Lab 850

Measurements of pH (Lab 850, Schott AG, Mainz, Germany), lactic acid production, and consequent lactose consumption were quantified throughout 6 days of fermentation, the latter two by ionic exchange in an HPLC System (Waters Corporation, Milford, MA, USA) equipped with a 717 plus Autosampler (Waters Corporation, Milford, MA, USA), a 515 HPLC Pump (Waters Corporation, Milford, MA, USA), and a Refractive Index Detector (RID) (486 Waters Corporation, Milford, MA, USA) for compound detection, as previously described [23 (link)]. Prior to injection, samples were centrifuged at 10,000 g (Eppendorf 5414D, Hamburg, Germany) for 10 min, and the supernatants were filtered through a Millipore membrane (Merck, Darmstadt, Germany) with a pore size of 0.2 µm. Samples were injected in a Schodex SUGAR SH1011 column (Waters Corporation, Milford, MA, USA), and separations were achieved at 50 °C, using 5 mM sulfuric acid as the mobile phase (isocratic elution) at a flow rate of 0.6 mL/min. Calibration curves were made with standard solutions (in 5 mM sulfuric acid) of lactose (Sigma-Aldrich, Netherlands) and lactic acid (Sigma-Aldrich, Netherlands). Peak integration was performed using the HPLC software Empower Pro (Waters Corporation, Milford, MA, USA).

Whey obtained after the manufacturing of cheese from a mixture of ewe, goat, and cow milk, containing 0.04% (m/v) NaCl, pH 6.65 (Lab 850, Schott AG, Mainz, Germany), and 30 g/L of lactose, quantified through ionic exchange in high-performance liquid chromatography (HPLC) as previously described [9 (link)], was used for fermentation assays.
Whey in natura was divided into aliquots of 500 mL and distributed into Erlenmeyer flasks and placed into an incubator at 37 °C for 6 days.

βhm was evaluated by the titration method as previously described (Edge et al., 2006 (link)). Briefly, 2–3 mg dry muscle (dm) was dissected from connective tissue and homogenized in a sodium fluoride containing buffer (33.3 μL 10 mM NaF·mg dm⁻¹). The homogenates were warmed to 37.0°C in a warm water bath. Basal pH was measured with a microelectrode (MI-410, Microelectrodes, Bedford, NH, USA) connected to a pH meter (Lab 850, Schott Instruments GmbH, Mainz, Germany) and subsequently adjusted to pH > 7.1 with sodium hydroxide (0.02 M NaOH). Then, via titration of 2 μL hydrochloric acid (0.01 M HCl) pH was stepwise adjusted until pH reached values below 6.1. βhm was expressed as mmol H⁺·kg dm⁻¹ required to decrease pH with a given unit. 23 samples were analyzed in duplicate. In accordance with previous reports (De Smet et al., 2016 (link)), the typical error of the βhm measurement was 5.4%.

Details of the titration method for analysis of βhm have been described elsewhere (Edge et al., 2006b (link)). Briefly, freeze-dried muscle samples (1.7–2.5 mg dm) were dissected from blood and connective tissue and homogenized on ice in a sodium fluoride containing buffer (33.3 μl 10 mM NaF per mg dm). The homogenates were warmed in a hot water bath at 37.4°C for 5 min. Basal pH measurement was performed with a glass microelectrode (MI-410, Microelectrodes, Bedford, NH, USA) connected to a pH meter (Lab 850, Schott Instruments GmbH, Mainz, Germany). The homogenates were first adjusted to pH ~7.2 with sodium hydroxide (0.02 M NaOH). Then a serial addition of 2 μL hydrochloric acid (0.01 M HCl) was titrated until a pH of ~6.1 was reached. After each titration, the homogenates were briefly vortexed to ensure a homogeneously mixed solution. The number of moles of H⁺ per kg dry muscle required to change pH from 7.1 to 6.5 was interpolated from the fitted titration trend line and expressed as mmol H⁺ per kg dm per unit pH as a unit for βhm. Each sample was measured in duplicate from which the mean was taken. The average CV as determined from the duplicate measures was 5.1%.

All luminescence measurements (luminescence spectra, excitation spectra and lifetimes) were carried out on a Cary Eclipse (Varian, Australia) luminescence spectrophotometer in the range (220–700 nm) equipped with a xenon flash lamp in a 1.00 cm quartz cell. The excitation and emission monochromator band widths were 5 nm. The excitation wavelength was set at 315 nm, and the luminescence was measured using the peak height at 545 nm. All measurements were performed at room temperature (21–23 °C) which should be kept at this level. Absorption spectra were recorded with a UV-2401 PC (Shimadzu, Japan) spectrophotometer.
A pH meter (Lab 850, Schott Instruments GmbH, Germany) was used for pH adjustment. HPLC chromatograms were obtained using an Agilent Technologies 1200 Series chromatograph with isocratic elution under the conditions given in the “Preparation of the lanthanide complex” section. Peak areas were automatically integrated by the Agilent software.