Browsing by Author "Gilbert EP"

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    Corrigendum to "Kinetics of pepsin-induced hydrolysis and the coagulation of milk proteins" (J. Dairy Sci. 105:990-1003)
    (Elsevier Inc on behalf of the American Dairy Science Association, 2023-11) Yang M; Ye A; Yang Z; Everett DW; Gilbert EP; Singh H
    In equation [3] (page 994), “1−” was placed incorrectly. The corrected equation reads as follows: [Formula Presented] The authors regret the error.
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    Effect of ingestion temperature on the pepsin-induced coagulation and the in vitro gastric digestion behavior of milk
    (Elsevier Ltd, 2023-05) Yang M; Ye A; Yang Z; Everett DW; Gilbert EP; Singh H
    Pepsin-induced protein coagulation occurs in the gastric environment when the milk pH is above the isoelectric point of casein proteins. In this study, the effect of milk temperature (4–48 °C) on the hydrolysis of κ-casein by pepsin and the consequent protein coagulation was studied at pH 6.0 for 120 min. Quantitative determination of the released para-κ-casein showed that both the κ-casein hydrolysis reaction rate constant and the pepsin denaturation rate constant increased with an increase in temperature. The temperature coefficient (Q10) of the specific hydrolysis of κ-casein was calculated to be ∼1.95. The coagulation process was investigated by the evolution of the storage modulus (Gʹ). At higher temperature, the milk coagulated faster but had a lower firming rate and Gʹmax with larger aggregates and voids were observed. The digestion behavior of the milk ingested at 4 °C, 37 °C, or 50 °C was investigated for 240 min in a human gastric simulator, in which the milk temperature increased or decreased to 37 °C (body temperature) over ∼ 60 min. The coagulation of the 4 °C milk was slower than for the 37 °C and 50 °C milk. The curd obtained from the 4 °C milk had a looser and softer structure with a significantly higher moisture content at the initial stage of digestion (20 min) which, in turn, facilitated the breakdown and hydrolysis of the caseins by pepsin. During the digestion, the curd structure became more cohesive, along with a decrease in moisture content. The knowledge gained from this study provides insight into the effect of temperature on the kinetics of pepsin-induced milk coagulation and the consequent digestion behavior.
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    Fibrillisation of faba bean protein isolate by thermosonication for process efficacy: Microstructural characteristics, assembly behaviour, and physicochemical properties
    (Elsevier Ltd, 2024-09) Hu Y; Cheng L; Gilbert EP; Loo TS; Lee SJ; Harrison J; Yang Z
    The effect of thermosonication (TS) (90 °C, 10–30 min) on the fibrillisation of faba bean protein isolate (FPI) was studied. The self-assembly behaviour, microstructural characteristics and techno-functional (gelation and emulsification) properties of FPI fibrils obtained from TS treatment were compared with those obtained from conventional prolonged heating (CH) at 90 °C up to 8 h. Compared to CH treatment, TS treatment was shown to significantly accelerate the formation of FPI fibrils with prominent β-sheet structures as revealed by Thioflavin T (ThT) fluorescence, Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD). The characteristics of fibril building blocks were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) to obtain the differences between TS and CH induced fibrillisation of FPI. Transmission electron microscopy (TEM) and small-angle neutron scattering (SANS) showed that 4 h CH and 10 min TS treatments resulted in the fibrils with similar radius (from 5 to 10 nm). Furthermore, SANS indicated that TS treatment induced the formation of an entangled FPI fibrillar network, which could lead to the observed viscoelastic properties of FPI at a high concentration (10 wt%). Finally, high internal phase O/W emulsions (HIPE, φ = 0.75) stabilised by 30 min TS induced FPI fibrils (3 wt%) demonstrated a stronger gel strength and smaller oil droplet size compared to those prepared with untreated FPI, suggesting a superior emulsification capability of FPI fibrils. This finding demonstrates that TS treatment is a promising and efficient method for fibrillisation of plant proteins with the resultant fibrils generating excellent gelation and emulsification properties.
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    Impact of thermosonication at neutral pH on the structural characteristics of faba bean protein isolate dispersions and their physicochemical and techno-functional properties
    (Elsevier Ltd, 2024-09) Hu Y; Cheng L; Gilbert EP; Lee SJ; Yang Z
    The effect of thermosonication (TS) (90 °C, 10–30 min) on faba bean protein isolate (FPI) at pH 7 was investigated. The microstructural and techno-functional properties of TS-treated FPI were compared with native FPI or FPI treated with conventional prolonged heating (CH, up to 8 h) at 90 °C. TS treatment effectively converted FPI to amorphous aggregates containing predominant β-sheet secondary structures, as determined by Thioflavin T (ThT) fluorescence and circular dichroism (CD). According to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), these amorphous aggregates could be formed by disulfide bonds. Additionally, TS treatment is efficient in disrupting large protein aggregates of FPI, thus improving their solubility. Both TS and CH treatments induced formation of viscoelastic FPI hydrogels, whose gel strength depends on the type and time of treatment. Hydrogels formation is likely to arise from the entanglement and interaction of protein aggregates as revealed by small angle neutron scattering (SANS) and scanning electron microscopy (SEM). TS-treated FPI was also used to prepare O/W emulsions and whose structural and physical properties were compared with those stabilised by untreated FPI. At all oil volume fractions (φ = 0.2, 0.5, and 0.7) and FPI concentrations (1, 3, and 5 wt %), emulsions stabilised by TS-treated FPI exhibited smaller oil droplet size, greater mechanical strength and superior stability compared to those stabilised by untreated FPI. The study suggests that TS treatment is promising in improving techno-functional properties of FPI; further studies are needed to exploit TS-treated plant proteins as a novel food ingredient in food product development.
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    Kinetics of pepsin-induced hydrolysis and the coagulation of milk proteins
    (Elsevier Inc and the Federation of Animal Science Societies on behalf of the American Dairy Science Association, 2022-02) Yang M; Ye A; Yang Z; Everett DW; Gilbert EP; Singh H
    Hydrolysis-induced coagulation of casein micelles by pepsin occurs during the digestion of milk. In this study, the effect of pH (6.7–5.3) and pepsin concentration (0.110–2.75 U/mL) on the hydrolysis of κ-casein and the coagulation of the casein micelles in bovine skim milk was investigated at 37°C using reverse-phase HPLC, oscillatory rheology, and confocal laser scanning microscopy. The hydrolysis of κ-casein followed a combined kinetic model of first-order hydrolysis and putative pepsin denaturation. The hydrolysis rate increased with increasing pepsin concentration at a given pH, was pH dependent, and reached a maximum at pH ~6.0. Both the increase in pepsin concentration and decrease in pH resulted in a shorter coagulation time. The extent of κ-casein hydrolysis required for coagulation was independent of the pepsin concentration at a given pH and, because of the lower electrostatic repulsion between para-casein micelles at lower pH, decreased markedly from ~73% to ~33% when pH decreased from 6.3 to 5.3. In addition, the rheological properties and the microstructures of the coagulum were markedly affected by the pH and the pepsin concentration. The knowledge obtained from this study provides further understanding on the mechanism of milk coagulation, occurring at the initial stage of transiting into gastric conditions with high pH and low pepsin concentration.
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    Probing structural modification of milk proteins in the presence of pepsin and/or acid using small- and ultra-small-angle neutron scattering
    (Elsevier Ltd, 2025-02) Yang M; Ye A; Yang Z; Everett DW; de Campo L; Singh H; Gilbert EP
    Acid- and pepsin-induced milk protein coagulation plays a crucial role in the gastric digestion of milk. Real-time structural evolution at a nano- (e.g. colloidal calcium phosphate (CCP) and micelle) and micro- (gel network) level of unheated and heated (85 °C for 30 min) bovine milk was examined under acidic conditions and at low and high concentrations of pepsin using ultra-small- and small-angle neutron scattering (USANS and SANS), small-amplitude oscillatory rheometry and confocal scanning laser microscopy. Milk was treated with glucono-δ-lactone (GDL), pepsin or a combination of GDL and pepsin to induce coagulation. Heat-treated milk showed a faster increase in elastic storage modulus (G′) and scattering intensity (USANS and SANS) compared with unheated milk when coagulated with GDL or the combination of GDL and pepsin. At pH 6.3, heat treatment retarded pepsin (1.10 U/mL)-induced milk coagulation, with slower increases in G′ and scattering intensity. At a high concentration of pepsin (2000 U/mL) that mimics the concentration found in the stomach, general proteolysis followed coagulation. Heat treatment retarded coagulation but accelerated curd proteolysis. This study demonstrates how time-resolved USANS and SANS can be used to investigate the structural evolution of protein coagulation and degradation under gastric environment conditions at nano- and micro-metre length scales.
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    Self-assembly and hydrogelation of a potential bioactive peptide derived from quinoa proteins
    (Elsevier BV, 2024-02) Cheng L; De Leon-Rodriguez L; Gilbert EP; Loo T; Petters L; Yang Z
    In this work the identification of peptides derived from quinoa proteins which could potentially self-assemble, and form hydrogels was carried out with TANGO, a statistical mechanical based algorithm that predicts β-aggregate propensity of peptides. Peptides with the highest aggregate propensity were subjected to gelling screening experiments from which the most promising bioactive peptide with sequence KIVLDSDDPLFGGF was selected. The self-assembling and hydrogelation properties of the C-terminal amidated peptide (KIVLDSDDPLFGGF-NH2) were studied. The effect of concentration, pH, and temperature on the secondary structure of the peptide were probed by circular dichroism (CD), while its nanostructure was studied by transmission electron microscopy (TEM) and small-angle neutron scattering (SANS). Results revealed the existence of random coil, α-helix, twisted β-sheet, and well-defined β-sheet secondary structures, with a range of nanostructures including elongated fibrils and bundles, whose proportion was dependant on the peptide concentration, pH, or temperature. The self-assembly of the peptide is demonstrated to follow established models of amyloid formation, which describe the unfolded peptide transiting from an α-helix-containing intermediate into β-sheet-rich protofibrils. The self-assembly is promoted at high concentrations, elevated temperatures, and pH values close to the peptide isoelectric point, and presumably mediated by hydrogen bond, hydrophobic and electrostatic interactions, and π-π interactions (from the F residue). At 15 mg/mL and pH 3.5, the peptide self-assembled and formed a self-supporting hydrogel exhibiting viscoelastic behaviour with G' (1 Hz) ~2300 Pa as determined by oscillatory rheology measurements. The study describes a straightforward method to monitor the self-assembly of plant protein derived peptides; further studies are needed to demonstrate the potential application of the formed hydrogels in food and biomedicine.