Extraction of Bacterial Metabolic Elicitors

The bacterial strain, stored at −80 °C in nutrient broth with 20% glycerol, was streaked onto nutrient agar (peptone 3 g·L⁻¹, beef extract 5 g·L⁻¹ and agar 15 g·L⁻¹ pH 7) plates and cultivated for 24 h at 28 °C. After 24 h of growth, bacterial cells were scraped off the plates into 10 L of sterile nutrient broth (peptone 3 g·L⁻¹ and beef extract 5 g·L⁻¹ pH 7) and incubated on a rotatory shaker at 28 °C and 180 rpm for 24 h.
Metabolic elicitors (released into the medium) were obtained by centrifuging the 10 L of N 21.4 culture at 2890× g during 20 min at 4 °C. Cells were discarded and the remaining supernatant was evaporated in a stove at 60 °C until obtaining 1 L. This concentrated supernatant was filtrated through a 0.2 µm nitrocellulose filter and extracted twice with a double volume of hexane (v/v). The extract was evaporated to dryness in a Buchi R-215 rotary evaporator at 50 °C [40 (link)]. The dry extract was weight (250 mg) and stored at 4 °C protected from light and humidity.
To obtain the control 1, the same procedure was followed as for extracting the metabolic elicitors from the bacterium, but while carrying out the entire process exclusively with the nutrient medium (peptone 3 g·L⁻¹ and beef extract 5 g·L⁻¹ pH 7), in the absence of bacterium.