Gram(+) Staphylococcus aureus (ATCC 25923) and Gram(–) Escherichia coli (ATCC 25922) were grown in 20 mL medium containing 30 g L−1 Todd-Hewitt-Bouillon (Roth) and 3 g L−1 yeast extract (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) at 37 °C overnight under constant agitation at 200 rpm on a shaking incubator (MaxQ 4450, Thermo Scientific, Marietta, OH, USA). Pathogenic yeast Candida albicans (Mya 273) was grown in 20 mL medium containing 32 g L−1 Peptone Casein (Sigma-Aldrich), 20 g L−1 yeast extract (Sigma-Aldrich), 5 g L−1 sodium chloride (VWR International, Vienna, Austria) and 5 mM sodium hydroxide (Fluka Analytical, Munich, Germany) at 37 °C overnight under constant agitation at 200 rpm on a shaking incubator (MaxQ 4450, Thermo Scientific, Marietta, OH, USA).
L 1 yeast extract
Manufactured by Merck Group
Sourced in Germany, United States
L-1 yeast extract is a laboratory reagent used as a growth supplement for microorganisms. It provides essential nutrients, including vitamins, amino acids, and other growth factors, to support the cultivation of various microbial cultures, such as yeasts and bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Maxq 4450, by Thermo Fisher Scientific (1 mentions)
Casein peptone, by Merck Group (1 mentions)
Nucleospin microbial dna kit, by Macherey-Nagel (1 mentions)
Anaerocult a, by Merck Group (1 mentions)
L 1 glucose, by Merck Group (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Avantor (1 mentions)
Mrs broth, by Carl Roth (1 mentions)
L 1 agar, by Merck Group (1 mentions)
L 1 tryptone, by Merck Group (1 mentions)
Brain heart infusion, by Thermo Fisher Scientific (1 mentions)
L cysteine, by Merck Group (1 mentions)
Nutrient broth medium, by Merck Group (1 mentions)
L 1 sucrose, by Merck Group (1 mentions)
Cellulose nitrate filter, by Sartorius (1 mentions)
L 1 nacl, by Merck Group (1 mentions)
Bixafen, by Merck Group (1 mentions)
Epoxiconazole, by Merck Group (1 mentions)
7 protocols using l 1 yeast extract
1
Cultivation of Pathogenic Microbes
2
Microbial Strain Cultivation and DNA Extraction
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The bacterial and yeast strains used in this study are shown in Table 1 . LAB strains were cultivated in De Man-Rogosa-Sharpe (MRS) broth (Carl Roth, Germany). The medium was supplemented with 10% filter-sterilized white table wine for cultivation of two strains of Lb. kefiranofaciens 5016 and 10550. Acetobacter (A.) orientalis and A. fabarum were cultivated in AAB broth [5 g L–1 yeast extract (Merck, Germany), 5 g L–1 bacto peptone (Merck, Germany), 5 g L–1 glucose (Merck, Germany), 1 g L–1 MgSO4 × 7 H2O (Sigma, Germany)] and yeasts were cultivated in YPG broth [10 g L–1 yeast extract, 20 g L–1 peptone (Merck, Germany), 20 g L–1 glucose]. All strains were incubated at 30°C under static aerobic conditions, except Lb. kefiranofaciens ssp., which were cultivated under severe oxygen-limited conditions (Anaerocult A®; Merck, Germany). The genomic DNA (gDNA) of bacteria and yeast species was extracted from exponentially growing cultures [24 h for all strains except Lb. kefiranofaciens ssp. and Kazachtania (Kz.) turicensis, which were cultivated between 4 and 5 days] by using a NucleoSpin® Microbial DNA kit (MACHEREY-NAGEL, Germany). Quality and concentration of extracted gDNA was measured with a NanoDrop ND-1000 spectrophotometer (Peqlab Biotechnologie, Germany).
3
Antimicrobial Activity of Microalgae Extract
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Test microorganisms including bacteria Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis, fungus Aspergillus niger and yeast Candida utilis were cultivated in standard LB growth medium (10 g L−1 tryptone (Sigma Aldrich; St. Louis, MO, USA), 5 g L−1 yeast extract (Sigma Aldrich; St. Louis, MO, USA), 5 g L−1 sodium chloride (Nin Saltwork; Nin, Croatia), 20 g L−1 agar (Sigma Aldrich; St. Louis, MO, USA)). One hundred microliters of overnight culture were spread on solid LB agar in Petri dishes. Fifty microliters of microalgae extract were applied on a sterile disk and placed on the surface of a solid LB medium with a test microorganism. Neomycin and nystatin at 50 mg mL−1 were used as a positive control, while 100% methanol was used as a negative control. Plates with bacterial and fungal strains were incubated at 30 °C for 24 and 48 h, respectively. Afterwards, inhibition zones around the disk were measured. For all test microorganisms, assays were done in duplicate.
4
Anaerobic Bacterial Growth Protocol
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All bacterial strains used in this study are listed in Table 1
5
Synthesis and Characterization of Biomass Compounds
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Sodium citrate, urea, and thiourea were purchased from Chemical Reagent Co. Ltd. (Tianjin, China). Xylose and glucose were purchased from Aladdin Ltd. (Shanghai, China). Nutrient broth (NB) medium (1 g L−1 yeast extract, 3 g L−1 beef extract, 5 g L−1 poly peptone, 10 g L−1 sucrose) was purchased from Sigma-Aldrich. All other chemicals were of analytical grade and used as received. Double distilled water was used in all experiments.
6
Air Sampling of Microbial Diversity
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Air samples were collected by impaction with a single-stage air sampler Microflow α (AQUARIA srl, Italy), holding 380 jets with a diameter of 1 mm. The sampler was placed in a central position of the cave at 1 m height. The impaction flow rate was set at 120 L min−1, and the airstream was directed on 90-mm-diameter agar Petri dishes containing different growth media, mainly Lysogeny Broth Agar (LBA) (10 g L−1 NaCl (Sigma-Aldrich, USA), 5 g L−1 yeast extract (Merck, Germany), 15 g L−1 agar (Difco, Michigan) and Plate Count Agar (PCA) (Difco, Michigan), or on cellulose nitrate filters with a pore size of 0.45 µm (Sartorius, Germany). Different sampling volumes, between 250 and 1000 L, were collected in duplicate. Filters were stored at −20 °C in sterile tubes until genomic DNA extraction.
7
Lipopeptide Antifungal Activity Assay
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Direct activity of the lipopeptides was assessed using the strain T01193 in clear and sterile flat-bottomed polystyrene 96-well plates (Iwaki, Asahi techno glass, Japan), according to Siah et al. (2010c) (link). Lipopeptides and mixtures were added to the medium at 50 °C following autoclaving. Plate wells were each filled with 150 μL of liquid glucose peptone medium (14.3 g L -1 dextrose (VWR), 7.1 g L -1 bacto peptone (Difco laboratories), and 1.4 g L -1 yeast extract (Merck)) supplemented with lipopeptides at 100, 33.3, 11.1, 3.7, 1.2, 0.4, 0.14, 0.05, 0.015, and 0.005 mg L -1 (final concentrations in 200 μL of medium). Aliquots of 50 μL containing 2.10 5 spores mL -1 of the Z. tritici strain T01193 were added to each plate well, according to Siah et al. (2010c) (link). Eight wells were used as replicates for each condition. In each microplate, noninoculated medium without lipopeptide, as well as inoculated medium without lipopeptide, were used as experimental controls. Two synthetic fungicides, bixafen (pyrazolecarboxamide) and epoxiconazole (triazole) (Sigma-Aldrich, France), were included in the assay as fungicide references. Plates were incubated for 6 days at 20 °C in the dark while being shaken at 140 rpm, after which fungal growth was measured using a plate reader (MRX, Dynex technologies) at 405 nm.
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