Hplc grade standards

The simple sugars profile was measured as described by Heredia-Olea et al. [30 (link)] and Alonso-Gómez et al. [31 (link)] with slight modifications. The samples were filtered through a polyvinylidene fluoride (PVDF) syringe filter (0.2 µm) and injected into high-performance liquid chromatography (HPLC) equipment (Waters HPLC Breeze model, Waters, Milford, MA, USA) with a refractive index detector (Waters 2414) kept at 50 °C. The chromatographic separation was achieved using an ion-exclusion column Phenomenex Rezex ROA-organic acid h+ (250 × 4.6 mm, 8 µm particle size, Phenomenex, Torrance, CA, USA) at 60 °C. The mobile phase consisted of a 5 mM H₂SO₄ solution with a 20 min isocratic flow rate of 0.4 mL min⁻¹ and with an injection volume of 10 μL. Glucose, maltose, and fructose quantifications were performed with calibration curves of HPLC-grade standards (Sigma-Aldrich, St. Louis, MO, USA).
Calibration curves for glucose (1–25 mg mL⁻¹), maltose (0.5–5 mg mL⁻¹), and fructose (1–25 mg mL⁻¹) of HPLC-grade standards (Sigma-Aldrich, St. Louis, MO, USA) were constructed for quantification purposes (data not shown).

The phenolic compounds were analyzed by HPLC (Waters 2695; C18 column (250 × 4.6 mm × 5 μm size particle) and the absorbance was measured at 280 nm by a Waters 2478 Dual λ Absorbance Detector. The mobile phase of acidified water containing 1% formic acid (A) and acetonitrile (B) was used. The setting procedure of the gradient was as follows: 0 min, 20% B; 17 min, 21.5% B; 17.5 min, 68% B; 40 min, 68.3% B; 41 min, 100% B; 51 min, 20% B, and held for 2 min. The volume of injection was 20 μL and the flow rate was 1.0 mL/min. All HPLC-grade standards (ferulic acid, epicatechin, catechins, phloridzin, phloretin, caffeic acid, rutin, and chlorogenic acid) were purchased from Sigma Chemicals. Identification of each peak was performed on the basis of comparing their retention times with their known standards. The concentration of phenolic contents was expressed as milligrams of each compound per liter of juice.

The pH differential method (Lee et al., 2005 (link)) was used to measure the total anthocyanin content. Approximately 1.5 g strawberry fruit was extracted with 15 mL pre-cooled extraction buffer (1% HCl-ethanol) on ice for 4 h, and then centrifuged (8000 × g, 4°C) for 25 min. The supernatant was measured for total anthocyanin content. The main anthocyanin pelargonidin-3-glucoside in strawberry fruits was determined by the HPLC method (Yonekura and Tamura, 2019 (link)). Anthocyanins were extracted using 1% HCl in a methanol solution, which was subsequently detected by a Silgreen ODS C18 column using an Agilent HPLC system with a DAD detector at 510 nm. The anthocyanins, listed as follows, were detected using a linear gradient eluent program: 5% formic acid in water as eluent A and methanol as eluent B, 100-0% A in B was used for 20 min, followed by 100% B for 5 min. A 10-µL sample was injected, the flow rate was set to 1 mL/min. The concentration of anthocyanins was quantified by comparing with the corresponding external standards. All HPLC-grade standards were purchased from Sigma (USA). Experiments were independently repeated three times.