Affinity purification and characterisation of chelating peptides from chickpea protein hydrolysates Cristina Torres-Fuentes, Manuel Alaiz, Javier Vioque ⇑ Instituto de la Grasa (C.S.I.C), Avda Padre García Tejero 4, 41012 Sevilla, Spain article info Article history: Received 3 February 2011 Received in revised form 23 March 2011 Accepted 28 April 2011 Available online 4 May 2011 Keywords: Chelating peptides Chickpea Protein hydrolysate Pepsin Pancreatin abstract A chickpea protein hydrolysate produced with pepsin and pancreatin was used for the affinity purifica- tion of chickpea chelating peptides. Three chelating peptide fractions were obtained after affinity chro- matography with immobilised copper. These peptide fractions showed a higher chelating activity and histidine contents than the original protein hydrolysate. Chelating activity was positively correlated with the histidine content of the purified fractions. Different subfractions were also obtained after gel filtration chromatography from the affinity purified peptide fractions. Some of these subfractions showed a higher chelating activity and histidine contents than the original fractions. These results suggest that a combi- nation of high His contents, around 20–30%, and small peptide size provide the best chelating activities. Thus sequential purification with affinity and gel filtration chromatography is a useful procedure for the purification of chickpea peptides with high chelating activity. These results show that a range of chelating peptides are generated during digestion of the chickpea proteins that, after metal chelation, may prevent the generation of reactive oxygen species (ROS) and favour metal absorption. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Chickpea (Cicer arietinum L.), is an important source of proteins for human nutrition in developing countries. Its protein content ranges from 15% to 25%, with a high nutritional quality. Chickpea is extensively grown in different parts of the world such as India, Mexico and the Mediterranean Region. About 20% of chickpea seeds are damaged during harvesting and processing, and is con- sidered as a by-product that is sold at low prices for livestock feeding (Ulloa, Valencia, & García, 1988). However, these seeds may be an interesting source of proteins for the production of protein hydrolysates (Clemente et al., 1999) and bioactive pep- tides (Megías et al., 2004). Bioactive peptides are small protein fragments, with beneficial biological activity, after they are released during gastrointestinal digestion or by previous in vitro protein hydrolysis (Gobbetti, Stepaniak, De Angelis, Corsetti, & Di Cagno, 2002; Pihlanto & Korhonen, 2003; Vioque et al., 2000). Bioactive peptides have been studied mainly in milk and derived products such as cheese or yogurt. In addition, their existence has been observed in other animal and plant proteins such as soy, rice, chickpeas and even in fungi. Bioactive peptides with different functions such as antihypertensive, immunomodulatory, opioid, antioxidant, hypocholesterolemic, or metal chelating activity have been de- scribed (Gobbetti et al., 2002; Vioque et al., 2000). Metals are capable of producing reactive oxygen species (ROS) causing damage to biomolecules, diseases such as cardiovascular disease, atherosis, cancer, neurological degenerative diseases, and others (Gaetke & Chow, 2003). In addition, ROS have a negative im- pact in flavour, texture, nutritive value, as well as shelf life of food products and, under extreme conditions, produce toxins. Chelating peptides may prevent this pro-oxidant effect by metal ion chela- tion. Also, the bioavailability of minerals represent a nutritional problem for different sectors of the population such as children, the elderly, and women in the first world. Food fortification with the addition of minerals in the form of salts has been proposed to reduce these deficiencies (Zhu, Glahn, Yeung, & Miller, 2006). However, this has some limitations such as low solubility, oxida- tive reactions and modification of the flavour and colour of the for- tified food. Thus the addition of minerals in the form of chelates may improve minerals bioavailability. Chelating peptides may be an alternative to increase mineral bioavailability. Thus, the positive effect of chelating phosphopeptides, derived from milk proteins, on the absorption of minerals such as calcium (Cross, Huq, & Reynolds, 2007), zinc (Miquel & Farre, 2007) and iron (Bouhallab et al., 2002) have been reported. Hence, chelating peptides, purified from vegetables protein hydrolysates, may also facilitate the bioavailability of these minerals. Bioactive peptides are usually purified from protein hydroly- sates by several chromatographic steps including FPLC gel filtration 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.04.103 ⇑ Corresponding author. Tel.: +34 954611550; fax: +34 954616790. E-mail address: jvioque@ig.csic.es (J. Vioque). Food Chemistry 129 (2011) 485–490 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem