Magnesium sulfate

Lactiplantibacillus plantarum subsp. plantarum ATCC14917, Lactobacillus delbruekii subsp. bulgaricus ATCC 11842, L. acidophilus ATCC4356 and Lactinocaseibacillus rhamnosus ATCC 7469 (Farmalabor, Canosa, Italy) were reactivated by culturing twice in 25 mL of MRS broth (meat peptone 10.0 gL⁻¹; dextrose 20.0 gL⁻¹; yeast extract 5.0 gL⁻¹; beef extract 10.0 gL⁻¹; disodium phosphate 2.0 gL⁻¹; sodium acetate 5.0 gL⁻¹; ammonium citrate 2.0 gL⁻¹; magnesium sulfate 0.1 gL⁻¹; manganese sulphate 0.05 gL⁻¹; Tween 80 1.0 gL⁻¹) (Thermo scientific, Waltham, MA, USA) at 37 °C for 18 h to obtain 10⁸ cells/mL. To prepare the inoculum, bacterial cultures were centrifuged and washed in sterile physiological solution (NaCl 8.5 gL⁻¹) and resuspended in 5 mL of the same solution. Fresh palm date fruits were purchased from a local supermarket, were boned, and 200 g of milled date fruit pulp, 100 g of cereals, 18 h cultures (final concentration > 10⁶ CFU/mL) and sterile water were mixed in a food processor mixer for 2 min. For the fermentation, the mixtures obtained were placed in plastic bags and incubated at 37 °C for 48 h. After this time, the pH was measured followed by sample drying up to 15% moisture content. Extrusion was accomplished using a laboratory single screw extruder S-45 (Metalchem Gliwice) after the fermentation time.