Enzymatic Fungal Growth Inhibition | BMC Microbiology

Test ingredients

The following commercially available compounds with antiseptic activity, climbazole (Symrise AG, Holzminden, Germany), piroctone olamine (Shanghai Cosroma Biotech Co., Ltd., Shanghai, China), selenium sulfide (Shanghai Cosroma Biotech Co., Ltd., Shanghai, China), ZnPy (Salicylates and Chemicals Pvt. Ltd., Hyderabad, Telangana, India), and propanediol caprylate (Symrise AG, Holzminden, Germany), were used as positive controls. Solutions containing these compounds are summarized in Table S1.

The enzymes chitinase (100 U/g, 30% – pure enzyme from Aspergillus niger70%– Magnesium sulfate) and chitosanase (200 U/g, 30% – pure enzyme from Aspergillus niger70%– Magnesium sulfate), obtained from Shaanxi Pioneer Biotech Co., Ltd., Xi’an, China, are promising agents for use as anti-dandruff agents. The original data are presented in Table S2.

Test microorganisms and growth conditions

The following fungal strains were used for the enzyme studies and as positive controls for the growth inhibition of Malassezia spp.: M. furfur CBS 1878, M. restricta CBS 7877, and M. globosa CBS 7966 were purchased from the Westerdijk Institute in Utrecht, Netherlands. The strains were cultured in Sabouraud dextrose agar and then incubated in CHROMagar Malassezia^® with Tween-40 and glycerol for visible growth at 37 °C for 72 h. An aliquot of the cell culture was diluted, and the cells were counted using a Neubauer chamber to ensure they reached the desirable inoculum concentration of 1.0 × 10⁶ colony-forming unit (CFU)/mL. All microbiological analyses were performed in a microbiology safety cabinet (Faster, Italy).

Antifungal screening by determining Log10CFU reduction

The decimal logarithm of the colony-forming units per milliliter (Log10CFU) reduction in fungi induced by the tested antifungal substances was used to evaluate their inhibitory effect on M. furfur, M. globosaand M. restricta. Briefly, following exposure of the fungal strains to the substances, they were quantitatively evaluated by determining the viable total colony counts. The test substances were prepared in water containing 0.3% (w/w) polysorbate 80, at final concentrations of 50.0, 30.0, 20.0, 10.0, 5.0, 2.5, and 1.25 mg/mL (equivalent to 5.00, 3.00, 2.00, 1.00, 0.50, 0.25, and 0.125% w/w). Polysorbate 80 was included in the aqueous solution to enhance the solubility and stability of the test substances, some of which exhibit limited water solubility. As a non-ionic surfactant, polysorbate 80 ensures homogeneous dispersion of the compounds. The benchmark antifungal drugs climbazole, piroctone olamine, ZnPy, propanediol caprylate and selenium sulfide, were used as controls. First, 1 mL mixture of the test microorganisms at a density of 1.0 × 10⁶ CFU/mL was cultured with 9 mL of each sample concentration.

The experimental procedure involved incubating the substances with a known suspension of M. furfur, M. restrictaand M. globosa on substrate culture medium (Mueller-Hinton broth) at 37 °C for 1 h, which is the average time required for the potential anti-dandruff activity of ingredients to exert its effects. Next, the viable fungal population was determined by counting the viable cells on an agar plate (CHROMagar Malassezia). A suspension of the microorganism inoculum without any test substance was used as the negative control, and all experiments were performed in triplicate. The results were expressed as a decimal logarithm of the reduction in colony-forming units per milliliter (log10CFU/mL) and then converted to antifungal efficacy as a percentage of inhibition. All analyses were conducted in triplicate for statistical data processing.

Cytotoxity evaluation of the tested substances by MTT test and fluorescence microscopy with live/dead staining

Cytotoxicity of substances was assessed using cell lines of non-immortalised fibroblasts in the MTT metabolic test. This method is based on the reduction reaction of the yellow tetrazolium salt (3’-(4,5-Dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide, MTT) by mitochondrial dehydrogenases of living cells to purple formazan crystals, which are insoluble in aqueous medium [34].

For the assay, cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal calf serum, L-glutamine and 1% penicillin/streptomycin in an atmosphere of 5% CO₂ at 37 °C until monolayer formation. To perform the test, 1000–3000 cells in 200 µl of culture medium are added to the wells of a 96-well plate depending on the proliferative potential. Cells are thoroughly suspended before introduction into the plate. The cell suspension was introduced into the wells of the plate using a multichannel pipette and sterile tips. The plates with cells were then incubated for 24 h in a CO₂ incubator for cell adhesion to the substrate. Using a multichannel pipette and sterile tips, aliquots of prepared solutions of compounds at the tested concentrations were added to the wells of the plate with cells. After addition of the test substances, the cells were cultured in a CO₂ incubator to maintain a stable physiological pH in the culture medium through a CO2-bicarbonate buffering system under standard conditions for 48 h. At the end of the incubation time, the culture medium with the test substances was removed from the plate. A mixture was prepared in a multi-channel pipetting tub: 9 ml of culture medium + 1 ml of MTT reagent (5 mg/ml in Hanks’ solution) per 96-well plate. The prepared reaction mixture was added in a volume of 100 µl into each well of the plate and incubated in a CO₂ incubator for 3.5 h.

At the end of the incubation time, the culture medium with the tested compounds was removed, 100 µl of dimethyl sulfoxide (DMSO) was added and incubated for 5–10 min. The resulting violet staining was detected at a wavelength of 550 nm on a Tecan infinite 200 Pro microplate reader (Switzerland).

The ability of chitinase and chitosanase to exert cytotoxic effect on cells was also evaluated by microscopy with counting the area of viable cells in ‘BCAnalyzer’, semi-automated tool for the quantification of cell monolayer from microscopic images [35] on fibroblast cell lines.

Quantitative PCR evaluation of Chitinase and Chitosanase antifungal effects on scalp mycobiome

The primary objective of this study was to characterize the effects of chitinase and chitosanase solutions on the scalp mycobiota of 18 volunteers, with a focus on mitigating the microbial factors associated with scalp flaking in conventionally individuals. The study aimed to elucidate the potential of these enzymatic treatments in addressing the fungal causes of dandruff formation.

A transparent aqueous solution containing 0.25% chitinase and 0.25% chitosanase was utilized in this study. The research protocol was approved by the Local Ethics Committee of Kazan (Volga Region) Federal University (Protocol No. 48, dated May 23, 2024) and was conducted in accordance with the ethical principles of the Declaration of Helsinki following written informed consent from all participants. The study enrolled 18 volunteers (9 females and 9 males), aged 18 to 60 years, who met the following exclusion criteria: pregnancy or lactation; kidney or liver diseases; severe autoimmune disorders within the last 5 years; chemical treatments such as perming, keratin straightening, bleaching, or permanent waving within the last month; hair coloring within the last 14 days; use of antifungal medications or supplements within the last 60 days; use of anti-dandruff shampoos within the last 60 days; history of skin diseases within the last 3 years; systemic retinoid use within the last 60 days; and use of hair loss treatments within the last 60 days. Participants completed a questionnaire and underwent a dermatological examination of the scalp prior to the study.

A 4 × 4 cm area was selected on the hairy part of the scalp. Pre-treatment samples were collected from these areas using sterile swabs and placed in microtubes with lysis buffer. Subsequently, 1 mL of the enzyme solution was added to this area. After a 3-hour incubation, post-treatment samples were collected using sterile swabs and transferred to microtubes with lysis buffer for further analysis.

Nucleic acids were extracted using the LIRA reagent (BioLabMix, Russia). Swabs collected from the scalp surface were placed in 1 mL of LIRA reagent and incubated for 10 min at room temperature. Chloroform (200 µL) (Sigma-Aldrich, USA) was added to the lysate, followed by vigorous shaking for 5 s and incubation for 5 min with intermittent manual mixing. The swab was removed, and the mixture was centrifuged at 10.000 × g for 10 min at 4 °C. The resulting phases—organic (lower), interphase, and aqueous (upper)—were separated. The aqueous phase was transferred to a new tube for RNA extraction, while the organic phase and interphase were retained for subsequent DNA isolation.

For RNA extraction, an equal volume of isopropanol (Sigma-Aldrich, USA) was added to the aqueous phase, mixed by inversion, and incubated for 10 min at room temperature. The sample was centrifuged at 12.000 × g for 10 min at 4 °C. The supernatant was discarded, and the pellet was washed with 1 mL of 80% ethanol (Sigma-Aldrich, USA). After centrifugation at 12.000 × g for 5 min at 4 °C, the supernatant was removed, and the pellet was air-dried for 10 min. RNA was resuspended in 15 µL of RNase-free water and stored at −20 °C.

For DNA extraction, the organic phase and interphase were combined, homogenized, and mixed with 300 µL of 96% ethanol. After inversion and incubation for 5 min at room temperature, the sample was centrifuged at 2.000 × g for 5 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in 1 mL of 0.1 M sodium citrate in 10% ethanol (pH 8.5). Following incubation for 30 min at room temperature and centrifugation, the pellet was washed with 1 mL of 80% ethanol, incubated for 20 min, and centrifuged again. The dried pellet was dissolved in 15 µL of 40 mM NaOH, neutralized with 0.5 µL of 1 M HEPES, and stored at −20 °C.

Quantitative PCR (qPCR) was performed using a BioRad CFX96 thermocycler (BioRad, USA) with the Biomaster HS-qPCR mix (BioLabMix, Russia) under manufacturer-recommended conditions [36]. The 50 µL reaction mixture contained 1× RT-qPCR buffer, 0.1 µM of each primer (Table S3), 0.1 µM dNTPs, 2.5% DMSO, 5% RT-qPCR mix, and DEPC-treated water. The ITS region of 26 S rRNA was used as reference gene. The primers were designed based on 26 S rRNA genes of Malassezia species.

The PCR protocol included an initial denaturation at 95 °C for 7 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 40 s, and elongation at 72 °C for 35 s [37]. Primer annealing temperatures were calculated using the Tm Calculator [38]. Real-time monitoring was performed after each elongation step.

qRT-PCR was performed using the BioRad CFX96 thermocycler (BioRad, USA) with the qOT-PCR SYBR Blue mix (BioLabMix, Russia), a one-step reverse transcription and real-time quantitative PCR mix containing the SYBR Green I fluorescent dye, following the manufacturer’s protocol. The 50 µL reaction mixture consisted of 1× RT-qPCR SYBR Blue buffer, 0.1 µM of each primer (Table S3), 0.1 µM dNTPs, 2.5% dimethyl sulfoxide (DMSO), 5% RT-qPCR mix, and DEPC-treated water. The primers were designed based on 26 S rRNA genes of Malassezia species.

The qRT-PCR protocol included a reverse transcription step at 45 °C for 30 min, followed by initial denaturation at 95 °C for 7 min and 40 cycles of denaturation at 95 °C for 30 s, primer annealing at 55 °C for 40 s, and elongation at 72 °C for 35 s. Primer annealing temperatures were calculated using the Tm Calculator ( Real-time fluorescence monitoring was conducted after each elongation step.

Following PCR amplification, cycle threshold (Ct) values were determined both before and after treatment for each sample, using specific probes for the reference gene and an intercalating dye for the target gene. The analysis was conducted in three sequential steps:

1. 1.

   Calculation of Transcriptional Changes: The changes in transcript levels for both the reference and target genes were calculated separately [39]:

   $$begin{aligned} :varDelta:Ct:left(reference:generight):=&:Ct:(pre-treatment): &-:Ct:(post-treatment) end{aligned}$$

   $$begin{aligned} :varDelta:Ct:left(target:generight):=:&Ct:(pre-treatment):&-:Ct:(post-treatment) end{aligned}$$

2. 2.

   Normalization of Target Gene Expression: The expression of the target gene was normalized relative to the reference gene using the formula:

   $$begin{aligned} :varDelta:varDelta:Ct:=:&varDelta:Ct:left(reference:generight):&-:varDelta:Ct:left(target:generight). end{aligned}$$

3. 3.

   Quantification of Relative Nucleic Acid Content: The relative change in nucleic acid content was determined using the formula (:{2}^{-varDelta:varDelta:Ct}) where

   $$begin{aligned} :varDelta:varDelta:Ct:=:&varDelta:Ct:left(target:sampleright):&-:varDelta:Ct:left(reference:sampleright). end{aligned}$$

   For ease of interpretation, the fold change in expression was calculated as (:{frac{1}{2}}^{varDelta:varDelta:Ct}) Values greater than 1 indicate a reduction in relative nucleic acid content post-treatment, while values less than 1 indicate an increase.

Statistical analysis

The research findings are presented as mean ± standard deviation, calculated from parallel replicates for each validation parameter. All experiments were performed in triplicate. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Microsoft Excel (Microsoft Corporation, version 2016, Redmond, WA, USA, www.microsoft.com), Student’s t-test with the software Statistica (StatSoft, Inc. STATISTICA, version 9.0, Tulsa, OK, USA, www.statsoft.com) and GraphPad Prism 6.0 (GraphPad Software, version 6.0, Boston, MA, USA, www.graphpad.com), which offers a comprehensive suite of statistical tools. This approach provides a robust method for assessing treatment-induced changes in gene expression and nucleic acid content. Results were considered statistically significant at p ≤ 0.05.

Metagenomic analysis of scalp Microbiome response to enzymatic treatment

The objective was to characterize the impact of chitinase-chitosanase enzymatic treatment on scalp bacterial composition in healthy volunteers.

The chitinase–chitosanase solution preparation, volunteer selection criteria, and scalp treatment protocol were identical to those used in the PCR experiments. A transparent aqueous solution containing 0.25% chitinase and 0.25% chitosanase was utilized in this study. The research protocol was approved by the Local Ethics Committee of Kazan (Volga Region) Federal University (Protocol No. 48, dated May 23, 2024) and was conducted in accordance with the ethical principles of the Declaration of Helsinki following written informed consent from all participants. The study enrolled 4 volunteers (2 females and 2 males), aged 18 to 60 years, who met the following exclusion criteria: pregnancy or lactation; kidney or liver diseases; severe autoimmune disorders within the last 5 years; chemical treatments such as perming, keratin straightening, bleaching, or permanent waving within the last month; hair coloring within the last 14 days; use of antifungal medications or supplements within the last 60 days; use of anti-dandruff shampoos within the last 60 days; history of skin diseases within the last 3 years; systemic retinoid use within the last 60 days; and use of hair loss treatments within the last 60 days. Participants completed a questionnaire and underwent a dermatological examination of the scalp prior to the study.

A 4 × 4 cm area was selected on the hairy part of the scalp. Pre-treatment samples were collected from these areas using sterile swabs and placed in microtubes. Subsequently, 1 mL of the enzyme solution was added to this area. After a 3-hour incubation, post-treatment samples were collected using sterile swabs and transferred to microtubes for further analysis.

DNA was extracted using the FastDNA™ SPIN Kit for Soil (MP Biomedicals, USA) according to the manufacturer’s protocol. Metagenomic libraries were prepared following the “Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System” protocol. The V3–V4 region of the 16S rRNA gene was amplified with the following primers: forward (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’) and reverse (5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’). Amplicons were indexed and sequenced on an Illumina MiSeq system (Illumina, USA) in 2 × 300 bp paired-end mode.

Raw sequences were quality-checked with FastQC, and low-quality reads plus adapter/linker sequences were removed using Trimmomatic [40]. Filtered reads were processed in QIIME 2 (v.2022.8) [41] and denoised with DADA2 algorithm [42]. Taxonomy was assigned using the SILVA 138 database clustered at 99% sequence identity [43]. Alpha diversity metrics (Shannon index, Simpson index, Faith’s phylogenetic diversity, and Chao1) were calculated to assess bacterial diversity.

Statistical analysis was performed in Microsoft Excel 2016 (Microsoft Corporation, Redmond, USA) and GraphPad Prism 6.0 (GraphPad Software, USA). Group differences were evaluated by multiple t-tests or the Mann–Whitney U test.