Jojoba Seed Meal Proteins Associated with Proteolytic and Protease Inhibitory Activities MADAN K. SHRESTHA, ² IRENA PERI, ²,‡ PATRICIA SMIRNOFF, ‡ YEHUDITH BIRK, ‡ AND AVI GOLAN-GOLDHIRSH* ,² Desert Plant Biotechnology Laboratory, Albert Katz Department of Dryland Biotechnologies, The Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boker Campus 84990, Israel, and Institute of Biochemistry, Food Science & Nutrition, The Hebrew University of Jerusalem, Rehovot 76100, Israel The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin- inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins. KEYWORDS: Jojoba meal protein; Simmondsia chinensis; solubility; protease inhibitory activity; digestibility INTRODUCTION Jojoba (Simmondsia chinensis (Link) Schneider) is a shrub indigenous to the Sonoran deserts of Arizona, California, and Mexico. The jojoba was introduced as a cultivated crop for the commercially useful liquid wax in its seeds. The meal remaining after oil extraction is rich in protein (1). The use of the meal proteins for food and feed is limited because it contains antinutritional factors such as simmondsin and its derivatives (2), heat labile trypsin inhibitors (3), and condensed tannins with antitryptic activity (4). Research has already been carried out to remove the antinutritional factors in order to utilize the jojoba proteins for animal consumption (1, 5-9). Jojoba proteins were fractionated according to solubility by Cardoso and Price (10). Abbott et al. (1) reported a process for preparation of a water- soluble protein isolate. The presence of several endopeptidases in jojoba seed was noted by Samac and Storey (3), and further fractionation of these enzymes was performed by Wolf and Storey (11). Recently it was suggested that a trypsin inhibitory activity might be responsible for growth retardation in rats fed jojoba proteins (9), but a more detailed analysis of jojoba meal proteins and their characterization is lacking. In addition, there is increasing interest in recovering the jojoba meal proteins for industrial, cosmetic, and food uses, as well as to avoid disposal problems and environmental hazards after oil extraction. In view of the recent interest in plant protease inhibitors as built-in defense mechanisms against stored-product insect proteases (12) and as possible cancer chemopreventive agents, the jojoba meal proteins offer a source for the biotechnological uses of proteases and protease inhibitors. In this paper we describe the isolation of major jojoba meal proteins, their in vitro digestibility, and the identification of serine protease inhibitory activity of a major 25 kDa jojoba meal protein, Joj25. MATERIALS AND METHODS Plant Material. Jojoba (Simmondsia chinensis (Link) Schneider) press-defatted meal was obtained by an industrial process (kindly provided by “Jojoba Israel”, Kibbutz Chatzerim). Fractionation of Meal Proteins. The meal proteins were extracted according to solubility by two methods (A, B) shown in Figure 1. In method A the starting material was used as obtained from the factory and in method B the meal was further defatted by solvent extraction as detailed in Figure 1B. Fractionation of the meal proteins was performed by submitting the albumins or globulins obtained by method B to column chromatography on Q-sepharose (Pharmacia Biotech Ab, Uppsala, Sweden). The proteins were eluted from the column by a stepwise gradient of 50 mM NaCl followed by 100 mM NaCl, and 200 mM salt, in 20 mM Tris/HCl buffer, pH 8.0. The eluate was monitored spectrophotometrically at 280 nm. Pooled protein fractions were dialyzed and lyophilized. The fraction containing Joj25 peak was applied to HPLC-Superdex 75 10/30 column (Pharmacia Biotech Ab, Uppsala, Sweden) and eluted by 50 mM phosphate buffer (NaH2PO4‚ H2O) pH 7.4, containing 150 mM NaCl. Molecular Weight Estimation. SDS-PAGE was performed ac- cording to Laemmli (13). The protein bands were either stained with Coomassie brilliant blue or electroblotted onto a nitrocellulose mem- * To whom correspondence should be addressed. Phone: 972-8-6596753. Fax: 972-8-6596742. E-mail: avigolan@bgumail.bgu.ac.il. ² Ben-Gurion University of the Negev. ‡ The Hebrew University of Jerusalem. 5670 J. Agric. Food Chem. 2002, 50, 5670-5675 10.1021/jf020161h CCC: $22.00 © 2002 American Chemical Society Published on Web 08/30/2002