H, S.K, and S.H.K, methodology: S.K, visualization: K.L. All authors have read and agreed to the published version of the manuscript.

Funding

The Basic Science Research Capacity Enhancement Project (Bio-Medical Engineering Core Facility and Support for Activating Joint Research) through a Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (grant nos. 2019R1A6C1010033 and 2021R1A6C103B392).

Data availability

All data are available in the articles or the supplementary materials.

Declarations

Conflict of interest

This study was supported by IL-YANG Pharmaceutical Co., Ltd.

Human and animal rights

This article does not contain any studies with human participants performed by any of the authors.