The wild-type A. fumigatus strain Af293 and Af293 with an agd3 knockout (Af293 ∆agd3) produced by Lee et al.5 (link) were used in this study. Unless otherwise noted, strains were grown and harvested on YPD agar (Fisher Scientific) at 37 °C as previously described65 (link). For growth in liquid medium, Brian medium4 (link), AspMM66 (link), and RPMI media 1640 (Wissent) were used as indicated. All strains and primers used in this study are summarized in Supplementary Table 2 .
Ypd agar
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YPD agar is a nutrient-rich growth medium used for culturing a variety of microorganisms, including yeasts and other fungi. It provides the necessary nutrients and support for the growth and propagation of these organisms in a laboratory setting.
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Lab products found in correlation
Potato dextrose agar (pda), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Wisent (1 mentions)
Chloramphenicol, by Roche (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Pro200, by PRO Scientific (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Mrs agar, by Thermo Fisher Scientific (1 mentions)
8 protocols using ypd agar
1
Characterization of A. fumigatus Strain Mutants
2
Aspergillus Strain Handling and Maintenance
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A. nidulans strain A26 (Fungal Genetics Stock Center, Kansas City, MO, USA) was used as the parent wild-type strain for all molecular manipulations. Other strains used in this study include A. fumigatus strain Af293 (a generous gift from P. Magee, University of Minnesota, St. Paul, MN, USA), the A. fumigatus Δuge3 mutant [10 (link)], ΔstuA mutant [41 (link)] and clinical isolates of A. flavus and A. niger (obtained from the McGill University Health Center, Montreal, QC, Canada) and A. nidulans isolates from CGD patients (generous gifts from A. Warris and S. Henriett, Radboud University Medical Center, Nijmegen, The Netherlands, and Adrian Zelazny US National Institute of Health, USA). Unless otherwise noted, A. fumigatus strains and A. flavus were maintained on YPD agar (Fisher Scientific), A. niger on potato-dextrose agar (Fisher Scientific), and A. nidulans strains on Aspergillus minimum medium agar [10 (link)] at 37°C. For growth in liquid medium, Brian medium [9 (link)] and phenol red-free RPMI 1640 (Wisent, Inc.) were used as indicated. All growth media for A. nidulans strains were supplemented with biotin (Fisher Scientific).
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A. nidulans strain A26 (Fungal Genetics Stock Center, Kansas City, MO, USA) was used as the parent wild-type strain for all molecular manipulations. Other strains used in this study include A. fumigatus strain Af293 (a generous gift from P. Magee, University of Minnesota, St. Paul, MN, USA), the A. fumigatus Δuge3 mutant [10 (link)], ΔstuA mutant [41 (link)] and clinical isolates of A. flavus and A. niger (obtained from the McGill University Health Center, Montreal, QC, Canada) and A. nidulans isolates from CGD patients (generous gifts from A. Warris and S. Henriett, Radboud University Medical Center, Nijmegen, The Netherlands, and Adrian Zelazny US National Institute of Health, USA). Unless otherwise noted, A. fumigatus strains and A. flavus were maintained on YPD agar (Fisher Scientific), A. niger on potato-dextrose agar (Fisher Scientific), and A. nidulans strains on Aspergillus minimum medium agar [10 (link)] at 37°C. For growth in liquid medium, Brian medium [9 (link)] and phenol red-free RPMI 1640 (Wisent, Inc.) were used as indicated. All growth media for A. nidulans strains were supplemented with biotin (Fisher Scientific).
3
Quantifying Fungal Burden in Mice
4
Microbial Enumeration During Cocoa Fermentation
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Ten grams of fermented cocoa bean sample, for each time or period of laboratory-scale fermentation, were subjected to 1:10 serial dilution, and the enumeration of cells was performed via plate count on YPD agar (Oxoid, Waltham, MA, USA) containing 100 mg/L of chloramphenicol, incubated at 25 °C for 3–4 days. LAB was enumerated on de Man Rogosa Sharpe (MRS) Agar (Oxoid, Waltham, MA, USA) containing 100 mg/L of cycloheximide at 30 °C for 3–4 days. Plates were incubated under modified atmosphere (76% N2, 4% O2, and 20% CO2). For the AAB enumeration, GYC (5% glucose, 1% yeast extract, 0.5% CaCO3, and 2.0% agar) supplemented with 7% ethanol and 100 mg/L of cycloheximide was used, incubating plates at 30 °C for 4–5 days. Plates were incubated in aerobiosis. Plate count was carried out in duplicate and results were expressed as mean and standard deviations of Log CFU/g. Ten colonies from the highest dilution plates for the presumptive LAB and AAB were isolated, purified and subjected to preliminary tests as morphology, Gram, catalase, oxidase, mobility, and acidification.
5
Monitoring Efficacy of Cleaning Treatments
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One very important aspect of any cleaning and biocleaning treatment is the monitoring of the effectiveness of the process. After the final drying step, in order to check the possible presence of residual yeast cells and/or microbial contaminants (bacteria and fungi), sterile swabs (Fissan, Milan, Italy) were used to ensure that no treatment residues were left on the artwork surfaces; the data were compared with analyses carried out before the treatments. Microbiological counts were performed using Potato Dextrose Agar for Micromycetes, such as yeast and fungi (YPD Agar, Oxoid), and Standard Plate Count Agar for aerobic heterotrophic bacteria (SPCA, Oxoid).
6
Isolation and Characterization of Probiotic Yeast Strains
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Three strains used in this study belong to the species Lthermotolerans (B13), K.unispora (M3-B3) and S. cerevisiae (2PV) coming from different un-anthropized environments and spontaneously processed foods. These strains were isolated, identified and characterized as probiotic and/or functional strains and tested in wort fermentation trials [23 (link),24 (link)]. All of the yeast strains were maintained at 4 °C for short-term storage in YPD agar (yeast extract 10 g/L, peptone 20 g/L, dextrose 20 g/L, (agar 18 g/L) (Oxoid, Basingstoke, UK) and in YPD broth supplemented with 80% (w/v) glycerol at −80 °C, for long-term storage.
7
Diverse Yeast Strains for Probiotic Research
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Forty-three strains used in this study belong to the genera Lachancea, Torulaspora, Metschnikowia, Pichia, Saccharomyces, Rhodotorula, Candida, Kazachstania, and Hanseniaspora were obtained from the Yeast Collection of (DiSVA) of the Polytechnic University of Marche (Italy) (Table 1). The strains coming from different un-anthropized environments or spontaneously fermented foods had been isolated, identified and characterized as probiotic and/or functional strains (Agarbati et al., 2020) (link). All the strains were identified through the sequencing of the D1/D2 domains of the 26S rDNA gene, as reported by Comitini et al. (2011) (link) and Solieri, Landi, De Vero, and Giudici (2006) (link). The S. cerevisiae commercial strain US-05 (Fermentis, Lesaffre, France) and commercial probiotic S. cerevisiae var. boulardii (Codex, Zambon, Italy) were used as control strains. All the yeast strains were maintained on yeast extract (10 g/L), peptone (20 g/L), dextrose (20 g/L) (YPD) agar (18 g/L) (Oxoid, Basingstoke, UK) at 4 • C, for short-term storage, and in YPD broth (without agar) supplemented with 80% (w/v) glycerol at -80 • C, for long-term storage.
8
Diverse Yeast Strains for Probiotic Research
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Forty-three strains used in this study belong to the genera Lachancea, Torulaspora, Metschnikowia, Pichia, Saccharomyces, Rhodotorula, Candida, Kazachstania, and Hanseniaspora were obtained from the Yeast Collection of (DiSVA) of the Polytechnic University of Marche (Italy) (Table 1). The strains coming from different un-anthropized environments or spontaneously fermented foods had been isolated, identified and characterized as probiotic and/or functional strains (Agarbati et al., 2020) (link). All the strains were identified through the sequencing of the D1/D2 domains of the 26S rDNA gene, as reported by Comitini et al. (2011) (link) and Solieri, Landi, De Vero, and Giudici (2006) (link). The S. cerevisiae commercial strain US-05 (Fermentis, Lesaffre, France) and commercial probiotic S. cerevisiae var. boulardii (Codex, Zambon, Italy) were used as control strains. All the yeast strains were maintained on yeast extract (10 g/L), peptone (20 g/L), dextrose (20 g/L) (YPD) agar (18 g/L) (Oxoid, Basingstoke, UK) at 4 • C, for short-term storage, and in YPD broth (without agar) supplemented with 80% (w/v) glycerol at -80 • C, for long-term storage.
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