ISOLATION AND CHARACTERIZATION OF ACTIVE METABOLITES PRODUCED FROM PROBIOTIC ISOLATES AGAINST DANDRUFF CAUSING MALASSEZIA

Dandruff is commonly known as seborrheic dermatitis, a common scaling condition, causing itching and discomfort. Anti-dandruff shampoos contain compounds harmful to the environment and humans; therefore, a solution with potential anti-dandruff activity with minimal or no harm is needed for an hour. This study aims to screen and identify the likely anti-dandruff probiotic organisms and characterize their active metabolites. Three morphologically different probiotic microorganisms were isolated from local dairy products and identified by gram staining. 16S rDNA of the isolates were amplified and sequenced for molecular identification. 16S rDNA sequences confirmed the isolates as Lactobacillus rhamnosus (98%), Enterococcus faecalis (98%), and Enterococcus faecium (97.65%). Phylogenetic analysis reveals that L. rhamnosus (MK951691) indicated high sequence similarity with L. paracasei. Similarly, E. faecium (MK951690) represented close similarity with E. faecalis (MK951689). A maximum zone of inhibition of 7 mm was recorded by the extracellular fractions of bacterial isolate L. rhamnosus, followed by the other two isolates of Enterococcus sp. The MIC value of the extracellular fraction of probiotic bacterial strains was optimized to be 100 mg/mL. GCMS analysis revealed that all three extracellular bacterial isolates had propionic acid, lactic acid, phenol, 2,4-bis(1,1-dimethyl ethyl), hexadecanoic acid, octadecanoic acid, and 3-isobutyl hexahydro pyrrolo [1,2-a]pyrazine-1,4-dione. In conclusion, these isolates are enriched with antifungal components, and hence, they could be considered an antifungal target against Malassezia furfur.


Screening and Characterization of Probiotic Strains
Commercial probiotic samples of curd, yogurt, and probiotic tablets were collected from the local market, Guduvancherry, Chennai, India. The samples were serially diluted and plated on MRS agar. The plates were incubated at 37°C for 48 h. Morphologically distinct colonies were selected and subjected to Scanning Electron Microscope analysis (SEM). Whole cells were used for SEM analysis by pelleting liquid cultures of all isolates. The cell pellet dissolved in PBS was placed on round glass coverslips and left to air dry. The fixed samples were coated with Au (Sputter-coater Quorum SC 7620) and viewed under SEM (TESCAN VEGA3,SBH,Tescan,Brno,Czech Republic).

Molecular Characterization of Bacterial isolates
The isolated strains active against M. furfur were selected and subjected to 16s rDNA amplification and sequencing. The PCR amplification of the 16srDNA region was performed in Eppendorf Master cycler nexus GX2 thermocycler (Germany) using the universal forward primer (5'AGAGTTTGATCCTGGCTCAG 3') and the reverse primer (5′ ACGGCTACCTTGTTACGACTT 3′) as reported by Weisburg et al., (1991). Colonies were subjected to the heat lysis method described by Alam et al., (1999) and used as a template for the PCR amplification reaction. The reaction conditions were initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min 30 sec, extension at 72 °C for 1 min 30 sec, and a final extension at 72 °C for 2 minutes. PCR products were visualized on 1 % agarose gel stained with ethidium bromide (0.5 μg/mL).

Sequencing and Phylogenetic analysis
The purified PCR products were sequenced by AgriGenome Labs Pvt. Ltd, Kochi, Kerala, India. The sequences obtained were then processed using BioEdit, a biological sequence alignment editor tool (http://www.mbio.ncsu.edu/bioedit/bioedit.html), which generated contig sequences for each primer pair (Hall, 1999). Lactococcus lactis (NR103878.1) and Myroides odoratimimus (KU382740.1) are considered as outgroups for this study. This contig sequence was then compared with the 16S ribosomal RNA sequences (Bacteria and Archaea) database of NCBI using BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and the most similar sequences were found out and sorted by the E score. The first fifteen similar sequences were aligned using ClustalW, and a phylogenetic tree was generated using MEGA7 software (Kumar et al., 2016).

Agar well diffusion assay
Intracellular and extracellular fractions of isolated probiotic strains were checked against M. furfur by agar well diffusion assay (Bulgasem et al., 2016). Emmons modified Sabouraud's agar plates, and the mid-log phase culture of M. furfur was swabbed on the plate. The agar plates were allowed to dry, and 9 mm diameter wells were made with sterile borer on inoculated plates. 100 µL of extracellular and intracellular fractions were loaded in the respective wells on the plates. The plates were incubated for seven days at 30 °C. The zone of inhibition (mm) was measured. This assay was performed in triplicates.

Minimum Inhibitory Concentration (MIC) assay
The microdilution method was performed with 96 microtiter plates to determine the minimum inhibitory concentration of cell-free Supernatants (CFS) of bacterial isolates (Leong et al., 2017). Different concentrations of lyophilized CFS as 10, 30, 50, 70, 90,100 µg/mL were dissolved in DMSO and used as test samples. Positive control contained 160 µL of Emmons modified Sabouraud's broth, 10 µL of climbazole drug (2 mg/mL), 10 µL of M. furfur, and 20 µL of resazurin dye (1 mg/mL). The negative control contained 170 µL of Emmons modified Sabouraud's broth, 10µL of M.furfur culture, and 20 µL of resazurin dye (6.75 mg/mL -1 ). The microtiter plate was incubated at 30 °C for 48 h. The minimum inhibitory concentration values for the extracellular fractions of isolates dissolved in DMSO were determined by measuring OD540 and OD590 using a multimode plate reader (BioTek, USA).

Gas Chromatography-Mass Spectrometry (GCMS) analysis
Culture supernatants of the isolates were extracted with ethyl acetate and subjected to GCMS analysis. These extracts were analyzed on an Agilent Technologies 7890B GC with FID. The software used for the study of extracted metabolites was Mass hunter software. The GC was mounted with an HP-5MS column with a length of 30 m, width -0.25 mm, and film thickness of 0.1µm. Helium was used as carrier gas flowing at 0.8 mL/min. 1µL of samples were injected with a split ratio of 1:100 in an injector kept at 250 °C. The temperature of the flame ionization detector was held at 240 °C. The temperature gradient was used for low boiling metabolites; the oven was held at 40 C for 2 min initially, then increased to 240 °C at a rate of 20 °C/min. For metabolites with higher boiling temperature, the oven initially held at 150 °C for 2 min, then increased to 240 °C at the rate of 20 °C/min. It was held at 240 °C for 10 min (Honore et al., 2016).

Morphological and Molecular Identification of the Isolates
A total of three probiotic organisms were isolated from the local dairy products. The organisms' morphological characteristics belong to the genus Lactobacillus (1) and Enterococcus (2). Further morphological characterization of these bacterial isolates was analyzed by SEM. SEM analysis showed that Enterococcus species (Fig.1A and B) were found to be cocci, and Lactobacillus species (Fig.1C) was a rod in shape. The DNA from the isolates were collected and subjected to molecular identification. The 16S rDNA gene of 1500 bp was amplified from all three bacterial isolates and confirmed on 1% agarose gel stained with ethidium bromide (0.5 μg/mL) (Fig.2). Following BLASTN analysis, similar sequences were retrieved from Genbank, and a phylogenetic tree was generated using the neighbor-joining method. These isolates showed high similarities with L. rhamnosus (MK951691) (98 %), E. faecium (MK951689) (97.65 %), and E. faecalis (MK951690) (98 %). All the sequences were deposited to Genbank with the accession numbers mentioned above. The phylogenetic analysis of L. rhamnosus indicated high sequence similarity with L. paracasei strain NBRC 15883 (Fig.3a). Smokvina et al., (2013) stated that L. paracasei and L. zeae and L. rhamnosus were closely related species E. faecium indicated high sequence similarity with E. faecalis (98 %) (Fig.3b). Castillo-Rojas et al. (2013) reported that E. faecalis and E. faecium have the highest sequence similarity, up to 84.3 %. M. odoratimimus and L. lactis were considered as outgroups.

Agar well diffusion assay
Probiotic organisms were isolated from local dairy products, including curd, yogurt, and probiotic tablets, and screened for antifungal activity against M. furfur. Our results showed that extracellular fractions of the probiotic isolates had an inhibitory effect against M. furfur. The zone of inhibition was recorded in diameters (mm), and the maximum inhibition of 7 ± 0.5 mm was observed by the extracellular fractions of bacterial isolate L. rhamnosus, followed by E. faecium (5 ± 0.3 mm) and E. faecalis (3.5 ± 0.2 mm) respectively ( Fig.4). Climbazole (50 mg/mL) used as a positive control, showing an inhibitory zone of 8 ± 0.2 mm. No zone of inhibition was confirmed by the intracellular fractions of the bacterial isolates, indicating that intracellular components do not affect the growth of M. furfur. Karami et al., (2017) reported that Lactobacillus sp. from dairy products were able to inhibit the growth of S. typhimurium (2.8 mm) and S. aureus (8 mm). L. rhamnosus showed the highest inhibitory activity on S. equinus, as reported by Masalam et al., (2018). A similar study was done by Nami et al., (2019) showed that the Enterococcus sp. had a strong inhibitory effect on E. coli (33.3 mm), S. flexneri (22 mm), B. subtilis (7.9 mm) and S. aureus (7.4 mm). The inhibition property of Enterococcus sp. could be due to the production of bacteriocin (Hassanzadazar et al., 2014).

Minimum Inhibitory Concentration (MIC) assay
The isolates were subjected to an antifungal susceptibility test using the microdilution method. The MIC50 value for the strains was measured by subtracting OD540 from OD590. The MIC for all three isolates were found to be 100 mg/mL (Fig.5). The standard drug, Climbazole, used in the study, had a MIC value of 2 mg/mL. Antimycotic activity of fluconazole was found to be 2 μg/mL.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis
The GCMS analysis of the antifungal probiotic extracts of three bacterial isolates consisted of propionic acid, lactic acid, hexadecanoic acid, Octadecanoic acid, phenol, 2,4-bis(1,1-dimethyl ethyl), and 3-Isobutyl hexahydro pyrrolo [1,2-a]pyrazine-1,4-dione (Table 1). Our study showed that L. rhamnosus and E. faecium produce all five extracellular volatile compounds. 3-Isobutyl Hexahydro Pyrrolo[1,2-a]pyrazine-1,4-dione was found to be a major antifungal active metabolite in E. faecalis. Similar to our study, propionic acid possesses antimicrobial activity against pathogenic bacteria S. typhimurium via inhibition and expression of genes in Salmonella (Saad et al., 2010). A recent study reported that lactic acid produced by CFS of L. plantarum and L. buchneri could inhibit P. nordicum by the production of Ochratoxin A (Guimaraes et al., 2018). Similarly, decanoic acid production by L. plantarum was already proven to inhibit food spoilage fungi A. fumigatus and a film-forming yeast Pichia kudriavzevii GY1. The inhibitory mechanism of decanoic acid could be due to the detergent-like properties that damage organisms' cellular membranes (Ryu et al., 2014). Moreover, the volatile compounds such as acetic acid, lactic acid, propionic acid, octadecanoic acid, and eicosanoic acid produced by L. pentosus showed activity against filamentous fungi (Lipińska et al., 2018). The phenol-2,4-bis (1,1-dimethyl ethyl) reported to inhibit sporulation in Alternaria solani (Gao et al., 2017). 3-Isobutyl Hexahydro Pyrrolo [1,2-a]pyrazine-1,4-dione is a major compound found in all three bacterial isolates. Moreover, this compound was already reported against rice pathogen Pyricularia oryzae (Awla et al., 2016). L. plantarum has been studied to show inhibitory activity against many fungal species through the production of phenolic compounds in a study by Valan Arasu et al., (2013).

CONCLUSION
The three different bacterial species like L. rhamnosus, E. faecalis, and E. faecium were isolated from dairy products and identified based on 16S rDNA sequences. The extracellular fractions of the isolates were shown potential probiotic activity against dandruff, causing M. furfur. All three strains produced more amounts of acidic metabolites that could be responsible for the production of antifungal activity. The overall result suggested that the identified components can be incorporated in anti-dandruff formulations as they have less harmful effects than the conventional anti-dandruff drugs. Further studies have to be carried out in the purification and stability analysis of the metabolites.

Conflict of interest:
The authors declare that they have no conflict of interest.