A New Approach to Producing High Yields of Pulcherrimin from Metschnikowia Yeasts

: Pulcherrimin, a red iron chelate, is produced by some yeasts and bacteria. It plays important ecological roles in many ecosystems, including growth control, bioﬁlm inhibition and photoprotection. In this study, ﬁfteen yeast strains of the genus Metschnikowia were characterized based on their production of pulcherrimin. Yeast pulcherrimin was isolated and its purity assessed using 1 H nuclear magnetic resonance spectroscopy. Under experimental conditions, pulcherrimin formation varied depending on both the tested strains and culture media. The best producers formed up to 240 mg / L of pulcherrimin in minimal medium with glucose as the carbon source, supplemented with 0.05% FeCl 3 and 0.1% Tween 80. This study presents a new approach to producing high yields of pulcherrimin from yeasts.


Introduction
Pulcherrimin (1,4-dihydroxy-3,6-bis(2-methylpropyl)pyrazine-1,4-diium-2,5-diol;iron) is a ferric chelate that may be synthesized by some yeast species of the genera Metschnikowia, Lipomyces, Kluyveromyces, and Dipodascopsis as well as by some bacteria of the genus Bacillus and Streptomyces [1][2][3][4][5][6]. Pulcherrimin depletes the free iron in the environment; therefore, pulcherrimin producers including Metschnikowia yeasts show strong antimicrobial activity. The antagonistic abilities of Metschnikowia strains have been found to be clearly correlated with the production of pulcherrimin [7]. According to Gore-Lloyd and co-workers, pigmentless M. pulcherrima mutants are less antagonistic than wild strains that produce more pulcherrimin [8]. The same authors identified the METSCH-Snf2 coding gene as responsible for not only the red pigmentation of the yeast, but also its strong antifungal activity. Pulcherrimin-producing Metschnikowia strains have the characteristics of an "ideal antagonist", such as the ability to survive under adverse environmental conditions and compete for nutrients and space. In wine fermentation, Metschnikowia strains modulate the population dynamics of the fermenting microbiota and improve the aromatic character of the final products [9][10][11]. These yeasts show antimicrobial action on undesired wild spoilage yeasts, such as Brettanomyces/Dekkera, Hanseniaspora and Pichia genera. Interestingly, the antimicrobial activity of Metschnikowia spp. does not have any influence on the growth of Saccharomyces cerevisiae [7, 12,13]. Therefore, selected strains may be used in controlled multi-starter fermentations with S. cerevisiae [10,12,14].
Metschnikowia yeasts are also known to inhibit the growth of molds, including phytopathogens [15][16][17][18][19]. Some yeast strains of the genus Metschnikowia have been recognized as plant protection agents against Fermentation 2020, 6, 114 2 of 7 fungal diseases by the European Food Safety Authority (EFSA) [20]. Preharvest application of these yeasts is becoming increasingly popular, since they colonize fruit surfaces and blemishes prior to the establishment of postharvest pathogens [21][22][23].
Pulcherrimin also seems to be an effective agent with other important ecological roles. Pulcherrimin formation can control the growth of bacterial biofilms [24]. Jayalakshmi and co-workers report that pulcherrimin has antioxidant properties [25]. According to Pourzand and co-workers, pulcherrimin shows significant photoprotection against UVA-induced damage and cell death [26]. Moreover, due to its inhibition of urease activity, pulcherrimin can be used in agriculture as a component of fertilizers with controlled availability [27]. Microorganisms that produce large quantities of pulcherrimin are therefore of great interest to biotechnologists.
The aim of our study was to evaluate pulcherrimin production by yeasts of the genus Metschnikowia. This work is the first paper to describe the ability of Metschnikowia yeasts to produce pulcherrimin in different culture media. The results could provide the basis for further work to optimize the conditions of pulcherrimin formation by yeast strains.

Yeast Strains
Fifteen strains of the Metschnikowia clade were used in this study. Five strains of M. pulcherrima were obtained from the National Collection of Yeast Cultures (Quadram Institute Bioscience, Norwich, United Kingdom) and the Culture Collection of Yeasts (Slovak Academy of Sciences, Bratislava, Slovakia). In addition, ten yeast strains isolated from Polish fruits and deposited in the LOCK Culture Collection (Lodz University of Technology, Lodz, Poland) were tested [18] (Table 1). The yeast cultures were stored on YPD agar slants (Merck Millipore, Darmstadt, Germany) at 4 • C.

Pulcherrimin Extraction and Purification
Pulcherrimin was obtained from the yeast cultures using the method described previously by Kluyver et al., with subsequent modification by Li et al. [1,28]. A 50 mL sample of each yeast culture was centrifuged at 4 • C and speed 5000× g for 10 min using a Centrifuge 5804R (Eppendorf, Hamburg, Germany). The pellet containing the yeast cells and pigment was treated with methanol (50 mL 99.8% methanol per 10 g of wet yeast biomass) at 4 • C. After overnight treatment, the yeast cells were centrifuged (4 • C, speed 5000× g, 10 min) and then washed twice with distilled water (25 mL). The yeast biomass was re-suspended in 2M NaOH and centrifuged (4 • C, speed 5000× g, 10 min). The pH of the supernatant was adjusted to 1.0 using 4M HCl, and the mixture was incubated at 100 • C for 30 min. The pigment precipitate was collected by centrifugation (4 • C, speed 8000× g, 20 min), and washed three times with 25 mL of distilled water. To obtain pure pulcherrimin, the steps of dissolution in NaOH and HCl precipitation were repeated three times. Finally, the pigment was collected by centrifugation and dried at 60 • C for 18 h (moisture analyzer MA 50.R, Radwag, Poland).
To determine the purity of the obtained red pigment, the 1 H NMR technique was used. The isolated product was dissolved (5% w/v) in alkaline deuterated water (

Quantitative Determination of Pulcherrimin
The purified pulcherrimin was dissolved in 2 M NaOH, and the absorption spectrum of the solution was measured using a Multiskan GO UV-light spectrophotometer (Thermo Fisher Scientific, Warsaw, Poland). A standard curve was made using different concentrations of purified pulcherrimin dissolved in 2M NaOH. The optical density of the solution was determined at 410 nm [1]. The amount of pulcherrimin in the yeast cultures was determined spectrophotometrically using the method described above.

Statistics
The production of pulcherrimin was presented as the mean ± SD of three separate experiments. The mean values results were compared using one-way repeated measures analysis of variance (ANOVA; OriginPro 8.1, OriginLab Corp., Northampton, MA, USA). The results obtained in minimal medium were compared with the YPD cultures. Values with different letters are statistically different; a: p ≥ 0.05; b: 0.005 < p < 0.05; c: p < 0.005.

Pulcherrimin Analysis
Purified and solubilized red pigment produced by the tested yeast strains exhibited maximum absorption spectra at three different wavelengths: 240, 280 and 410 nm (Figure 1a). These data are Fermentation 2020, 6, 114 4 of 7 consistent with those for pulcherrimin produced by yeasts and bacteria [1,30]. A wavelength of 410 nm was used for spectrophotometric determination of the pulcherrimin.

Pulcherrimin Formation
The tested Metschnikowia strains were characterized by various levels of pulcherrimin formation. The production of the red pigment was dependent on the yeast strain and culture medium. For almost all the tested strains, pulcherrimin formation was significantly (p < 0.05) higher in the minimal medium than in the rich YPD broth (Figure 2). The best producers-NCYC 2321 and NCYC 747, as well as LOCK D9 and LOCK D10 strains-showed pulcherrimin production in the range of 192-198 mg/L. However, supplementation of the minimal medium with 0.1% (v/v) Tween-80 improved pulcherrimin formation to 230-240 mg/L, especially in the case of the best producers, NCYC 2321 and LOCK D10. Tween-80 is known to significantly increase the permeability of cell walls. Therefore, it is often used as a stimulatory agent in various fermentation processes [33,34]. To the best of our knowledge, there is only one previous report concerning the yield of pulcherrimin from yeast cultures. Pourzand and co-workers state in their poster that around 150 mg/L of pulcherrimin was produced from a 10 L culture of a strain of M. pulcherrima [26]. In the case of bacteria, B. licheniformis strain produced about 53 mg/L of pulcherrimin [28]. However, production of the pigment was dependent on the culture medium and culturing conditions. Under optimized Proton nuclear magnetic resonance is often used to characterize structural properties of various molecules [29,31]. The 1 H NMR spectrum of the isolated red pigment dissolved in alkaline D 2 O is presented in Figure 1b. The 1 H NMR profile contains all the signals typical for this compound (CH 3 , CH 2 and CH, belonging to isobutyl groups) [32]. The small satellite low-field peaks observed for CH 3 and CH protons may be tentatively ascribed to the presence of conformational isomers. The large solvent peak is typical for analyses carried out on solutions in D 2 O. It contains a residual amount of ordinary hydrogen isotope (protium), which results in characteristic resonance at about 4.8 ppm. It also indicates traces of H 2 O/DOH, which are always present in D 2 O. The signals are widened due to the presence of hydrogen bonds.

Pulcherrimin Formation
The tested Metschnikowia strains were characterized by various levels of pulcherrimin formation. The production of the red pigment was dependent on the yeast strain and culture medium. For almost all the tested strains, pulcherrimin formation was significantly (p < 0.05) higher in the minimal medium than in the rich YPD broth (Figure 2). The best producers-NCYC 2321 and NCYC 747, as well as LOCK D9 and LOCK D10 strains-showed pulcherrimin production in the range of 192-198 mg/L. However, supplementation of the minimal medium with 0.1% (v/v) Tween-80 improved pulcherrimin formation to 230-240 mg/L, especially in the case of the best producers, NCYC 2321 and LOCK D10. Tween-80 is known to significantly increase the permeability of cell walls. Therefore, it is often used as a stimulatory agent in various fermentation processes [33,34].
To the best of our knowledge, there is only one previous report concerning the yield of pulcherrimin from yeast cultures. Pourzand and co-workers state in their poster that around 150 mg/L of pulcherrimin was produced from a 10 L culture of a strain of M. pulcherrima [26]. In the case of bacteria, B. licheniformis strain produced about 53 mg/L of pulcherrimin [28]. However, production of the pigment was dependent on the culture medium and culturing conditions. Under optimized environmental conditions with the supplementation of glucose, (NH 4 ) 2 SO 4 and Tween-80, pulcherrimin production was improved substantially, reaching 331 mg/L. Melvydas and co-workers report that by changing the composition of the agar media, as well as the growth time, temperature, and the mode of yeast inoculation, various patterns of pulcherrimin formation can be obtained [2]. In addition, certain amino acids may play crucial roles in the synthesis of pulcherrimin [35]. The concentration of iron ions in the fermentation medium can also affect the formation of pulcherrimin. Lachance suggests that the highest production of pigment occurs when the iron concentration is slightly inhibitory [36]. These data stimulate further research on the optimization of conditions for pulcherrimin formation.
The tested Metschnikowia strains were characterized by various levels of pulcherrimin formation. The production of the red pigment was dependent on the yeast strain and culture medium. For almost all the tested strains, pulcherrimin formation was significantly (p < 0.05) higher in the minimal medium than in the rich YPD broth (Figure 2). The best producers-NCYC 2321 and NCYC 747, as well as LOCK D9 and LOCK D10 strains-showed pulcherrimin production in the range of 192-198 mg/L. However, supplementation of the minimal medium with 0.1% (v/v) Tween-80 improved pulcherrimin formation to 230-240 mg/L, especially in the case of the best producers, NCYC 2321 and LOCK D10. Tween-80 is known to significantly increase the permeability of cell walls. Therefore, it is often used as a stimulatory agent in various fermentation processes [33,34]. To the best of our knowledge, there is only one previous report concerning the yield of pulcherrimin from yeast cultures. Pourzand and co-workers state in their poster that around 150 mg/L of pulcherrimin was produced from a 10 L culture of a strain of M. pulcherrima [26]. In the case of bacteria, B. licheniformis strain produced about 53 mg/L of pulcherrimin [28]. However, production of the pigment was dependent on the culture medium and culturing conditions. Under optimized

Conclusions
Yeast strains capable of producing high yields of pulcherrimin are of great interest to biotechnology. In this study, we identified a red pigment produced by the strains of the genera Metschnikowia as pulcherrimin, using spectrometric and spectrophotometric analysis. Pulcherrimin production was found to be dependent on the strain and culture medium. Minimal medium with glucose, ferric ions and Tween-80 improved the yield of pulcherrimin in comparison to the rich medium YPD. Under these conditions, the maximum yield of pulcherrimin was over 230 mg/L. However, further research is needed to the optimize the production of pulcherrimin pigment by yeast strains.
Author Contributions: Conceptualization, E.P. and D.K.; methodology, E.P., B.K. and M.N.; investigation, E.P., B.K. and M.N.; writing-original draft preparation, E.P. and D.K.; writing-review and editing, D.K. All authors have read and agreed to the published version of the manuscript.