Lc 10ad

NE and 5-HT concentrations were determined by HPLC coupled with an electrochemical detector (INTRO, Antec Leyden, The Netherlands). Separation was performed by a LC18 reverse phase column (hypersil, 150 × 3 mm, ODS 5 μm; Thermoscientific, Milan, Italy). The detection was accomplished by a Unijet cell (BASi, Kenilworth, U.K.) with a 6 mm diameter glassy carbon electrode at a working potential of 0.65 V vs. Ag/AgCl. The mobile phase used was 85 mM CH₃COONa, 0.8 mM octane sulfonic acid, 0.3 mM EDTA, 15 mM NaCl, methanol 6%, in distilled water, buffered at pH 4.85. The flow rate was maintained by an isocratic pump (Shimadzu LC-10 AD, Kyoto, Japan) at 1 ml/min. Data were acquired and integrated using Chromeleon software (version 6.60, Dionex, San Donato Milanese, Italy).

The cellulose, hemicellulose and lignin contents were determined based on the standard NREL procedure. A SEM JSM-740F (JEOL LTD, Japan) was used to observe the WS surface shape before and after the NaOH pre-treatment to indirectly investigate the effect of NaOH pre-treatment on the WS surface and the enzymatic hydrolysis process. The sugar samples were analysed by an HPLC instrument (LC-10AD, SHIMADZU, Kyoto, Japan) equipped with a refractive index detector (RID-10A, SHIMADZU, Kyoto, Japan). An Aminex HPX-87 P column (Bio-Rad, USA) with a safeguard column operated at 80 °C using pure-grade water as the mobile phase (0.6 mL/min) was utilized for the separation^{15 (link)}. The RS yield was calculated according to the stoichiometric relationship represented by Eq. (1). $${\text{Y}}_{\text{RS}}\left(\%\right)={\text{C}}_{\text{RS}}/{\text{M}}_{\text{WS}},$$ where Y_RS is the actual RS yield, C_RS is the RS concentration in the saccharification liquid, and M_WS is the initial mass of the WS.

The phenolic profile of the carob pulp and its derived products was obtained with an HPLC system from Shimadzu (Kyoto, Japan), equipped with a photodiode array detector (PDA) (SPD-M20A), a pump (LC-10AD), an autosampler (SIL-20AHT), and a thermostat column compartment (CTO-10ASVP). Chromatographic separation conditions were performed according to the procedure described by Kumar et al. [43 (link)]. The PDA detector was set at 280 nm for the determination of all polyphenolic compounds. The Venusil XSP C18 (Radnor, PA, USA) analytical column (150 × 4.6 mm, 5 μm) was equipped with a pre-column composed of the same material and kept constant at 25 °C. Milli Q water (mobile phase A) and 0.02% (v/v) TFA in acetonitrile (mobile phase B) were used as solvents with a flow rate of 1 mL/min. A gradient elution program was used, starting at 80% A for the first five min; mobile phase A was decreased linearly to 60% at 8 min and then to 50% at 12 min. Finally, mobile phase A increased linearly to 60% until 17 min and then returned to the initial composition at 21 min, where it remained constant until 25 min. Twenty microliters of each extract were injected into the system and assayed in triplicate.