Formation of amyloid fibrils from ovalbumin under Ohmic heating

Abstract

Ohmic heating (OH) is an alternative sustainable heating technology that has demonstrated its potential to modify protein structures and aggregates. Furthermore, certain protein aggregates, namely amyloid fibrils (AF), are associated with an enhanced protein functionality, such as gelation. This study evaluates how Ohmic heating (OH) influences the formation of AF structures from ovalbumin source under two electric field strength levels, 8.5 to 10.5 and 24.0-31.0 V/cm, respectively. Hence, AF aggregate formation was assessed over holding times ranging from 30 to 1200 sunder various environmental conditions (3.45 and 67.95 mM NaCl, 80, 85 and 90 °C, pH = 7). AF were formed under all conditions. SDS-PAGE revealed that OH had a higher tendency to preserve native ovalbumin molecules. Furthermore, Congo Red and Thioflavin T stainings indicated that OH reduces the amount of AF structures. This finding was supported by FTIR measurements, which showed OH samples to contain lower amounts of beta-sheets. Field flow fractioning revealed smaller-sized aggregates or aggregate clusters occurred after OH treatment. In contrast, prolonged holding time or higher treatment temperatures increased ThT fluorescence, beta-sheet structures and aggregate as well as cluster sizes. Ionic strength was found to dominate the effects of electric field strength under different environmental conditions.

Keywords: Amyloid fibril formation; Cross-beta sheet structures; Field flow fractioning; Moderate electric fields; Protein aggregation; Thioflavin T.

Fig. 1

SDS-PAGE results showing the ovalbumin band at approx. 40 kDa at various temperatures, holding times (HOLD), heating methods (OH = Ohmic heating, COV = conventional heating) and ionic strength: (a) at low ionic strength and (b) at high ionic strength. MM = molecular marker, ctrl. = untreated ovalbumin.

Fig. 2

Congo Red (CR) spectral shift assay results for 0,5 wt% ovalbumin solutions at pH 7 heated at various temperatures, holding times, heating methods and ionic strengths: (a) at low ionic strength, and (b) at high ionic strength. Straight lines: Ohmic heating (OH) at short HOLD, dotted lines: conventional heating (COV) at short HOLD, dashed lines: OH at long HOLD, dotted and dashed lines: COV at long HOLD. Temperatures are indicated via colors as shown.

Fig. 3

Thioflavon T (ThT) staining results of 0,5 wt% ovalbumin solutions at pH 7 heated over 1200 s holding time via Ohmic heating (OH) and conventional heating (COV) and ionic strengths as indicated. Heating temperatures are (a) 80 °C, (b) 85 °C, and (c) 90 °C. Untreated ovalbumin solutions showed ThT fluorescence intensity values of approx. 3600–4400 AU.

Fig. 4

Representative field flow fractioning chromatographs of heated of 0,5 wt% ovalbumin at pH 7 and 85 °C via Ohmic or conventional heating (OH, COV). Protein solutions heated at low ionic strength and high ionic strength are shown in (a), (b), (c), and (d), (e), (f), respectively, via different heating methods as indicated. Proteins solutions heated at low ionic strength for various holding times (HOLD) as indicated are shown in (g), (h) and (i). UV = ultraviolet light detector, FL = fluorescence detector (in combination with Thioflavin T staining), MALS = Multi angle light scattering detector.

Fig. 5

Transmission electron micrographs (at 20,000× magnification) of 0,5 wt% ovalbumin solutions at pH 7 heated conventionally at 85 °C for 1200 s: heated (a) at low ionic strength, and (b) at high ionic strength. Scale bars represent 100 nm length.

Fig. 6

Representative second derivate FTIR amide I region spectra of 0,5 wt% ovalbumin solutions heated under various conditions. (a) full amide I region spectra of proteins solutions heated at short holding time under conditions as indicated, ctrl. = untreated ovalbumin. (b) low-frequency beta-sheet region of conventionally heated samples at 85 °C at low ionic strength. (c) high-frequency beta-sheet region of ohmically heated samples at low ionic strength after short holding time (HOLD). (d) low-frequency beta sheet region of conventionally heated samples at 85 °C after short HOLD (left) and long HOLD (right).

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