626 pump

GC-MS was performed on a QP5050A system (Shimadzu Corp., Kyoto, Japan) using an Equity-5 column (0.25 mm i.d. × 30 m, 0.25 μm film thickness; Supelco, Inc., Bellefonte, PA, USA). The HPLC consisted of a 626 pump with 996 photodiode array detector (Waters Corp., Milford, MA) equipped with a C-18 column (Inertsil ODS-3, 5 μm, 250 × 4.6 mm ID; GL Sciences, Inc., Tokyo, Japan). ¹H and ¹³C NMR spectra were recorded with tetramethylsilane as the internal standard using JNM-EX270 (270 MHz) and JNM-LA500 (500 MHz) NMR spectrometers (JEOL Ltd., Tokyo, Japan). High resolution mass spectra were obtained with a JMS-T100LC AccuTOF mass spectrometer (JEOL). The seeds used in the present study have been previously described6 (link). They were planted at a depth of 5 mm in pots (8 × 8 × 6 cm deep) containing sand and then incubated in an illuminated growth chamber (FLI-301N; Tokyo, Rikakikai, Co., Ltd., Tokyo, Japan) under a 16-h light/8-h dark cycle at 22 °C.

The content of rosmarinic acid was measured according to Chizzola et al. [36 (link)] using a Waters HPLC system consisting of a 626 pump, a 600S controller, a 717plus autosampler, a column oven operated at 25 °C, and a 996 diode array detector (Waters S.A.S, Saint-Quentin, France). The separation was carried out on a Symmetry C18, 5.0 μm particle size, 4.6 × 150 mm column. The mobile phase used was 1% acetic acid/acetontrile 85:15 (solvent A) and methanol (solvent B). The analysis started with a solvent ratio of A/B of 9:1, and a linear gradient was performed to reach 100% B within 30 min. The flow rate was 1.0 mL/min and the injection volume, 20 μL.
The quantification of rosmarinic acid was done using the external standard method by preparing seven calibration standards ranging from 3.9 to 500 μg/mL and recording the calibration curve at 330 nm. A calibration line with the correlation coefficient R² 0.997 could be established.
The following measurements of polyphenols and antioxidative activity were based on colorimetric reactions and were adapted to be measured with an iMark microplate reader (BioRad, Hercules, CA, USA). On a microplate four replicates of each sample or standard were carried out as described by Chizzola et al. [37 (link)].

A volume of 10 mL of the culture supernatants was purified through C18 Maxi-Clean 10 g cartridges (Alltech). Compared to the lipopeptide HPLC analysis protocol, all the volumes used were adapted. A sample of 100 μL of the lipopeptide mixture concentrated at 250 mg/L was charged in the HPLC (Online Degaser, 717 Autosampler, 660S Controller, 626 Pump, 996 PhotoDiodeArray, Waters Corp., Milford, MA, USA). The column used was a C18 (5 μm, 300 × 10 mm, ACE), working at a flow rate of 3 mL/min. The mycosubtilins were separated with an acetonitrile–water–trifluoroacetic acid solvent, 35:65:0.1, v/v/v for 54 min, followed by a 6 min rinsing step with acetonitrile–trifluoroacetic acid solvent, 100:0.1, v/v. As for lipopeptides analysis, the retention time and second derivative of the absorption spectrum between 200 and 400 nm were used to identify the eluted molecules (Millenium 32 Software, Waters).