Tailoring the Techno-Functional Properties of Fava Bean Protein Isolates: A Comparative Evaluation of Ultrasonication and Pulsed Electric Field Treatments

Abstract

The fava bean protein isolate (FBPI) holds promise as a sustainable plant-based protein ingredient. However, native FBPIs exhibit limited functionality, including unsuitable emulsifying activities and a low solubility at a neutral pH, restricting their applications. This study is focused on the effect of ultrasonication (US) and pulsed electric fields (PEF) on modulating the techno-functional properties of FBPIs. Native FBPIs were treated with US at amplitudes of 60-90% for 30 min in 0.5 s on-and-off cycles and with PEF at an electric field intensity of 1.5 kV/cm with 1000-4000 pulses of 20 μs pulse widths. US caused a reduction in the size and charge of the FBPIs more prominently than the PEF. Protein characterization by means of SDS-PAGE illustrated that US and PEF caused severe-to-moderate changes in the molecular weight of the FBPIs. In addition, a spectroscopic analysis using Fourier-transform infrared (FTIR) and circular dichroism (CD) revealed that US and the PEF induced conformational changes through partial unfolding and secondary structure remodeling from an α-helix to a β-sheet. Crystallographic and calorimetric determinations indicated decreased crystallinity and lowered thermal transition temperatures of the US- and PEF-modified FBPIs. Overall, non-thermal processing provided an effective strategy for upgrading FBPIs' functionality, with implications for developing competitive plant-based protein alternatives.

Keywords: fava bean protein isolate; plant-based proteins; protein functionality; pulsed electric fields; ultrasonication.

Figure 1

Particle size distribution of FBPIs treated with US and PEF at varying intensities. FBPI: fava bean protein isolate; US: ultrasonication; PEF: pulsed electric fields; CON-FBPI: native FBPI without any treatment; US-FBPI-60: FBPI ultrasonicated at 60% amplitude; US-FBPI-70: FBPI ultrasonicated at 70% amplitude; US-FBPI-80: FBPI ultrasonicated at 80% amplitude; US-FBPI-90: FBPI ultrasonicated at 90% amplitude; PEF-FBPI-1000: FBPI subjected to PEF treatment for 1000 pulses; PEF-FBPI-2000: FBPI subjected to PEF treatment for 2000 pulses; PEF-FBPI-3000: FBPI subjected to PEF treatment for 3000 pulses; and PEF-FBPI-4000: FBPI subjected to PEF treatment for 4000 pulses.

Figure 2

SDS-PAGE protein patterns of FBPIs treated with US and PEF at varying intensities in (a) the absence and (b) presence of β-mercaptoethanol. Lane M: molecular weight marker; Lane 1: CON-FBPI; Lane 2: US-FBPI-60; Lane 3: US-FBPI-70; Lane 4: US-FBPI-80; Lane 5: US-FBPI-90; Lane 6: PEF-FBPI-1000; Lane 7: PEF-FBPI-2000; Lane 8: PEF-FBPI-3000; and Lane 9: PEF-FBPI-4000. For the caption, please see Figure 1.

Figure 3

Fourier-transform infrared (FTIR) spectra of FBPIs treated with US and PEF at varying intensities. For the caption, please see Figure 1.

Figure 4

(a) Circular dichroism (CD) spectra and (b) relative content of secondary structure components in FBPIs treated with US and PEF at varying intensities. For the caption, please see Figure 1.

Figure 4

(a) Circular dichroism (CD) spectra and (b) relative content of secondary structure components in FBPIs treated with US and PEF at varying intensities. For the caption, please see Figure 1.

Figure 5

X-ray diffraction (XRD) spectra of FBPIs treated with US and PEF at varying intensities. For the caption, please see Figure 1.

Figure 6

Differential scanning calorimetry (DSC) thermograms of FBPIs treated with US and PEF at varying intensities. For the caption, please see Figure 1.