Effects of cationic property on the in vitro antioxidant activities of pea protein hydrolysate fractions
Introduction
Functional foods and nutraceuticals have the potential to improve the human health status as well as support economic growth in rural communities. A major focus of the functional foods and nutraceuticals market is antioxidants, as damages caused by reactive oxygen species is considered to be the basis for the progression of aging and several diseases including chronic kidney disease (Oberg et al., 2004), cardiovascular disease, cancer and neurodegenerative diseases (Seifried, Anderson, Fisher, & Milner, 2007). A wide variety of food protein sources including animal (Li, Jiang, Zhang, Mu, & Liu, 2008), plant (Zhang et al., 2008, Udenigwe et al., 2009) and marine (Theodore, Raghavan, & Kristinsson, 2008) have been identified to possess antioxidant peptides. Food protein-derived bioactive peptides have been shown to have strong antioxidant properties, with the ability to scavenge hydroxyl radicals (˙OH), superoxide anion radicals (O2˙−), hydrogen peroxide (H2O2) and the chelation of metal ions. However, the structure–function relationships between peptide characteristics and antioxidant activity have not been fully elucidated (Je, Qian, Lee, Byun, & Kim, 2008). It is known that amino acids such as histidine, cysteine, tyrosine, phenylalanine and tryptophan, and increased total amino acid hydrophobicity contribute to the increase in the antioxidant capacity of food protein-derived peptides (Peña-Ramos et al., 2004, Erdmann et al., 2008, Pownall et al., 2010). However, there is a need to provide more information on the relationships between the net charge properties of amino acid components of peptides and the ability of the peptides to scavenge free radicals and act as effective antioxidants.
Pea seed is considered a significant source of protein, consisting of about 25% protein by weight and has comparable amino acid profile to other commonly consumed legumes (Iqbal, Khalil, Ateeq, & Sayyar Khan, 2006). Yellow pea seed is an important commodity to Canada's economy, specifically to the economy of the Prairie Provinces, Manitoba, Saskatchewan and Alberta as Canada is the world's largest producer and exporter of pea seed (Pulse Canada, 2010). The abundance of yellow field peas in Canada is presently driving some research investigations (Li and Aluko, 2005, Li and Aluko, 2006) that explore other benefits of pea seed protein-derived peptides beyond their basic nutritional uses. A previous study has shown that pea protein hydrolysates (PPH) contain low molecular weight (< 3 kDa) peptides with various antioxidant activities that were dependent on the amounts of hydrophobic and aromatic amino acid constituents (Pownall et al., 2010). The objectives of this study were to separate the < 3 kDa PPH on a cation-exchange chromatography column, collect fractions based on their retention properties, and determine the effects of amino acid composition on various antioxidant properties. Antioxidant properties of the peptide fractions were compared with those of reduced glutathione (GSH), since the latter is a physiologically relevant antioxidant peptide. Findings from the present study could contribute to a better understanding of the structure–function relationship of food protein-derived antioxidant peptides.
Section snippets
Materials
Pea protein isolate was a gift from Nutri-Pea Ltd, Portage La Prairie, Manitoba, Canada. Thermolysin (from Bacillus thermoproteolyticus rokko), pyrogallol (1,2,3-trihydroxybenzene) 2,2-diphenyl-1-picrylhydrazyl (DPPH), Triton X-100, hydrogen peroxide, potassium ferricyanide, GSH, ammonium thiocyanate, linoleic acid, 1,10-phenanthroline and 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4″-disulphide acid sodium salt (ferrozine) were purchased from Sigma Chemicals (St. Louis, MO). All other
Cation-exchange column fractionation and amino acid profiles of peptides
The < 3 kDa PPH was fractionated using cation-exchange column accompanied by collection into five fractions that eluted at different times and hence possess differences in net cationic charge (Fig. 1). The principle of cation-exchange chromatography is based on affinity of the peptides for the stationary phase. The binding affinity of the peptides will depend on their net charges, which is in turn dependent on the constituent amino acids and ultimately pH of the mobile phase (Aluko, 2004). The
Conclusions
Overall, the results showed that antioxidant properties of peptides could be influenced by their cationic properties, though the effects of other properties of the amino acids cannot be totally excluded. Fractionation according to cationic properties could help concentrate active peptides as evident by the increased superoxide scavenging properties of the peptide fractions when compared to the un-fractionated protein hydrolysate. In contrast such fractionation according to charge was actually
Acknowledgments
The research was supported by the Advanced Foods and Materials Network of Centres of Excellence (AFMNet NCE) Canada, and Natural Sciences and Engineering Research Council of Canada (NSERC). The authors would like to thank Nutri-Pea Ltd, Portage La Prairie, Manitoba for the donation of the pea protein isolate.
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