Abstract
The thesis aimed to understand the effect of pH combined with either heat or pulsed electric fields (PEF) processing on protein aggregation and subsequent pepsin-pancreatin hydrolysis of ovomucin-depleted egg white (OdEW) solutions. The OdEW hydrolysates obtained from the simulated in vitro gastrointestinal digestion were assessed for their antioxidant and anti-inflammatory activity through in vitro experiments.
OdEW solutions (10% v/v, pH 4, 5, 7, and 9) were exposed to either isothermal heating at 50-85°C for 10 min or PEF at 1.4-1.8 kV/cm under low (259-288 kJ/kg), intermediate (453-503 kJ/kg), and high (653-695 kJ/kg) specific energy input. Solution turbidity was spectrophotometrically determined and distribution of proteins in soluble and insoluble fractions of OdEW solutions was analyzed using qualitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). OdEW proteins were more prone to both heat- and PEF-induced protein aggregation at pH 5 than at other pH values studied. OdEW solutions showed no or minimal turbidity at low or intermediate PEF energy inputs. At the highest PEF energy input, OdEW solutions varied in their turbidity which was similar or lower than solution turbidity after heat treatment at 60°C, depending on solution pH. Heat treatment at 65°C and above resulted in s higher turbidity in the OdEW solutions.
Upon simulated gastrointestinal digestion, protein digestibility of untreated and treated OdEW solutions was determined by measuring the production of peptides during hydrolysis. PEF treatment at 695 kJ/kg (1.7 kV/cm) at pH 4 significantly enhanced the digestibility of OdEW proteins. Heat treatment did not affect protein digestibility at 60°C but significantly enhanced the digestibility of OdEW proteins at 80°C. Furthermore, OdEW solutions at pH 4, exposed to PEF at 695 kJ/kg (1.7 kV/cm) or heat at 80°C (10 min), had a similar protein digestibility, with PEF treatment resulting in a much lower solution turbidity. Qualitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to monitor changes in proteolysis patterns of major OdEW proteins following either heat or PEF treatment. Ovotransferrin was more susceptible to pepsin hydrolysis than lysozyme. Ovalbumin showed the highest proteolysis resistance to both pepsin and pancreatin. Qualitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS) was utilized to identify both intact proteins and hydrolyzed protein fragments during the digestion. Ovalbumin produced several proteolysis fragments, and a ~ 40 kDa fragment showed comparable digestive stability to intact ovalbumin, persisting till the end of pancreatin hydrolysis.
Antioxidant activity of the OdEW hydrolysates was determined using 2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC), 2,2-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and Ferric reducing antioxidant power (FRAP) methods. PEF treatment of OdEW solutions at pH 4 and 695 kJ/kg (1.7 kV/cm) enhanced the antioxidant activity of OdEW hydrolysates, to similar or even higher levels than heat treatment at 80°C (10 min), by the DPPH or ORAC method. PEF (1.7 kV/cm, 695 kJ/kg) or heat (80°C, 10 min) treatments of OdEW solutions at pH 4 significantly enhanced the anti-inflammatory activity of OdEW hydrolysates. At a concentration of 1 mg/mL, OdEW hydrolysates showed 35.9% and 35.5% inhibition of interleukin-8 (IL-8) production of lipopolysaccharide-stimulated HT-29 cells due to prior heat and PEF treatment, respectively.
This study demonstrated that PEF treatment of the thermolabile OdEW solutions had advantages over conventional heat treatment as it enabled the retention of high solution clarity, while significantly enhancing the gastrointestinal digestion of OdEW proteins. PEF treatment provides the potential for wider utilization of OdEW including the use in protein-fortified drinks with desirable qualities such as high visual clarity and good digestibility.