Sil 20a xr

The fermentation broth was centrifugated and properly diluted. The concentrations of glucose and ethanol were detected using a biosensing analyzer (SBA-40C, Shandong Academy of Sciences, China). The quantification of DM-II and PPD were conducted using a SHIMADZU LC20A system (Shimadzu, Kyoto, Japan) equipped with LC-20ADXR liquid chromatograph and SIL-20AXR auto-sampler. Chromatographic separation of PPD was carried out at 30 °C on a Poroshell 120 EC-C18 column (4.6 × 250 mm, 4 μm, Agilent). DM-II and PPD were separated by using 10% water and 90% acetonitrile. The injection volume was 10 μL, and the flow rate was kept at 1.0 mL/min.

Purity was determined by reversed-phase analytical HPLC (see Supplementary Figs 7–9) using a Phenomenex Gemini C18 column (100 × 4.6 mm), with 5 μm particle size and 110 Å pore size, connected to a HPLC system equipped with an autosampler (Shimadzu SIL-20A XR), degasser (Shimadzu DGU-20A3) and a high-pressure gradient system comprising two LC-20AD XR pumps (Shimadzu). Separation was performed at a flow of 1 ml min⁻¹ and using a 20-60% acetonitrile linear gradient in water containing 0.1% NH₄OH. All eluents and additives were HPLC grade. Peptides, cyanine dye-coupled peptides and eventual contaminants were detected using a PDA (Shimadzu SPD-M20A), acquiring a full ultraviolet-visible spectrum between 200 and 750 nm at any time point. Main chromatogram peaks were collected and product mass confirmed by ESI mass spectrometry using a LCQ Deca XP Max (Thermo Finnigan) ion-trap mass spectrometer. For that, samples were manually injected bypassing the column at a flow rate of 0.20 ml min⁻¹ and positive ion mass spectra were acquired in standard enhanced mode.

The extracts were reconstituted with absolute methanol to a concentration of 1 mg/mL and analyzed by HPLC–PDA in a Shimadzu unit with an LC-20AD pump and a CTO-10AS VP column oven, coupled to an SPD-M20A Diode Array Detector. Separation was obtained using an ACE 3 C18 column (150 mm × 4.6 mm, 3 µm particle size) with an ACE3 C18 analytical pre-column. Compounds were eluted with methanol 0.1% acetic acid (LC–MS grade) (MeOH): MiliQ water 0.1% acetic acid, with a gradient of 80:100% over 15 min, 100% MeOH over 5 min, and 100:80% for 10 min, with a flow of 0.5 mL/min. The results were analyzed at a UV wavelength of 210 nm. The stock solutions of the ethanolic extracts were injected at 1 mg/mL, carrying out a 10 µL injection through an automatic injector (SIL-20A XR, Shimadzu, Kyoto, Japan). All extracts were dissolved in 100% MeOH for injection. The identification of the products was carried out through a comparison between the retention time and UV spectrum of standards of the pure product, previously injected. The percentage expressed in the table of the products is the relative percentage of each product of the whole extract, excluding the peaks of the sample with a percentage lower than 0.011%.