Pure reference standards of FLBZ, reduced-FLBZ (R-FLBZ) and hydrolyzed-FLBZ (H-FLBZ) used to develop the analytical methodology were kindly provided by Janssen Animal Health (Beerse, Belgium). Oxibendazole (OBZ), used as internal standard, was obtained from Schering Plough (Kenilworth, NJ, USA). HPLC grade acetonitrile and methanol were from Sintorgan S.A. (Buenos Aires, Argentina) and J.T. Baker (New Jersey, USA), respectively. HPBCDs were from ISP Pharmaceuticals (Cavasol, Cavitron, New Jersey, USA). Low viscosity grade sodium CMC was purchased from Anedra (Buenos Aires, Argentina). Tween 80 was purchased from Biopack (Buenos Aires, Argentina).
Tween 80
Manufactured by Biopack
Sourced in Argentina
Tween 80 is a non-ionic surfactant commonly used in laboratory applications. It is a polyoxyethylene sorbitan monooleate that serves as an emulsifier, dispersant, and wetting agent.
Automatically generated - may contain errors
Lab products found in correlation
Oxibendazole, by Merck & Co (2 mentions)
Sodium acetate, by Biopack (1 mentions)
Gallic acid, by Merck Group (1 mentions)
Sodium carbonate, by Biopack (1 mentions)
Manganese sulphate, by Merck Group (1 mentions)
Fluorescein sodium salt, by Merck Group (1 mentions)
Malt extract agar mea, by Merck Group (1 mentions)
Victor3 model, by PerkinElmer (1 mentions)
Spark 20m multimode microplate reader, by Tecan (1 mentions)
2 2 azobis 2 methylpropionamidine dihydrochloride aaph, by Merck Group (1 mentions)
2 7 dichlorodihydrofluorescein diacetate h2dcf da, by Merck Group (1 mentions)
Potassium dihydrogen phosphate, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Dipotassium hydrogen phosphate, by Merck Group (1 mentions)
V 630, by Jasco (1 mentions)
Disodium hydrogen phosphate, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Folin ciocalteau reagent, by Merck Group (1 mentions)
5 protocols using tween 80
1
Analytical Methodology for FLBZ Detection
2
Standardized Blueberry Botrytis Infection
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B. cinerea BAFC 3003 strain was provided by BAFC Culture Collection (Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina) (http://www.inmibo.exactas.uba.ar ). Isolates were incubated on malt extract agar (MEA) (Merck, Germany) during 14 days at 25 ± 1 °C; then, conidia were harvested by washing the culture in a Tween 80 (Biopack, Argentina) detergent solution (0.05 % v/v) in peptone water (0.1 % w/v) and gently shaken on a vortex mixer. The final conidia concentration (approximately 107 conidia/mL) was determined with a Neubauer counting chamber (Exacta, Germany).
Blueberry surface was decontaminated by immersing the fruit in a sodium hypochlorite solution (200 mg.L−1) for 2 min and then, were rinsed three times with sterile water and dried with towel paper. A spot-inoculation method was used; 10 μl of the conidia suspension were inoculated in a small injury made at the picking scar. Finally, inoculated blueberries were kept at 21 ± 2 °C overnight in a laminar air flow cabinet to promote mould adhesion (Contigiani et al., 2020a ).
B. cinerea incidence was evaluated daily by visual inspection, as previously described for native mycobiota. Untreated inoculated fresh fruit (FF(BC)) was considered as control. Three replicates of 20 blueberries (n = 60) were analysed for each condition.
Blueberry surface was decontaminated by immersing the fruit in a sodium hypochlorite solution (200 mg.L−1) for 2 min and then, were rinsed three times with sterile water and dried with towel paper. A spot-inoculation method was used; 10 μl of the conidia suspension were inoculated in a small injury made at the picking scar. Finally, inoculated blueberries were kept at 21 ± 2 °C overnight in a laminar air flow cabinet to promote mould adhesion (Contigiani et al., 2020a ).
B. cinerea incidence was evaluated daily by visual inspection, as previously described for native mycobiota. Untreated inoculated fresh fruit (FF(BC)) was considered as control. Three replicates of 20 blueberries (n = 60) were analysed for each condition.
3
Spectroscopic Analyses of Bioactive Compounds
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Colour measurements were made with a hand-held tristimulus reflectance spectrocolorimeter (Minolta Co., Model CM-508-d, Japan). For total anthocyanin and phenolic compound contents, and antioxidant capacity (TEAC assay), a UV- VIS spectrophotometer (model V-630, JASCO, Japan) was used. For the determination of antioxidant capacity evaluated by ORAC assay, fluorescence measurements were made by means of a Multilabel Microplate Reader (Perkin Elmer, Victor3 model, USA). Fluorescence measurements in Caenorhabditis elegans assays were performed in a Spark 20M Multimode Microplate Reader (Tecan, NC, USA).
AAPH (2,2′-Azobis (2-methylpropionamidine) dihydrochloride, ABTS (2,2 -azinobis (3-ethylbenzothiazoline)-6-sulfonate), fluorescein sodium salt, gallic acid (GA), Trolox (C14H18O4) and 2,7 – dichlorodihydrofluorescein diacetate (H2DCF-DA) were purchased from Sigma-Aldrich (St. Louis, USA). Ethyl alcohol, sodium acetate, sodium carbonate, and Tween 80 were from Biopack (Buenos Aires, Argentina). Folin Ciocalteau reagent, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, hydrochloric acid, potassium chloride, sodium chloride, sodium hydroxide, manganese sulphate, and malt extract agar (MEA) were purchased from Merck (Darmstadt, Germany).
AAPH (2,2′-Azobis (2-methylpropionamidine) dihydrochloride, ABTS (2,2 -azinobis (3-ethylbenzothiazoline)-6-sulfonate), fluorescein sodium salt, gallic acid (GA), Trolox (C14H18O4) and 2,7 – dichlorodihydrofluorescein diacetate (H2DCF-DA) were purchased from Sigma-Aldrich (St. Louis, USA). Ethyl alcohol, sodium acetate, sodium carbonate, and Tween 80 were from Biopack (Buenos Aires, Argentina). Folin Ciocalteau reagent, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, hydrochloric acid, potassium chloride, sodium chloride, sodium hydroxide, manganese sulphate, and malt extract agar (MEA) were purchased from Merck (Darmstadt, Germany).
4
FLBZ Analytical Methodology Development
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Pure reference standards of FLBZ, reduced-FLBZ (R-FLBZ) and hydrolyzed-FLBZ (H-FLBZ) used to develop the analytical methodology were kindly provided by Janssen Animal Health (Beerse, Belgium). Oxibendazole (OBZ), used as internal standard, was obtained from Schering Plough (Kenilworth, NJ, USA). HPLC grade acetonitrile and methanol were from Sintorgan S.A. (Buenos Aires, Argentina) and J.T. Baker (Phillipsburg, New Jersey, USA), respectively. HPBCDs were from ISP Pharmaceuticals (Cavasol, New Jersey, USA). Low viscosity grade sodium CMC was purchased from Anedra (Buenos Aires, Argentina). Tween® 80 was purchased from Biopack (Buenos Aires, Argentina).
5
Determining Antimicrobial Activity of Essential Oils
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The MIC of EO and ERY was performed by microdilution in broth using 96-well polystyrene microtiter plates with Mueller Hinton broth (MHB) (Biokar Diagnostics, France). A 0.5% of Tween 80 (Biopack, Argentina) was added to enhance the EO dissolution. The broth pH was adjusted to 7.4, 6.5 and 5.0 by addition of hydrochloric acid 1N (Anedra, Argentina), emulating pH conditions of extracellular and intracellular level sites. The ERY (Parafarm, Argentina) range of concentrations analyzed (applying a scheme of two-fold serial dilution) were between 1024 and 0.007 μg/mL. EO concentrations tested were between 50 and 0.1 μL/mL. In both cases each well was inoculated with a final bacterial concentration of 5 × 105 CFU/mL. Microplates were incubated at 35°C for 18–24 h. MIC was established as the lowest concentration which inhibits the bacterial growth. Each determination was done in triplicate. Positive and negative controls contained MHB with Tween 80 (0.5%) were included in the test.
The MBC was determined by inoculation spreading of 25 μL from each well showing no evident bacterial growth (after establishing the MIC) in nutritive agar plates for colony counting after incubation at 35°C for 18–24 h. The MBC was established as the first antimicrobial concentration which produce the fall of 99.9% from the initial inoculum.
The MBC was determined by inoculation spreading of 25 μL from each well showing no evident bacterial growth (after establishing the MIC) in nutritive agar plates for colony counting after incubation at 35°C for 18–24 h. The MBC was established as the first antimicrobial concentration which produce the fall of 99.9% from the initial inoculum.
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