Kiwifruit (Actinidia deliciosa) extract shows potential as a low-cost and efficient milk-clotting agent Milica M. Grozdanovic a, * , Lidija Burazer b , Marija Gavrovic-Jankulovic a a Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentskitrg 12,11000 Belgrade, Serbia b Allergy Department, Institute of Immunology and Virology, VojvodeStepe458,11152 Belgrade, Serbia article info Article history: Received 25 October 2012 Received in revised form 12 February 2013 Accepted 2 March 2013 abstract Actinidin, a cysteine protease accounting for more than 50% of the soluble proteins in kiwifruit pulp, has shown promise as a milk-clotting agent. In this study, the potential use of kiwifruit pulp extract as a clotting agent was investigated. It was shown that three kiwifruit extracts made from the pulp of a single fruit have significantly different levels of active actinidin, depending on the extraction buffer employed. Kiwifruit extract prepared at pH 5.0 had the best milk-clotting properties, with a nearly 30% better ratio of clotting activity to proteolytic activity than purified actinidin. This extract produced a casein coagulum clearly separated from the whey proteins, and was shown to be stable at room temperature for up to two months. This extract has the potential to be employed as an efficient and low-cost milk-clotting agent in the production of dairy products. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Milk coagulation is a basic step in cheese manufacturing and is most commonly achieved by addition of chymosin (rennet), a milk- clotting enzyme preparation obtained from the stomach contents of the unweaned calf (Lopes, Teixeira, Liberato, Pais, & Clemente, 1998). The worldwide increase in cheese production, alongside with the reduced supply of calf rennet and higher prices, have led to an increase in the demand for alternative sources of milk co- agulants (Cavalcanti, Teixeira, Lima Filho, & Porto, 2004; Katsaros, Tavantzis, & Taoukis, 2010). In addition, the use of animal rennet has consumer constraints due to religious reasons (e.g., Judaism and Islam), diet (vegetarianism), or bans on genetically engineered food (Roseiro, Barbosa, Mames, & Wilbey, 2003). For these reasons, enzymes extracted from plants have become a subject of growing interest in dairy technology. Milk-clotting enzymes have been identified in various plant species; unfortunately, many of these enzyme preparations were found to be unsuitable because they produce extremely bitter cheeses (Egitoa et al., 2007). Both the flavour and texture of cheeses are affected by the high proteolytic activity of plant proteases, which leads to the production of short peptides that are responsible for bitterness in dairy products (Lo Piero, Puglisi, & Petrone, 2011). Moreover, the toxin content of many of these plant extracts constrains their use in the food industry (Silva & Malcata, 2005). Many of the proteolytic enzymes that have been studied for their milk-clotting ability belong to the group of acidic proteases, which includes chymosin. These include the commercially used microbial milk-clotting aspartic proteases from Mucor miehei, Mucor pusillus and Cryphonectria parasitica. Pepsin-like acid proteases are also derived from Aspergillus spp., Rhizopus spp., and recently from Thermomucorindicae-seudaticae N31 (Lo Piero et al., 2011; Sumantha, Larroche, & Panday, 2006). Aspartic proteases with a pH optimum of 5.1 and milk-clotting activity have also been isolated from the flowers of Cynara cardunculus (Heimgartner et al., 1990). Lo Piero et al. (2011) evaluated the applicability of purified actinidin as a plant-derived milk coagulant. Actinidin (EC 3.4.22.14) is a cysteine protease from kiwifruit (Actinidia deliciosa) with a wide pH activity range (4e10) and wide substrate specificity (Arcus, 1959; Boland & Hardman, 1972; McDowall, 1970). Lo Piero et al. (2011) showed that actinidin has the ability to form milk clots and that the enzyme is fully compatible with conditions used in cheese manufacture (optimum activity at 40e42  C, mildly acidic pH values). Analysis of hydrolysis products produced by actinidin showed that the preferred substrate for this enzyme is b-casein, followed by k-casein, which is hydrolysed into a small number of larger peptides (Lo Piero et al., 2011). Saha and Hayashi (2001) re- ported that dairy products clotted with the use of kiwifruit juice actinidin develop less off-flavour notes which are attributed to bitter peptides occurring when the fig protease ficin (or papaya * Corresponding author. Tel.: þ381 11 3336663. E-mail address: mgrozdanovic@chem.bg.ac.rs (M.M. Grozdanovic). Contents lists available at SciVerse ScienceDirect International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj 0958-6946/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.idairyj.2013.03.001 International Dairy Journal 32 (2013) 46e52