Production, Characterization, and Antimicrobial Activity of Mycocin Produced by Debaryomyces hansenii DSMZ70238

The present study was conducted to estimate the antimicrobial activity and the potential biological control of the killer toxin produced by D. hansenii DSMZ70238 against several pathogenic microorganisms. In this study, the effects of NaCl, pH, and temperature, killer toxin production, and antimicrobial activity were studied. The results showed that the optimum inhibitory effect of killer toxin was at 8% NaCl, and the diameters of clear zones were 20, 22, 22, 21, 14, and 13 mm for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pyogenes, Candida albicans, and Candida neoformans, respectively. The largest inhibition zones were observed at pH 4.5 with inhibition zone of 16, 18, 17, 18, 11, and 12 mm for the same microorganisms. The results also showed that 25°C is the optimal temperature for toxin killing activity against all targeted microorganisms. In addition, the activity of killer toxin significantly inhibited the growth of fungal mycelia for all target pathogenic fungi and the percentages of inhibition were 47.77, 48.88, 52.22, and 61.11% for Trichophyton rubrum, Alternaria alternata, Trichophyton concentricum, and Curvularia lunata, respectively. The results showed the highest growth rate of D. hansenii DSMZ70238 under condition of 8% NaCl concentration, pH 4.5, and 25°C for 72 h.


Introduction
Debaryomyces hansenii is one of the most common yeast species in nature and food such as dairy products, including soft, brine, and hard cheese [1][2][3]. D. hansenii is a halotolerant species and it can be grown in medium supplemented with up to 25% sodium chloride [4]. In addition, it can survive at pH levels ranging between 3 and 10 and low levels of water activity [5]. It has a halo-xerotolerance trait and shows a broad spectrum of carbon source assimilation and fermentation [6]. The genus Debaryomyces Lodder et Kreger-van Rij is a genus of yeasts that belongs to the family of Saccharomycetaceae. In general, most of D. hansenii strains are haploid and can be reproduced vegetatively by forming of multilateral budding [3] via sexual reproduction (heterogamous conjugation) leading to short diplophase and then by meiosis and ascospore formation. These yeasts are distributed as commensals in the healthy persons but can be pathogenic microorganisms and cause a disease called candidemia after disturbance occurs in the host defense barriers [7,8]. Many species of yeast have been reported to produce toxic proteins called (exotoxin), also named mycocins, against several pathogenic yeasts such as Candida albicans. D. hansenii has been reported to produce strong and active toxic proteins or glycoproteins, as killer toxins. These antimicrobial substances can play an important role in the inhibition growth of different genera of yeasts. The production of killer toxin was firstly described in Saccharomyces cerevisiae in 1963; after that the activity of killer toxin has been reported in 100 yeast species belonging to more than twenty genera [9]. Mode of action of these mycocins may be belonging to several mechanisms that involve DNA damage and inhibition of DNA replication, 2 International Journal of Microbiology attacking the cell membranes of target yeast cells and pores formation. This can lead to changes in the permeability of the cytoplasmic membrane and loss of potassium ions and energy in the form of ATP or arrest the cell cycle of yeast cells at the G1 phase [10][11][12].
Killer toxins produced by D. hansenii showed antimicrobial efficiency towards several yeast species, especially the opportunistic pathogenic Candida spp. [13,14]. D. hansenii was used as a biological control agent against some molds such as Penicillium digitatum and P. italicum [6,15] and phytopathogenic fungi such as Mucor circinelloides, Fusarium proliferatum, and F. subglutinans that infect Zea mays L. grains [16]. It was also used as a bioprotective agent against some dairy molds including Aspergillus sp., Byssochlamys fulva, B. nivea, Cladosporium sp., Eurotium chevalieri, P. candidum, and P. roqueforti [17]. However, studies in the field of the antibacterial activity of mycotoxin produced by D. hansenii against Gram positive and negative bacteria are very rare. One of these studies [18] evaluated the antimicrobial activity of D. hansenii toward Clostridium tyrobutyricum and C. butyricum. In this work, the main objectives are production, detection, and testing of the killer activity of mycocin produced by D. hansenii DSMZ70238 against several types of microorganisms, including pathogenic yeasts, bacteria, and fungi.

Microorganisms and Its
Maintenance. D. hansenii DSMZ-70238 strain was obtained from the German Collection of Microorganisms and Cell cultures (DSMZ) and used to produce the killer toxin. Yeast cells were grown and maintained in Yeast Extract-Peptone Dextrose (YEPD) agar slants medium containing 2.0% glucose, 2.0% peptone, 1.0% yeast extract, and 2.0% agar at 4 ∘ C. Pathogenic strains of Candida albicans, Cryptococcus neoformans, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Streptococcus pyogenes, Trichophyton rubrum, Trichophyton concentricum, Alternaria alternata, and Curvularia lunata were used. These microorganisms were obtained from laboratories of microbiology, Department of Biology, Faculty of Science For Women, University of Baghdad. They were originally isolated from clinical samples. Identification and characterization were done according to biochemical, morphological, and physiological tests. All isolates were subcultured and maintained on selective media for further use.

Production of Killer
Toxin. D. hansenii DSMZ70238 was grown for 72 h at 25 ∘ C in 250 ml Erlenmeyer flasks with 100 mL of the YEPD broth medium at pH 4.5. The cultures were aerobically incubated with gentle shaking (130 rpm). The biomass was separated from the supernatant by centrifugation at 5000 ×g for 10 min at 4 ∘ C. The free cell supernatant was adjusted to a final glycerol concentration of 15% and concentrated to a volume of 15 mL by using ultrafiltration with appropriate device size and membrane (Vivaspin56, 5-kDacutoff membrane PES, Sartorius, Germany). This partially purified concentrated supernatant was used as a concentrated killer toxin after sterilization through a 0.45 m pore-size membrane Millipore filters [19].

One-Dimensional SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
. SDS-PAGE was performed in order to identify the produced killer toxins. A 12% resolving gel was prepared by adding 30% polyacrylamide, 10% [w/v] SDS, Tris-HCl pH 8.8, 10% [w/v] ammonium persulfate (APS) N,N,N,N tetramethylethylenediamine (TEMED), and water. The mixture was poured into the clean glass and allowed to settle. After gel assemble, a 6% stacking gel was prepared from the same components but with Tris-HCl pH 6.8 instead of Tris-HCl pH 8.8. The mixture was poured above the resolving gel with 1.0 mm comb and left to solidify. The produced killer toxin was prepared by mixing with 6x SDS loading dye (

Antifungal and Antibacterial Activity of Killer Toxin.
The killer toxin activity of D. hansenii toward sensitive yeast strains was assessed by using the agar diffusion well method [20]. YEPD-methylene blue agar was prepared by adding 0.003% methylene blue to the YEPD agar medium dissolved in the 10 mM citrate/phosphate buffer and pH 4.5 for yeast strains. The medium was seeded with sensitive yeast (C. albicans, C. neoformans) at a final density of 1 × 10 5 cells ml/mL before being poured. Wells were cut in the medium using a sterile cork borer (5 mm diameter) and agar plugs removed with a sterile scalpel. Finally, 100 L of concentrated killer toxin extract was added to each well and incubated at 25 ∘ C for 48 h. The existence of a halo zone around the wells was recorded and measured. The assay was performed in triplicate [19,21].
The cells of pathogenic bacteria were grown in nutrient broth for 24 h with gentle shaking (120 rpm) at 37 ∘ C, and a 100 l of 1 × 10 5 cells was spread out on Muller Hinton Agar. Wells in the size of 5 mm were cut by using sterile cork borer, and up to 100 l of concentrated killer toxin extract was added to each well. The plates were incubated at 37 ∘ C for 24-48 h and the diameter of the clear zone around the wells was measured [22]. The assay was repeated three times from three independent biological replicates.

Antagonistic Activity of D. hansenii against Pathogenic
Fungi. The antagonistic effect of D. hansenii was evaluated on four pathogenic fungi strains, including two strains of human pathogens (T. rubrum and T. concentricum) and two plant pathogens (A. alternata and C. lunata). The activity of D. hansenii was performed by agar plate inhibition assay, as described by Núñez et al. [23] using PDA medium. Five mm International Journal of Microbiology 3 of mycelia was inoculated on one side of PDA medium plate, while, on the other side, 100 L of 1 × 10 8 cell/mL suspension of D. hansenii was spread out onto the medium. All plates were incubated for 7 days at 28 ∘ C. Three biological replicates from three independent technical replicates were conducted, the fungal colony diameter was measured, and the inhibition of radial growth was calculated as follows: where (control) is the average diameter of fungal colonies in the absence of D. hansenii and (treatment) is the average diameter of fungal colonies in cultured plates. The killer toxin activity was evaluated against sensitive yeasts and pathogenic bacterial strains using YEPD-methylene blue agar and Muller Hinton Agar prepared as described above. The clear zones were measured and used as the mean zone of inhibition activity. Yeast growth under different conditions was monitored by measurement of OD 600 of each condition in comparison to the relevant media as a control (blank). The growth was observed every two hours starting from OD 600 ≈ 0.1 to approximately OD 600 ≈ 2.5.

Statistical
Analysis. The effect of several growth factors on the killer toxin production and antimicrobial activity was analyzed statistically using One-way ANOVA to determine significant differences in mean values of treated samples as compared with controls. The significance levels were set at < 0.05 using Duncan's range test.

Production of Killer Toxin from D. hansenii. The D.
hansenii broth culture was centrifuged and the supernatant was collected for killer toxin isolation. The concentrated killer toxin was subjected and electrophoresed on 12% SDS-PAGE. The result indicates that the molecular weight of the killer toxin was estimated to be 22.000 Da according to the corresponding standard ladder (Figure 1). The present study is focused on the determination of the antimicrobial activity of killer toxin and biological control of D. hansenii DSMZ70238 against several pathogens. This yeast is able to produce killer toxin (killer protein) with low molecular protein probably with a 23-kDa portion and encoded by chromosomal genes [13]. From the above result, we found that the D. hansenii secreted killer toxin with a molecular weight was about 22 KDa. In order to determine the activity of killer toxin produced by of D. hansenii, partially purified and concentrated supernatant was tested using different types of pathogenic microorganisms. The results showed high activity of killer toxin against all tested pathogenic bacteria S. aureus, E. coli, K. pneumonia, and S. pyogenes and moderate activity against sensitive yeasts C. albicans and C. neoformans. The clear zone around wells was measured and considered as an indicator for killer activity. The effect of NaCl concentration on the activity of killer toxin was examined using the agar diffusion well bioassay. Figure 2 shows the mean diameters of the inhibition zones under different concentrations of NaCl. The optimum inhibitory effect against all pathogens is acquired with 8% NaCl; the clear zone diameters were 20, 22, 22, 21, 14, and 13 mm for S. aureus, E. coli, K. pneumonia, S. pyogenes C. albicans, and C. neoformans, respectively. The concentration of 2% NaCl showed the lowest antimicrobial effect on the tested pathogenic microbes with inhibition zone diameter from 17 to 12 mm, compared to the control. In addition, Figure 2 shows the effect of NaCl concentration on the activity of killer toxin produced by D. hansenii against all pathogens.
To investigate the effect of pH on the production of killer toxin and its antimicrobial activity, the killer toxin production medium was adjusted with different pH values 4.5, 5.5, 6.5, and 7. Figure 2 shows the effect of partially purified and concentrated killer toxin on the antimicrobial activity. At optimal pH 4.5, the largest inhibition zones were observed 16,18,17,18,11, and 12 mm for S. aureus, E. coli, K. pneumonia, S. pyogenes C. albicans, and C. neoformans, respectively, whereas under the rest of pH values the toxin varied in its ability to reduce the growth of testing pathogens. The results also showed that the neutral medium reduced  the killer activity of the concentrated killer toxin, with the lowest inhibition zone observed (5 mm) at pH 7.0 for all tested pathogens. Figures 3(b) and 3(c) show the effect of pH on the killer activity of the concentrated killer toxin against pathogenic S. aureus and C. albicans. The stability, the activity, and the production of all characterized killer toxins are strongly dependent on the temperature of incubation and the pH values [2,4,14]. Moreover, some yeast produce highly active killer toxin under high concentration of salts [21,24]. In our study, the optimal conditions for killer toxin production from D. hansenii DSMZ70238 were investigated; we found that the killer toxin produced by this yeast exhibits more killer activity against pathogenic yeasts and bacteria when the concentration of NaCl is 8%. Previous studies showed that the production, activity, and stability of the killer toxin produced by yeast are increased with the increasing of salt concentration. A study by Hau et al. [24] showed that the addition of 3% NaCl in the production medium gave the highest killer activity of the crude killer toxin produced by Mrakia frigida 2E00797 against the pathogenic yeast Metschnikowia bicuspidata WCY, while the purified killer toxin showed the highest killer activity when treated with 3.0% of NaCl [25]. The marine yeast Williopsis saturnus WC91-2 produced killer toxin that had the maximum killing activity against pathogenic yeast M. bicuspidate WCY when the medium was supplemented with 10% NaCl [21]. However, little information is known about the mechanisms that affect the production and killing activity of killer toxin in the presence of NaCl [12]. The optimum temperature of the killer toxin activity was determined by incubating the yeast under different temperatures 15, 20, 25, and 30 ∘ C. After 72 h incubation time, the results showed that the 25 ∘ C is the optimal temperature for toxin killing activity as demonstrated by largest inhibition zones 35,36,32,31,8, and 6.5 mm for S. aureus, E. coli, K. pneumonia, S. pyogenes C. albicans, and C. neoformans, respectively. Figure 4 shows that the smallest inhibition zone was observed at 15 ∘ C with value ranging from 6 to 5 mm in diameter. Figures 4(b)-4(e) show the inhibition zones within the wells filled with partially purified and concentrated toxin produced at different temperatures against and C. albicans and C. neoformans, S. aureus, and S. pyogenes.
The ability of killer toxin to inhibit the pathogenic microorganisms was examined under two different conditions. The yeast culture was grown aerobically under temperatures ranging from 20 to 25 ∘ C and at pH values ranging from 4 to 4.5. The results showed that the produced killer toxin did not exert any activity against pathogens (data not shown).
Several studies revealed that the killer toxins are well produced and stabilized under low pH values and low temperature [26,27]. Kluyveromyces siamensis HN12-1 produced high amount of killer toxin and showed a remarkable activity against the pathogenic yeast M. bicuspidata WCY in crab when incubated at 25 ∘ C and low pH 4.4-4.0 [28]. In a study performed by Hau et al., [24], the antimicrobial activity of the crude killer toxin was investigated against pathogenic yeast M. bicuspidata WCY; the results showed a high killer activity when it grew under acidic condition pH 4.5 and at 15 ∘ C, whereas Wang et al. [21] showed that the purified killer toxin produced by the marine yeast W. saturnus WC91-2 had high antimicrobial activity against pathogenic yeast M. bicuspidata WCY under low value of pH (3-3.5) and at 16 ∘ C. The killer activity of killer toxin produced by D. hansenii isolated from cheese was investigated against pathogenic C. albicans and C. tropicalis, and the optimal temperature and pH values were evaluated. The results of this study revealed that the killing activity of killer toxin is mainly affected by the growth conditions like temperature and pH [2].
Our study was performed to assess the activity of killer toxin against Gram (+) and Gram (−) pathogenic bacteria; however, there are fewer studies about the activity of killer toxin against pathogenic bacteria and killer toxin mechanism that mediated the inhibition of Gram (+) bacterial growth [29].

In Vitro Antagonistic Activity of D. hansenii against Fungal
Pathogens. Table 1 and Figure 5 show the antagonistic activity of D. hansenii against 4 fungal pathogenic strains studied in vitro, two human pathogens, T. rubrum and T. concentricum, and two plant pathogens, A. alternata and C. lunata. The results showed that D. hansenii significantly inhibited the fungal mycelial growth for all tested pathogens and the percentage of inhibition was 47.77, 48.88, 52.22, and 61.11% for T. rubrum, A. alternata, T. concentrcum, and C. lunata, respectively.
The antagonistic ability of D. hansenii against pathogenic fungi was previously mentioned by Grzegorczyk et al. [30]. In this study, the in vitro and in vivo activity of D. hansenii KI2a, D. hansenii MI1a, and Wickerhamomyces anomalus BS91 was examined against Monilinia fructigena and Monilinia  hansenii was monitored in culture media supplemented with different NaCl concentrations (2, 4, and 8%). In Figure 6(a), the results showed that the growth rate was enhanced by the addition of different NaCl concentrations. The effect of temperature on the growth curve of this yeast was also studied. The D. hansenii culture was incubated under different 6 International Journal of Microbiology  The maximum level of growth rate was observed at 25 ∘ C ( Figure 6(b)). The results also showed that this yeast is unable to grow at 37 ∘ C. The ability of D. hansenii to grow in the YEPD medium at different pH values 4.5, 5.5, 6.5, and 7 was determined ( Figure 6(c)), and the results indicated that the highest growth was observed at pH 4.5 and that the growth decreased with increasing pH values.
Basically, killer toxin acts mainly on the surface of the yeast cells by special mechanism that includes the interaction with the cell wall components and causes the inhibition of the -glucan synthesis. Alternatively, it may hydrolyse the -glucan in the cell wall of target cells or block the DNA synthesis and cleavage of tRNA. Finally, ion leakage occurs due to the formation of channels in the cytoplasmic membrane and blocking of calcium uptake [10,19,31]. Several studies were pointed out on the use of yeasts as biocontrol agents against pathogens like fungal pathogens and using yeast as an alternative solution to synthetic of fungicides [32][33][34]. In this study, we used this yeast to determine the antifungal activity against four species of fungal pathogens T. rubrum, T. concentricum, A. alternata, and C. lunata. Our results showed that this yeast shows high efficiency in the reduction of radial growth of tested pathogens and the values of antifungal activity ranged between 48.44 and 62.66%. Researchers working in the field of biological control recognized that different yeast species can play important roles in the reduction of disease and prevention of the infection. They can also play a lethal role for pathogens and decrease the colonization of host tissue [32,35,36]. Previous study investigated that the zygocin killer yeast Zygosaccharomyces bailii has killing activity against human, phytopathogenic yeasts, and filamentous fungus Fusarium oxysporum using a standard agar diffusion method [37]. The results showed a significant inhibition of mycelial growth. Moreover, Pichia membranifaciens and Sporobolomyces roseus were investigated to be good controller for plant pathogenic Penicillium expansum and they could be promising alternative biocontrol agents of the growth of this pathogen [38]. The antagonistic activity of yeasts has been attributed to several mechanisms that include (1) competition for nutrients, especially sugars and iron, (2) changes of pH medium through growth-coupled ion exchange or by production of organic acid, (3) tolerance to high concentrations of ethanol [39], and (4) production of antimicrobial compounds, such as killer toxin (mycocin) [40,41].

Conclusion
Our results revealed that the D. hansenii DSMZ70238 secreted killer toxin, and the antimicrobial activity of killer toxin and its optimizing conditions were studied. The results proved that the optimal killer activity was obtained at high concentration of NaCl, low pH, and mesophilic temperature. Moreover, results showed that this yeast has a good role in the reduction of radial growth of tested pathogenic bacteria and biological control against our studied fungi. Also the growth curve of D. hansenii DSMZ70238 was affected by NaCl, pH concentration, and temperature, and the optimal growth conditions were estimated.

Conflicts of Interest
The authors declare that they have no conflicts of interest.