Advanced vortex mixer zx3

Water absorption capacity (WAC) was determined as described by Rodriguez-Ambriz et al. [24 (link)]. A 100 mg protein sample was mixed with 1 mL distilled water and vortexed (Advanced Vortex Mixer—ZX3, VELP Scientifica, Usmate Velate, Italy) for 30 s. The resulting suspension was incubated at room temperature (23 °C) for 30 min and centrifuged for 20 min at 1800× g (MPW-251, Med. Instruments, Warsaw, Poland). The supernatant was decanted for 10 min at a 45-degree angle. WAC was calculated by dividing the weight of the absorbed water (g) by the weight of the protein sample (g).
Oil absorption capacity (OAC) was determined by the method of Lin and Zayas [25 (link)]. Each protein sample (100 mg) was mixed with 1 mL sunflower oil and vortexed (Advanced Vortex Mixer—ZX3, VELP Scientifica, Usmate (MB), Italy) for 30 s. The mixture was incubated at room temperature (23 °C) for 30 min and subsequently centrifuged at 13,600× g for 10 min (MPW-251, Med. Instruments, Warsaw, Poland). The supernatant was decanted and drained for 20 min at a 45-degree angle. OAC was calculated by dividing the weight of the absorbed oil (g) by the weight of the protein sample (g).
The influence of NaCl on WAC and OAC of the protein isolates was evaluated by adding the salt to the test systems to final concentrations of 0.03 or 0.25 M wherever needed.

Each sample was placed in a sterile 50 mL falcon tube, to which 45 mL of sterile Ringer’s solution was added. The tube was vortexed (ZX3 Advanced Vortex Mixer, Velp Scientifica, Usmate Velate, Italy) at 2200 rpm for 90 sec. Successively, this solution was serially diluted (1:10) in Ringer’s solution. For the enumeration of E. coli [30 (link)] and L. innocua [28 (link)], 100 μL of the selected dilutions were spread-plated on MacConkey agar with crystal violet (Microbiol diagnostici, Cagliari, Italy) and BHI agar (Microbiol diagnostici, Cagliari, Italy), respectively. Plates were incubated for 24 h at 37 °C in an incubator (Memmert, Schwaback, Germany) and then enumerated. For mesophilic and psychrophilic natural microflora, non-inoculated samples were similarly analyzed by pour-plating in plate count agar (PCA, Microbiol diagnostici, Cagliari, Italy), in 1 mL of solution. The plates were then incubated at 30 °C for 72 h and at 10 °C for 120 h, for mesophilic and psychrophilic bacteria, respectively. Results are expressed as log CFU/g.

The extraction of polyphenols was performed according to the method described by Alahyane et al. [14 (link)], with slight modifications. Briefly, 1 g of the date powder was mixed with 30 mL of ethanol/water solution (80/20, v/v), and the extraction was performed at 25 °C for 2 h with a VDRL 711 orbital shaker (Asal S.r.l., Milan, Italy) under constant rotatory agitation at 60 rpm. All extracts were centrifuged at 2800× g for 10 min at 4 °C, and the supernatants were then collected and filtered through a 0.45-µm nylon membrane filter. The samples were stored in amber vials at −18 °C. All extractions were done in triplicate.
For biscuit samples, fat was removed with hexane using a solid–liquid extraction protocol. Briefly, 1 g of the biscuit was mixed with 5 mL hexane using a ZX3 advanced vortex mixer (Velp Scientifica, Milan, Italy) for 5 min. All samples were centrifuged at 2800× g for 10 min at 4 °C. These operations were repeated two times. Thereafter, hexane was evaporated with nitrogen, and ethanolic extractions were performed as previously described for the fruit samples.