Please cite this article in press as: Esti, M., et al., Immobilized pineapple stem bromelain activity in a wine-like medium: Effect of inhibitors. Food Bioprod Process (2013), http://dx.doi.org/10.1016/j.fbp.2013.12.001 ARTICLE IN PRESS FBP-460; No. of Pages 6 food and bioproducts processing x x x ( 2 0 1 3 ) xxx–xxx Contents lists available at ScienceDirect Food and Bioproducts Processing j ourna l ho me page: www.elsevier.com/locate/fbp Immobilized pineapple stem bromelain activity in a wine-like medium: Effect of inhibitors Marco Esti a , Ilaria Benucci a,∗ , Katia Liburdi a , Anna Maria Vittoria Garzillo b a Department for Innovation in Biological, Agro-Food and Forest Systems, University of Tuscia, via S. Camillo de Lellis, 01100 Viterbo, Italy b Department of Ecological and Biological Sciences, University of Tuscia, via S. Camillo de Lellis, 01100 Viterbo, Italy a b s t r a c t The catalytic activity of stem bromelain, covalently immobilized on chitosan beads, was characterized towards a synthetic substrate (Bz-Phe-Val-Arg-pNA), in a wine-like acidic medium containing wine inhibitors (ethanol, sulphur dioxide, grape skin and seed tannins), at their average concentration range. For all tested substances, the inhibition constant (K i ) values of immobilized bromelain were significantly higher than the corresponding values obtained in a previous work for free enzyme, thus indicating that direct covalent immobilization on chitosan beads makes protease more resistant to the inhibition effect. Immobilized protease was affected by ethanol inhibition (competitive type) only if present at abnormal concentra- tion for real wine. Grape skin and seed tannins exerted uncompetitive inhibition to the same extent, with a K i value close to 1 g l -1 gallic acid eq . The effect of free sulphur dioxide on immobilized bromelain changed respect to free enzyme, both becoming uncompetitive inhibitor and dramatically lowering its strength. These results show that immobilized stem bromelain could have productive biotechnological applications in wine- making, even though further studies will be necessary to test its proteolytic activity towards wine proteins in real matrices. © 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Pineapple stem bromelain; Covalent immobilization; Wine inhibitors; Wine-like medium 1. Introduction The addition of adsorbent materials, such as bentonite, is a non-specific common practice, applied in wine industry for the prevention of protein haze. Despite its effectiveness, bentonite fining adversely affect the quality of treated wine, through the removal of colour, flavour and texture compounds (Waters et al., 2005). The possibility of stabilizing white wine by acid proteases, able to hydrolyze specifically proteins and inert respect to other wine compounds, appears of great interest. Neverthe- less, currently, enzyme application, in free form, is limited by the increased request of additive-free food, prevailing in some European countries (Feijoo-Siota and Villa, 2011). For this rea- son, growing attention has been given to the application of proteases immobilized on solid support, which could be apply ∗ Corresponding author. Tel.: +39 0761357426; fax: +39 0761357498. E-mail address: ilaria.be@tiscali.it (I. Benucci). Received 14 February 2013; Received in revised form 4 November 2013; Accepted 2 December 2013 for wine protein stabilization, by a continuous treatment, in different bioreactor configurations with no enzyme resid- ual contamination. Stem bromelain, a cysteine proteinase extracted from the stem of pineapple plant, has been immo- bilized on various supports (organic and inorganic) and by different methods (Seo et al., 1998; Tan et al., 2008; Shi et al., 2007; Krajewska, 2004; Mahmood and Saleemuddin, 2007; Gupta et al., 2007). Enzyme immobilization, achieved by fixing a biocatalyst to or within solid supports, is of great interest for applications in the food industry. Compared with their free disposable forms, immobilized enzymes avoids product contamination in the food reaction mixture, allowing their reusability (Yodoya et al., 2003). Moreover, immobilization by covalent linkage could induce enzyme structure modification to overcome the inhibitory 0960-3085/$ – see front matter © 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fbp.2013.12.001