Degree Type


Date of Award


Degree Name

Doctor of Philosophy


Animal Science

First Advisor

Dong Uk Ahn


Egg yolk phosvitin, as a highly phosphorylated protein shows high metal chelating, antioxidant and emulsifying properties. The phosphorylated serine moieties of phosvitin play major roles with regard to its extraordinary functionalities. Phosvitin is considered as a nutritionally negative protein since it forms insoluble complexes with the bivalent cations, and it shows potential allergenecity and resistance to enzymatic digestion. However, if it is fragmented, the peptides containing phosphates (phosphopeptides) can greatly enhance the mineral bio-availability, thus its negative attributes can be avoided effectively. The aims of this study were 1) to investigate effective pre-treatment(s) for improved enzymatic hydrolysis of phosvitin, 2) to evaluate the chemical digestion of phosvitin, 3) to find out the functional properties of phosvitin and its hydrolysates, and 4) to characterize the peptides of phosvitin hydrolysates using MS-MS based approach.

Pre-treated phosvitin with SDS (sodium dodecyl sulfate), SDS + heat, acid + heat, alkali+heat, and heat alone were subjected to circular dichroism to evaluate the secondary structural changes of the protein. Enzymatic digestion of phosvitin was done with trypsin, pepsin, alpha-chymotrypsin, Multifect ® P-3000, alkalase (Bacterial alkaline protease: Protex 6L), and thermolysin (from Bacillus thermoproteolyticus Rokko). Consecutive digestion of phosvitin with trypsin and pepsin, Multifect ® P-3000 & alpha-chymotrypsin, and alkalase & alpha-chymotrypsin were also performed. All the enzymatic digests of phosvitin were subjected to SDS-PAGE analysis. The chemical hydrolysis (acid and alkaline) of phosvitin was also performed for high level of hydrolysis of phosvitin as a novel approach to produce functional peptides. Phosvitin and the enzymatic hydrolysates of thermolysin, Multifect ® P-3000 and alkalase of phosvitin and pre-treated phosvitin (100 oC, 1h) were evaluated for their antioxidant and mineral-binding activities. The acid and alkali hydrolysates of phosvitin were also evaluated for their functionalities. The identification and characterization of phosphopeptides present in different hydrolysates were carried out using LCMS/MS (Liquid chromatography-Tandem Mass Spectrometry) and MALDI (Matrix-Assisted Laser Desorption Ionization) approaches.

Significant changes in -sheet and coil structures of phosvitin were caused by SDS with and without heat treatments. Phosvitin underwent alkaline hydrolysis at 70 to 85 oC at pH 13, which implied the decomposition of protein. Heat treatment at 100 oC for 20 to 60 minute significantly affected the secondary structures of phosvitin. Phosvitin subjected to heat treatment at 100 oC for 1 h showed visible differences in SDS-PAGE compared to the control sample.

Among the six enzymes tested, Multifect ® P-3000 and alkalase showed higher proteolytic digestion than the other four enzymes tested. Thermolysin was better than the other three enzymes tested. Consecutive use of enzymes did not show any further digestion of the hydrolysates obtained from the first enzyme. Even though SDS was capable of denaturing phosvitin, it did not improve the enzymatic digestion of phosvitin. Combination of alkali (pH 12) and heating at 85 oC for 30 min resulted in higher degree of enzymatic digestion possibly due to dephosphorylation of phosvitin. Pre-treatment of phosvitin at 100 oC for 1h clearly showed improved enzymatic digestion of phosvitin. Based on these results, the effective enzymatic digestion of phosvitin for bioactive peptide production can be achieved by pre-treating phosvitin at 100 oC for 1 h followed by hydrolysis using Multifect ® P-3000 or alkalase. Phosvitin showed higher level of hydrolysis in alkaline than acidic conditions. Incubation of phosvitin in 0.075 N NaOH solution at 37 oC for 1 h, and 2 N HCl at 60 oC for 6 h partially hydrolyzed phosvitin. But 0.1 N NaOH at 37 oC for 3 h or 3 N HCl at 60 oC for 6 h incubation was needed for the complete hydrolysis of phosvitin.

Phosvitin and its hydrolysates showed a powerful antioxidant effect in iron-induced oxidation in oil emulsion system. Both Fe3+-binding and Cu2+-binding efficiencies of phosvitin decreased with increasing levels of phosvitin in the assays. Hydrolysates of phosvitin showed different levels of Fe3+-binding and Cu2+-binding activities. Phosvitin significantly reduced the solubility of calcium in solution, but phosvitin hydrolysates increased calcium solubility. Preparation of hydrolysates using pre-treated phosvitin did not help in improving the antioxidant or mineral-binding capacities of hydrolysates compared with those without pre-treatments.

Increasing degree of hydrolysis in phosvitin either by HCl or NaOH negatively affected their antioxidant and Fe3+-chelating capacities. However, hydrolyzing phosvitin with 0.1 N NaOH, 2 N HCl, or trypsin after 0.05 N NaOH treatment significantly increased Cu2+-chelating activity. The acid hydrolysates of 3 N and 6 N HCl significantly increased the solubility of Ca2+ in sodium phosphate buffer (pH 6.8) compared to the negative control implying the possibility of using acid hydrolysates for functional phosphopeptides as mineral supplementing agents.

The amino acid sequences and structural information observed in this study shows the possibility of using phosvitin derived phosphopeptides in the area of bioactive/functional peptides production. However, more studies have to be implemented in order to discover the physiological mechanisms and biological effect in vivo.


Copyright Owner

Chandima Himali Samaraweera Mudiyanselage



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156 pages