1169Protein extraction of Pereskia aculeata leaves – effects of ultrasounds and Ohmic heating on their physicalchemical and acid gelling properties

Naaman Francisco Nogueira Silva1*, Lucas Sales Queiroz2, Livia Andrade Santiago3 , Ipek Altay2, Lou Robert2, Yuri Kominami4 , Isabelle Cristina de Oliveira Neves1, Aberham Hailu Feyissa2, Rodolphe Marie5 , Jens Slot2, Mohammad Amin Mohammadifar2, Federico Casanova2

1Center of Natural Sciences, Federal University of São Carlos (UFSCar), Buri, SP, 18290-000, Brazil.
2Research Group for Food Production Engineering, National Food Institute, Technical University of Denmark, Søltofts Plads, 2800 Kongens Lyngby, Denmark.
3Department of Food Science and Nutrition, School of Food Engineering, University of Campinas, 13083-862 Campinas, Brazil.
4Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 1138657, Japan.
5Department of Health Technology, Technical University of Denmark, Ørsteds plads, 2800 Kongens Lyngby, Denmark

Leaf proteins are gaining increasing attention as sustainable, nutrient-rich alternatives to animal and legume proteins. Among them, the leaves of Pereskia aculeata Miller (PAM) stand out due to their high protein content (14.3–29.0% dry basis) and favorable nutritional profile. This study pursued two objectives: (i) to extract PAM leaf proteins (PAM-LP) from PAM leaf flour (PAM-LF) by alkaline solubilization followed by isoelectric precipitation, and (ii) to investigate how ultrasound (US), Ohmic heating (OH), and conventional thermal treatment (TT) influence the physicochemical and acid-gelling properties of the resulting PAM protein concentrate (PAM-PC). PAM-LF was dispersed in 0.1 M NaOH, centrifuged, and the recovery supernatant was acidified to pH 3.0. This step was followed by multiple centrifugations in which the supernatants were replaced with acidified water (pH 3.0). The final pellet was solubilized in water, adjusted to pH 7.0, and freeze-dried to obtain the PAM-PC. Aqueous suspensions of PAM-PC (2% w/w protein) were then treated by US (20 kHz, 80% of 550 W, 5-s on/off pulses, 10 min), OH (31 V/cm to reach 90 °C for min, kept by 10 pulses), or TT (90 °C for 5 min in sealed bottles). The resulting samples - Control, US, OH, and TT - were evaluated for zeta potential, isoelectric point, particle-size distribution, protein solubility, viscosity, and thermal behavior. Acid-induced gelation was assessed by adding glucono-δ-lactone (GDL) and analyzing pH kinetics, oscillatory rheology (amplitude and frequency sweeps), confocal microscopy, and water-holding capacity. The extraction procedure yielded 27% protein recovery and increased protein content from 15.3% in the flour to 55.4% in the concentrate. The amino acid profile confirmed that PAM-PC retained the favorable essential amino acid composition of PAM-LF and showed a higher proportion of sulfur-containing essential amino acids. Although PAM-LF is rich in calcium, 97% of this mineral was removed during extraction. SDS-PAGE revealed molecular masses of proteins consistent with RuBisCO (approximately 15 and 55 kDa), and FTIR spectroscopy showed a predominance of β-sheet structures. PAM-LP exhibited a negative charge of ≈ −35 mV at neutral pH and a low isoelectric point (≈ pH 2.0). Particle-size measurements indicated three particle populations in Control and US samples, whereas OH and TT suspensions contained only larger fractions (≈ 5 and 45 µm), reflecting heat-induced aggregation. Nevertheless, protein solubility was remarkably high (>92%) across all treatments. All samples displayed similar denaturation temperatures near 80 °C. Compared with US, both OH and TT increased viscosity, reduced denaturation enthalpy, and enhanced acid-gel elasticity. Indeed, acid-induced gels displayed gel-like (“weak-gel”) behavior, with elastic moduli exceeding viscous moduli and both moduli depending on frequency (Figure 1). Confocal microscopy supported this profile, revealing dispersed protein aggregates instead of a continuous network. Water-holding capacity of the gels was low for all samples, consistent with their weak-gel nature, the low protein concentration used (2% w/w), and final pH of the (~4.1), well above the isoelectric point of PAM-LP (~pH 2.0). Overall, PAM-LP showed efficient extractability, high solubility, and the ability to form acid gels, reinforcing their potential for incorporation into diverse food applications.

Acknowledgement

: This work was supported by the São Paulo Research Foundation (FAPESP) under the Grant 2022/10274-0

Acid gelation kinetics after GDL addition
Figure 1 (A–D) Acid gelation kinetics after GDL addition: storage modulus (G′) is shown as red circles (Control), blue squares (US), black diamonds (OH), and green triangles (TT); the corresponding loss modulus (G″) is shown as inverted grey triangles. (a–d) Frequency sweep tests: G′ (open symbols) and G″ (solid black symbols) are represented using the same color code (Control: red circles; US: blue squares; OH: black diamonds; TT: green triangles). The secondary Y-axis displays the loss tangent (tan δ = G″/G′) as a function of frequency. Lines are only a guide to the eye.