Ringer solution

In order to select the optimal fermentation conditions for each of the six substrates considered, a preliminary screening based on 179 trials was carried out (Figure 1). These trials took into account the combination of several factors: (i) type of substrate, (ii) bacterial strains inoculated both as single cultures or in co-culture, (iii) initial inoculum size, (iv) time of fermentation, and (v) type of stabilization after fementation, in particular pasteurization (TT) or refrigeration (NTT). Pasteurization was conducted for 2 min at 100 ^°C for tomato exctract, while tomato puree and tomato juices were treated for 10 min at 100 ^°C; refrigeration was set up at 4 ^°C.
A first sorting was made on the basis of the results of increase of viable cells after fermentation. Bacterial concentration was determined after inoculum (T₀) and at the established times of fermentation. Ten-fold dilutions in Ringer solution (Oxoid, Basingstoke, UK) were plated in MRS agar (Oxoid), followed by incubation for 48 h in aerobic condition at 37 ^°C. The pH of all samples was also measured by pH meter (Mettler Toledo, Switzerland) in duplicate at the beginning and at the end of fermentation.
At the end of the selection step, 16 fermentation conditions were chosen for each type of juices, 8 of which fermented and pasteurized, and 8 fermented and refrigerated.

Cherry juice, inoculated with starters, was analyzed before and after fermentation (48 h) and after the storage period (12 days). Cultivable cells were determined using the standard plate count agar method as follows: decimal dilutions of samples were carried out in Ringer solution (Oxoid, Milan, Italy) and plated on MRS agar, then incubated at 30 °C (L. plantarum) and 37 °C (L. rhamnosus, L. casei and L. paracasei) for 48 h under anaerobic condition. The pH of samples was measured using a pH metre (Mettler Toledo, Greifensee, Switzerland). Plate count and pH measurement was carried out in triplicate.

Before fermentation, the frozen cultures were revitalized twice in MRS broth (Oxoid) (inoculum of 3% v/v) incubated overnight at 37 °C under suspended conditions (for the LAB) and in NB incubated overnight at 30 °C under shaking (for B. subtilis). Afterwards, LAB and B. subtilis were inoculated (3% v/v) in MRS and NB and incubated at specific temperatures for each species (for 16 h) in order to obtain a concentration of 9 log cfu/mL. The grown cell cultures were collected by centrifugation (12,857× g for 10 min at 4 °C), washed twice in Ringer solution (Oxoid, Milan, Italy), and resuspended in sterile bidistilled water. HE I was rehydrated with 75% of water and then inoculated individually with each bacterial suspension in order to obtain a final concentration of 7 log cfu/mL. The microbial concentration was evaluated just after the inoculum (T0), after 24 h (T1) and 72 h (T2) of fermentation at the optimal temperature for each strain. Ten-fold serial dilutions in Ringer (Oxoid) were plated on MRS agar or nutrient agar (NA) (for LAB and B. subtilis, respectively) and then incubated for 72 h in aerobic conditions at the optimal temperature for each strain. Fermentation was carried out in duplicate, and for each sample, time analyses were performed in duplicate. Average values ± standard deviations are reported. After the process, fermented seaweeds were lyophilized.

Water extracts from the silage or grain (day 0) samples were prepared by homogenizing 25 g of sample in 225 ml of sterile solution (Ringer Solution, Oxoid, Hampshire) in an industrial blender for 1 min. Then, the extract was filtered through a double layer of sterile gauze, and the pH was measured with the aid of a potentiometer (Tecnal, SP, Brasil).
A 15 mL aliquot of the extract was filtered through Whatman 54 filter paper (Whatman, Florham, NJ) and packaged in tubes containing 100 μL of H₂SO₄ 50%, for further analysis of ammoniacal nitrogen (NH₃-N)¹⁸ , water soluble carbohydrates (WSC)^{19 (link)}, lactic acid (LA), acetic acid (AA), butyric acid (BA), and ethanol (ETA) by high-performance liquid chromatography (HPLC, Dionex Corporation, Sunnyvale, CA, USA)²⁰ .

A first screening to establish the susceptibility of A. apis to EOs was carried out using the paper disc agar diffusion (PDD) method. From an SDA agar plate, a mycelium disc of 6 mm in diameter was taken, dissolved in Ringer solution (Oxoid) and vortexed for 5 min. Subsequently, 1 mL of the fungal suspension was overlaid on the SDA plate. After plate solidification, sterile filter paper discs (6 mm in diameter, Oxoid, Milan, Italy), saturated with each pure essential oil (n = 10), were placed over the plate surface (PDD method). The cultures were then incubated at 30 °C for 5 days. Blank discs and synthetic antifungal nystatin were used as negative and positive controls, respectively. The diameter (mm) of fungal growth inhibition was measured (disk diameter included). An EO activity level higher than 20 mm was classified as high activity. All the experiments were carried out in triplicate for each essential oil.

The amines TMA, DMA, and the internal standard, n-propylamine hydrochlorides, were acquired from BDH and Merck. Sodium chloride (NaCl, 99.5%) and sodium hydroxide (NaOH) were obtained from PanReac (Barcelona, Spain). Ultra-pure water was obtained from a Milli-Q^® system (Millipore, Burlington, MA, USA), and helium of purity 5.0 was used as the GC carrier gas, supplied by Air Liquide (Alges, Portugal). The SPME holder for the manual sampling of SPME fiber and the respective fibers, namely the divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS), with a 50/30 mm film thickness, carboxen/polydimethylsiloxane (CAR/PDMS) with a 75 mm film thickness, and polydimethylsiloxane/divinylbenzene (PDMS/DVB) with a 65 mm film thickness, were purchased from Supelco (Bellefonte, PA, USA). Ringer solution (Oxoid, Basingstoke, Hampshire, UK) was used for microorganism transference. Inoculations were made in iron agar, nutrient agar (Oxoid, Basingstoke, Hampshire, UK), and MacConkey agar (PanReac AppliChem, Darmstadt, Germany). For physical evaluation, a Torrymeter 295 (Distell, West Lothian, Scotland, UK) was used.

In this study we used chemical and reagent such a CaCl₂·2H₂O, MgSO₄·7H₂O, acetic acid (glacial) 100% anhydrous for analysis, glutaraldehyde, dimethyl sulfoxide (DMSO) for analysis, ethanol absolute for analysis, NaCl, and Tris-Cl obtained from Merck, Germany. Reagents and other chemicals used as a Ringer solution (Oxoid, Great Britain), 5.25% of hypochlorite (SC Johnson, Indonesia), crystal violet, and powdered gelatine (Sigma-Aldrich, USA).