Labmaster aw

The content of water, protein, ash and NaCl were analysed according to AOAC [23 –26 ], while fat was evaluated in accordance with ISO 3433:2008 [27 ]. Active acidity (pH) was obtained by conductometric measurements using a pH metre (CP-411, Elmetron, Poland). Water activity was measured using the LabMaster-aw (Novasina AG, Switzerland). All analyses were performed in triplicate.

Water activity (a_w) of the medium was set between 0.99 and 0.75 by substitution of water with 0–50% glycerol (v/v). A volume of 23 ml of medium was poured in Petri dishes with vents (Greiner, Bio-One B. V., Alphen aan de Rijn, The Netherlands) and left to solidify for 24 h on the bench with the lid closed. The a_w of control (non-inoculated) plates of the different glycerol-agar mixtures was determined before and after growth experiments using a Novasina labmaster-a_w (Novasina, Lachen, Switzerland) and samples with a diameter of 5 cm. The a_w changed only marginally during 3 weeks of storage (ranging between a decrease of 0.01 a_w unit in 5% glycerol plates and an increase of 0.03 units in 50% glycerol plates).
Agar plates were inoculated with 3 μl spore solution containing 1 x 10⁶ spores ml ^-1 harvested from 7-days-old MEA-grown cultures. Conidia were collected in 10 mM ACES, 0.02% Tween 80. The spore suspension was filtered over sterile glass wool to remove hyphal fragments. Spores were counted using a Bürker-Türk haemocytometer and the suspension was diluted to 1 x 10⁶ spores ml ^-1.

Before the heat treatment, uninoculated frass was subjected to measurement of pH, water activity (a_w) and moisture content. To measure the pH, a digital pH meter (Portamess 911, Knick, Berlin, Germany, with SI analytics electrode, Mainz, Germany) was used at room temperature, after addition of 17 mL of demineralized water to 10 g of frass according to the method of [22 (link)]. A water activity meter (LabMaster a_w, Novasina, Lachen, Switzerland) was used to determine the water activity (i.e., the amount of water available for microbial growth), after water activity and temperature (25 °C) were stable for 5 min. Moisture content (i.e., the total amount of water present) was determined by calculating the difference in weight of 5 g frass before and after overnight oven drying at 105 °C. All measurements of pH, a_w and moisture content were performed in triplicate.

Beads were produced by dripping the respective encapsulation suspension with a sterile syringe with cannula (0.90 × 40 mm Sterican, B. Braun Melsungen AG, Melsungen, Germany) by means of a syringe pump (Cole-Parmer, Vernon Hills, USA) into a cold sterile 180 mM CaCl₂ solution while stirring. The pumping speed was set to 4 mL/min. Beads cured for 20 min before being separated from the gelation bath and rinsed with ultra-pure water. Drying was performed in two steps. First, moist beads were dried under a laminar flow at room temperature [23 ± 2 °C, approx. 30% relative humidity (RH)] for 24 h and subsequently further dried in a self-constructed fluidized bed dryer at 37 ± 2 °C and approx. 10% RH for 10–15 min until the desired water activity of 0.2–0.3 was reached, unless stated otherwise. The water activity was monitored with a water activity meter (LabMASTER-aw, Novasina AG, Lachen, Switzerland) at 25 °C. Water activity and temperature stable times were set to 2 min and 1 min, respectively.

A complete factorial design was set up to study the effects of NaCl and temperature. Growth trials with four replicates of each G. candidum strain were performed with fifty combinations of experimental conditions according to the following factor levels:
Storage temperature (°C): 6, 8, 12, 15, 18, 21, 25, 30, 34, 37
NaCl (%):                            0, 1, 3, 5, 7 (w/v)
Standard growth SMA medium, which was acidified with 10 mL/L lactic acid (Sigma-Aldrich, St. Louis, MO, USA) to pH 5.5, was used in the experiments. The a_w of the medium was adjusted with 1, 3, 5 or 7% sodium chloride (Sigma-Aldrich, St. Louis, MO, USA) and measured after sterilization with Novasina LabMaster-a_w (Novasina, Lachen, Switzerland). The inoculum was prepared, and the growth experiments were carried out according to Koňuchová and Valík [21 ].
The diameters of G. candidum colonies (d) were measured using a Vernier calliper (150 × 0.02 mm, Sinochem Jiangsu, Nanjing, China) in two orthogonal directions per plate without opening the dishes, and the final values were calculated according to previous work.

The moisture content of GCs was determined after dehydration of 1 g sample to a constant weight using the oven at 60 °C (Memmert UN30, Schwabach, Germany). The water activity of samples was measured at 25 °C using a water activity meter (Lab Master-aw, Novasina, Switzerland).