Production and New Extraction Method of Polyketide Red Pigments Produced by Ascomycetous Fungi from Terrestrial and Marine Habitats
"> Figure 1
<p>Fungal biomass production (mean in g·L<sup>−1</sup> ± s.d.) in a submerged culture of the ascomycetous fungi. (<b>A</b>) Biomass production obtained in DMD (Defined Minimal Dextrose broth) submerged culture; (<b>B</b>) in PDB (Potato Dextrose Broth) submerged culture; (<b>C</b>) in YCD (Yeast Casamino Dextrose broth) submerged culture; Culture conditions: under illumination (<span style="color:red">∎</span>: red) and in the dark (<span style="color:grey">∎</span>: grey); s.d.: standard deviation; Strain identification: <span class="html-italic">4464 =</span> ❶ <span class="html-italic">Penicillium purpurogenum rubisclerotium; 30570 =</span> ❷ <span class="html-italic">Talaromyces</span> spp. <span class="html-italic">(marine isolate)*</span>; <span class="html-italic">531 =</span> ❸ <span class="html-italic">Fusarium oxysporum; 30555 =</span> ❹ <span class="html-italic">Trichoderma atroviride*; 4890= Penicillium purpurogenum; 2226 = Dreschlera cynodontis; 3684 = Penicillium erythromellis</span>; <span class="html-italic">T22 = Trichoderma harzianum</span>; <span class="html-italic">4158 = Penicillium oxalicum</span>; <span class="html-italic">5511= Aspergillus repens</span>; <span class="html-italic">PA9= Talaromyces verruculosus*</span>; <span class="html-italic">3404= Trichoderma harzianum</span>; <span class="html-italic">3391= Paecylomyces farinosus; B34 = Aspergillus sydowii*</span>; <span class="html-italic">3531 = Trichoderma polysporum</span>; (* strains collected from marine biotopes of La Reunion island’s reef flat).</p> "> Figure 1 Cont.
<p>Fungal biomass production (mean in g·L<sup>−1</sup> ± s.d.) in a submerged culture of the ascomycetous fungi. (<b>A</b>) Biomass production obtained in DMD (Defined Minimal Dextrose broth) submerged culture; (<b>B</b>) in PDB (Potato Dextrose Broth) submerged culture; (<b>C</b>) in YCD (Yeast Casamino Dextrose broth) submerged culture; Culture conditions: under illumination (<span style="color:red">∎</span>: red) and in the dark (<span style="color:grey">∎</span>: grey); s.d.: standard deviation; Strain identification: <span class="html-italic">4464 =</span> ❶ <span class="html-italic">Penicillium purpurogenum rubisclerotium; 30570 =</span> ❷ <span class="html-italic">Talaromyces</span> spp. <span class="html-italic">(marine isolate)*</span>; <span class="html-italic">531 =</span> ❸ <span class="html-italic">Fusarium oxysporum; 30555 =</span> ❹ <span class="html-italic">Trichoderma atroviride*; 4890= Penicillium purpurogenum; 2226 = Dreschlera cynodontis; 3684 = Penicillium erythromellis</span>; <span class="html-italic">T22 = Trichoderma harzianum</span>; <span class="html-italic">4158 = Penicillium oxalicum</span>; <span class="html-italic">5511= Aspergillus repens</span>; <span class="html-italic">PA9= Talaromyces verruculosus*</span>; <span class="html-italic">3404= Trichoderma harzianum</span>; <span class="html-italic">3391= Paecylomyces farinosus; B34 = Aspergillus sydowii*</span>; <span class="html-italic">3531 = Trichoderma polysporum</span>; (* strains collected from marine biotopes of La Reunion island’s reef flat).</p> "> Figure 2
<p>Volumetric production of extracellular pigments (mean in meqv. carmine pigment per liter ± s.d.) by the ascomycetous fungi in submerged culture. (<b>A</b>) Volumic production of extracellular pigments obtained in DMD (Defined Minimal Dextrose broth) submerged culture; (<b>B</b>) in PDB (Potato Dextrose Broth) submerged culture; (<b>C</b>) in YCD (Yeast Casamino Dextrose broth) submerged culture; Culture conditions: in the dark (grey) and under illumination (in color; color in figure for each strain indicates the shade of the fungal culture filtrate after 7 days of fermentation); s.d.: standard deviation; ❶ <span class="html-italic">Penicillium purpurogenum rubisclerotium</span> (4464); ❷ <span class="html-italic">Talaromyces spp.</span> (marine isolate 30570); ❸ <span class="html-italic">Fusarium oxysporum</span> (531); ❹ <span class="html-italic">Trichoderma atroviride</span> (marine isolate 30555). For other strain identification (numbers in horizontal axis), see <a href="#jof-03-00034-f001" class="html-fig">Figure 1</a>. The yield of extracellular pigments was estimated according to a calibration curve of standard carmine solution by measuring the absorbance at 276 nm (λ<sub>max</sub> of carmine) of the colored culture filtrate (after blank subtraction).</p> "> Figure 3
<p>Color coordinates projected in the CIE L*a*b* colorimetric system of the culture filtrates after seven days of cultivation of the ascomycetous fungi. (<b>A</b>) Color coordinates of extracellular pigment extracts obtained in PDB (Potato Dextrose Broth) submerged culture; (<b>B</b>) in DMD (Defined Minimal Dextrose broth) submerged culture; (<b>C</b>) in YCD (Yeast Casamino Dextrose broth) submerged culture; Culture conditions: under illumination (-l), and darkness (-d). Strain identification: ❶ <span class="html-italic">Penicillium purpurogenum rubisclerotium</span> (4464); ❷ <span class="html-italic">Talaromyces spp.</span> (marine isolate 30570); ❸ <span class="html-italic">Fusarium oxysporum</span> (531); ❹ <span class="html-italic">Trichoderma atroviride</span> (marine isolate 30555); <span class="html-italic">Penicillium purpurogenum</span> (4890).</p> "> Figure 3 Cont.
<p>Color coordinates projected in the CIE L*a*b* colorimetric system of the culture filtrates after seven days of cultivation of the ascomycetous fungi. (<b>A</b>) Color coordinates of extracellular pigment extracts obtained in PDB (Potato Dextrose Broth) submerged culture; (<b>B</b>) in DMD (Defined Minimal Dextrose broth) submerged culture; (<b>C</b>) in YCD (Yeast Casamino Dextrose broth) submerged culture; Culture conditions: under illumination (-l), and darkness (-d). Strain identification: ❶ <span class="html-italic">Penicillium purpurogenum rubisclerotium</span> (4464); ❷ <span class="html-italic">Talaromyces spp.</span> (marine isolate 30570); ❸ <span class="html-italic">Fusarium oxysporum</span> (531); ❹ <span class="html-italic">Trichoderma atroviride</span> (marine isolate 30555); <span class="html-italic">Penicillium purpurogenum</span> (4890).</p> "> Figure 4
<p>(<b>A</b>) Cultures in PDA of the marine isolate <span class="html-italic">Talaromyces</span> spp. 305_70; (<b>B</b>) liquid extracts obtained after a six-stage pressurized liquid solvent extraction of the mycelium cultivated in PDB submerged culture; (<b>C</b>–<b>E</b>) chromatograms* of the overall compounds detected by HPLC-DAD in the extracts. <span class="html-italic">Captions</span>: PDA (Potato Dextrose Agar); PDB (Potato Dextrose Broth); HPLC-DAD: high-performance liquid chromatogram combined with photo-diode array detection; Rt.: retention time; * the analysed samples were extracted with different solvents. <a href="#jof-03-00034-t002" class="html-table">Table 2</a> reports on all of the identified or assumed compounds detected in all the different samples and, in particular, the 50% aqueous methanolic extract was the most representative, as shown in <a href="#jof-03-00034-f004" class="html-fig">Figure 4</a>D. Only compound 1 (Rt. 1.71 min), not tentatively identified, was detected in the intracellular aqueous extract (<a href="#jof-03-00034-f004" class="html-fig">Figure 4</a>C). The main compound 4 (Rt. 30.97 min), identified as <span class="html-italic">N</span>-threoninerubropunctamine, was detected mainly in the 50% aqueous methanolic extract (<a href="#jof-03-00034-f004" class="html-fig">Figure 4</a>D), and compound 8 (Rt. 69.78 min), identified as ergosterol, was detected mainly in 100% methanolic extract (<a href="#jof-03-00034-f004" class="html-fig">Figure 4</a>E).</p> "> Figure 5
<p>UV-visible absorption (<b>A1</b>, <b>B1</b>, <b>C1</b>, <b>D1</b>, <b>E1</b>) and mass spectra (<b>A2</b>, <b>B2</b>, <b>C2</b>, <b>D2</b>, <b>E2</b>) of the identified or assumed compounds detected in intracellular extracts of the marine isolate <span class="html-italic">Talaromyces</span> spp. with reference to the chromatograms shown in <a href="#jof-03-00034-f004" class="html-fig">Figure 4</a>: glycylrubropunctatin <b>3</b>, <span class="html-italic">N</span>-threoninerubropunctamine <b>4</b>, <span class="html-italic">N</span>-glutarylrubropunctamine <b>6</b>, Monascorubramine <b>7</b>, and ergosterol <b>8</b>. Compounds <b>1</b>, <b>2</b> and <b>5</b> are red azaphilone-derivative pigments not tentatively identified.</p> "> Figure 6
<p>Chemical structures of the identified or assumed compounds detected in the present study in intracellular extracts of the marine isolate <span class="html-italic">Talaromyces</span> spp. Formula and calculated nominal masses are shown in parentheses.</p> "> Figure 7
<p>(<b>A</b>) Cultures in PDA of the strain <span class="html-italic">Penicillium purpurogenum rubisclerotium</span> LCP4464; (<b>B</b>) liquid extracts obtained after a six-stage pressurized liquid solvent extraction of the mycelium cultivated in PDB submerged culture; (<b>C</b>–<b>E</b>) chromatograms * of the overall compounds detected by HPLC-DAD in the different extracts. <span class="html-italic">Captions</span>: PDA (Potato Dextrose Agar); PDB (Potato Dextrose Broth); HPLC-DAD: high-performance liquid chromatography combined with photo-diode array detection; Rt.: retention time; *the analysed samples were extracted with different solvents. <a href="#jof-03-00034-t002" class="html-table">Table 2</a> reports the compounds detected in all the different samples and, in particular, the 50% aqueous methanolic extract was the most representative, as shown in <a href="#jof-03-00034-f007" class="html-fig">Figure 7</a>D. Only compound <b>9</b> (Rt. 1.57 min; not tentatively identified) was detected in the aqueous extract (<a href="#jof-03-00034-f007" class="html-fig">Figure 7</a>C), the main compound <b>10</b> (Rt. 22.05 min; not tentatively identified) was detected mainly in the 50% aqueous methanolic extract (<a href="#jof-03-00034-f007" class="html-fig">Figure 7</a>D), and ergosterol 8 (Rt. 69.78 min) was detected mainly in 100% MeOH extract (<a href="#jof-03-00034-f007" class="html-fig">Figure 7</a>E).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Fungal Strains
2.2. Fermentation and Biomass Production
2.3. Quantitative Colorimetric Analysis of Extracellular Extracts
2.4. UV-Visible Spectrophotometry and Extracellular Polyketide Metabolites Quantification
2.5. New Extraction Method of Intracellular Polyketide Pigments
2.6. Absorbance and HPLC-DAD Analyses
2.7. UHPLC-HRMS Analyses
3. Results
3.1. Biomass and Polyketide Extrolites Production Capacities under Various Growth Conditions
3.2. New Extraction Procedure and Nature of the Polyketide Red Pigments Produced by Talaromyces spp. Marine Isolate and P. purpurogenum rubisclerotium Terrestrial Isolate
4. Discussion
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Fungal Strain | Broth | Biomass | Extracellular Pigments | a-Value (CIELab) | Intracellular Pigments Extracted | |
---|---|---|---|---|---|---|
(g/L) | (meqv. Carmine/L) | (Red Color) | (meqv/g Biomass) or (meqv/L Culture) | |||
30570 | in DMD | 6.2–7.1 | 18–31 | 10.8–13.4 | 23.8–24.7 | 148–176 |
in PDB | 5.0–5.5 | 208–278 | 63.8–65.8 | 49.4–69.8 | 247–384 | |
in YCD | 1.6–2.0 | - | 5.3–5.6 | 44.3–48.5 | 71–97 | |
4464 | in DMD | 8.4–8.5 | 1–189 | 0.7 – 0.9 | 57.9–83.2 | 487–707 |
in PDB | 1.0–1.3 | 685–1118 | 49.9–51.5 | 69.2–116.0 | 90–116 | |
in YCD | 1.6–1.7 | 666–680 | 42.8–43.5 | 22.5–71.2 | 36–121 |
Compound No. R.t. (mn) | λmax (nm) | Solvent | Tentative Identification | Mol. Peak (m/z) | |
---|---|---|---|---|---|
IE of Talaromyces spp. (305_70) | |||||
1 | 1.71 | 215, 244, 276, 418, 514, 524 | water | n.i. | n.d. |
2 | 26.07 | 246, 276, 425, 512 | MeOH | n.i. | 488 [M + H]+ |
3 | 29.60 | 245, 274, 421, 518 | MeOH | Glycylrubropunctatin | 416 [M + H]+ |
4 | 30.97 | 245, 274, 424, 521 | MeOH | N-threoninerubropunctamine | 456 [M + H]+ |
5 | 32.66 | 218, 250, 287, 424, 546 | MeOH | n.i. | 498 [M + H]+ |
6 | 38.04 | 246, 273, 427, 521 | MeOH | N-glutarylrubropunctamine | 484 [M + H]+ |
7 | 43.95 | 245, 272, 424, 519 | MeOH | Monascorubramine | 381 [M + H]+ |
8 | 69.78 | 272, 282, 293 | MeOH | Ergosterol | 393 [M + H]+ |
IE of Penicillium purpurogenum rubisclerotium (4464) | |||||
8 | 69.78 | 272, 282, 293 | MeOH | Ergosterol | 393 [M + H]+ |
9 | 1.57 | 216, 292, 492 | MeOH | n.i. | n.d. |
10 | 22.05 | 262, 322, 430 | MeOH | n.i. | n.d. |
11 | 23.50 | 284, 389 | MeOH | n.i. | n.d. |
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Lebeau, J.; Venkatachalam, M.; Fouillaud, M.; Petit, T.; Vinale, F.; Dufossé, L.; Caro, Y. Production and New Extraction Method of Polyketide Red Pigments Produced by Ascomycetous Fungi from Terrestrial and Marine Habitats. J. Fungi 2017, 3, 34. https://doi.org/10.3390/jof3030034
Lebeau J, Venkatachalam M, Fouillaud M, Petit T, Vinale F, Dufossé L, Caro Y. Production and New Extraction Method of Polyketide Red Pigments Produced by Ascomycetous Fungi from Terrestrial and Marine Habitats. Journal of Fungi. 2017; 3(3):34. https://doi.org/10.3390/jof3030034
Chicago/Turabian StyleLebeau, Juliana, Mekala Venkatachalam, Mireille Fouillaud, Thomas Petit, Francesco Vinale, Laurent Dufossé, and Yanis Caro. 2017. "Production and New Extraction Method of Polyketide Red Pigments Produced by Ascomycetous Fungi from Terrestrial and Marine Habitats" Journal of Fungi 3, no. 3: 34. https://doi.org/10.3390/jof3030034