External standard

5-HT concentrations were determined, as previously described (Amat et al., 1998 (link)), by HPLC with electrochemical detection. The system consisted of an ESA 5600A Coularray detector with an ESA 5014B analytical cell and an ESA 5020 guard cell. The column used was an ESA MD-150 (C-18; 3 μm; 150 × 3.2 mm), which was maintained at 40°C, and the mobile phase was the ESA buffer MD-TM. The analytical cell potentials were kept at −100 and +200 mV, and the guard cell was kept at +220 mV. Dialysate (25 μl) was injected with an ESA 542 Autosampler that kept the dialysates at 6°C. The 5-HT detection limit was 30 fg. External standards (Sigma-Aldrich) were run each day to quantify 5-HT by means of peak height and using ESA software.

Individual carotenoids were extracted by using 5 mg of two-mode dry biomass for conventional extraction (benchmark extraction) or 50 mg from supercritical fluids (SF) extracts. Saponification of samples was then performed, and a tricomponent solution was added in all the samples as described by Cerón-García et al. [67 (link)]. This tricomponent solution was composed of ethanol:hexane:water in a proportion of 77:17:6 v/v/v and contained 0–60% w/w potassium hydroxide [68 ]. The supernatant was transferred into an amber vial for chromatographic analysis. Subsequently, individual carotenoids were separated and identified by using the HPLC system (Jasco Inc, Tokyo, Japan). It was equipped with a quaternary pump (PU-2089 s Plus), diode array detector, and RP-18 column (Lichrosphere, 5 µm × 150 mm) by using a method described by Cerón-García et al. [67 (link)]. In the mobile phase, solvent A was water/methanol (2:8, v/v), solvent B was acetone/methanol (1:1, v/v), and the detection wavelength was 450 nm at 25 °C of column temperature. External standards (Sigma-Aldrich) and their corresponding calibration curves were used to identify and quantify individual carotenoids such as lutein, zeaxanthin, violaxanthin, astaxanthin, and β-carotene. It was performed in triplicate (n = 3).

The culture supernatants were thawed on ice, diluted, and passed through 0.45 μm membrane syringe filters (Millipore; Merck-Millipore, Burlington, MA, USA). A high-performance liquid chromatograph, Agilent Technologies 1200 series (Agilent Technologies, Santa Clara, CA, USA), was used to determine the concentration of glycerol (GLY; [g L⁻¹]) and metabolites (erythritol, ERY; mannitol, MAN; citric acid, CA; α-ketoglutaric acid, α-KG [g L⁻¹]) contained in the culture liquid. The apparatus was equipped with a refractive-index detector (G1362A) and a Rezex ROA-Organic Acid H+ column (Phenomenex, Torrance, CA, USA). Operating conditions were as follows: 0.005 N H₂SO₄ as eluent at a flow rate of 0.6 [mL min⁻¹]; the column temperature was set at 40 °C. External standards (purchased from Sigma-Aldrich) were used for identification and quantification of the peaks areas in chromatograms, which were analyzed using ChemStation for LC 3D software (Agilent).

FLAB strains were sub-cultured in modified FYP broth containing fructose (0.5% w v⁻¹) and mannitol (1% w v⁻¹) as carbon sources. Sub-cultured cells were harvested, washed as described above, and inoculated on Mannitol Yeast extract Peptone (MYP) agar (identical to FYP medium but fructose was replaced by mannitol) supplemented with 0.5% CaCO₃ (w v⁻¹). Plates were incubated at 30 °C for 48 h. Mannitol consumption was evaluated by measuring the size of the clearance zone surrounding the colonies, which indirectly indicates the hydrolysis of CaCO₃ reacting with organic acids synthesized by bacteria.
Mannitol consumption and deriving metabolites were also investigated during FLAB growth in MYP broth. Cells were cultivated in FYP broth until the late exponential growth phase was reached (ca. 18 h), harvested as described above, and re-suspended at the initial cell density of ca. 7 Log CFU mL⁻¹ into a MYP broth. Cells were cultured at 30 °C for 72 h under aerobic condition in shaking (200 rpm) flasks with baffles. Organic acids and mannitol concentrations were determined by HPLC as described above, using external standards (Sigma-Aldrich). The pH was measured by a Crison pH-meter (Model 507, Crison, Milan, Italy).