Purification and Characterization of Antibacterial Activity against Phytopathogenic Bacteria in Culture Fluids from Ganoderma lucidum
Abstract
:1. Introduction
2. Results
2.1. Determination of Bioactive Culture Fluid Stability
2.2. Solubility Determination of Bioactive Compounds
2.3. Proteinase K Does Not Affect Antibacterial Activity
2.4. Size Determination of the Bioactive Compounds
2.5. HPLC Analysis
HPLC-APCI-MS Analysis
2.6. GC-MS Analysis
3. Discussion
4. Materials and Methods
4.1. Microbial Strains and Culture Conditions
4.2. Production of Bioactive Culture Fluids
4.3. Antibacterial Activity
- (1)
- The agar dilution method (poisoned agar) [25,26] was used to evaluate the antibacterial activity in two assays: culture fluid stability and Proteinase K sensitivity. The method consisted of incorporating in a 1:1 ratio the treatment (treated culture fluids) into a molten twofold concentrated NA medium and poured into 90 × 15 mm petri plates. The inoculation (15 μL) of the bacterial test at a concentration of 107 colony forming units (CFU)/mL was spotted onto the agar plate surface. Plates were incubated at 28 °C for 24 h. A mix of potato dextrose broth and twofold concentrated NA medium in a 1:1 ratio was poured into petri dishes to confirm the growth of the bacteria (control treatment). Plates were incubated at 28 °C for 24 h. Results were recorded as (+) = growth inhibition, (±) = partial growth inhibition, and (−) = no growth inhibition. Each experiment was repeated three times.
- (2)
- The disk diffusion method [25,27] was used to evaluate antibacterial activity using the following assays: solubility of the bioactive compounds (testing different solvents), dialisis tubing, and bioactive fractions in the gel exclusion chromatography and HPLC. The method consisted of loading onto the paper disks (9 mm diameter) separately four times with 100 μL of the culture fluid samples and drying them between each application under aseptic conditions. Loaded disks were then evenly distributed on the bacterial lawn of NA plates. Bacterial lawns were made by spreading 100 μL of bacterial suspensions at 107 colony forming units (CFU)/mL. Plates were sealed with parafilm and incubated at 28 °C. After 24 h, inhibition zone diameters were recorded. Each experiment was repeated three times.
4.4. Determination of Bioactive Culture Fluid Stability
4.5. Solubility of Antibacterial Compounds
4.6. Proteinase-K Sensitivity
4.7. Size Determination
4.7.1. Dialysis Tubing
4.7.2. Gel Exclusion Chromatography
4.8. Reverse Phase HPLC Separation of Methanol Soluble Bioactive Fraction
HPLC-APCI-MS Analysis of the Bioactive Fraction
4.9. GC-MS Analysis of the Antibacterial Fraction
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Bacterial Species | Fresh Culture Fluids | 90-Day-Old Culture Fluids | Boiled Culture Fluids | PDB/NA Control ** |
---|---|---|---|---|
Acidovorax avenae | + * | + | + | − |
Agrobacterium rhizogenes | + | + | + | − |
Agrobacterium tumefaciens | + | + | + | − |
Brenneria quercina | ± | ± | − | − |
Burkholderia cepacia | − | − | − | − |
Erwinia carotovora subsp. carotovora | + | + | + | − |
Pseudomonas fluorescens | − | − | − | − |
Pseudomonas syringae pv. syringae | + | + | + | − |
Rathayibacter tritici | ± | ± | − | − |
Xanthomonas campestris pv. campestris | + | + | + | − |
Bacterial Species | Proteinase K Assay * | Dialysis Tubing Assay ** | ||
---|---|---|---|---|
Treated CF | Non-Treated CF | Tubing CF | Beaker Diffused CF | |
Acidovorax avenae | + *** | + | - | + |
Agrobacterium rhizogenes | + | + | - | + |
Agrobacterium tumefaciens | + | + | - | + |
Brenneria quercina | NT | NT | - | ± |
Acidovorax avenae | + | + | - | + |
Erwinia carotovora subsp. carotovora | + | + | - | + |
Pseudomonas syringae pv. syringae | + | + | - | + |
Pantoea herbicola | ± | ± | - | ± |
Xanthomonas campestris pv. campestris | + | + | - | + |
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Robles-Hernández, L.; Salas-Salazar, N.A.; Gonzalez-Franco, A.C. Purification and Characterization of Antibacterial Activity against Phytopathogenic Bacteria in Culture Fluids from Ganoderma lucidum. Molecules 2021, 26, 5553. https://doi.org/10.3390/molecules26185553
Robles-Hernández L, Salas-Salazar NA, Gonzalez-Franco AC. Purification and Characterization of Antibacterial Activity against Phytopathogenic Bacteria in Culture Fluids from Ganoderma lucidum. Molecules. 2021; 26(18):5553. https://doi.org/10.3390/molecules26185553
Chicago/Turabian StyleRobles-Hernández, Loreto, Nora A. Salas-Salazar, and Ana C. Gonzalez-Franco. 2021. "Purification and Characterization of Antibacterial Activity against Phytopathogenic Bacteria in Culture Fluids from Ganoderma lucidum" Molecules 26, no. 18: 5553. https://doi.org/10.3390/molecules26185553