248 JOURNAL OF FOOD SCIENCE—Volume 63, No. 2, 1998 Properties of Films Produced by Cross-linking Whey Proteins and 11S Globulin Using Transglutaminase M. YILDIRIM and N.S. HETTIARACHCHY ABSTRACT Transglutaminase (TG) was used to produce films from whey protein isolate, soybean 11S globulin and a mixture of the two (1:1, wt/wt). Solubility of TG cross-linked films was lower than that of control films at pH 3, 4, 6 and 8. Solubility of control films in 6.6M urea and in 10% SDS was higher than that of TG cross-linked films. Hydrolysis of control and TG cross-linked films by trypsin and -chymotrypsin was simi- lar after 24h incubation. Mean thickness of films ranged from 69 to 77 μ m and there were no differences among thick- nesses. Average tensile strength values of TG cross-linked films were two times greater than those of the homologous controls. Key Words: cross-linking, whey protein, 11S globulin films, transglutaminase INTRODUCTION TRANSGLUTAMINASE (TG) (PROTEIN-GLUTAMINE -GLUTAMYL transferase, E.C. 2.3.2.13) catalyzes the introduction of -( - glutamyl)-lysine cross-links into proteins via an acyl transfer reac- tion. The -carboxyamide group of glutamine serves as the acyl do- nor (amine acceptor) and the -amino group of lysine serves as the acyl acceptor (amine donor) (Folk, 1980). This reaction can intro- duce covalent cross-linkages between proteins as well as peptides and primary amines. When the -amino groups of lysine residues in proteins act as acyl acceptors, -(-glutamyl)-lysine bonds are formed both intra- and inter-molecularly (Folk, 1980; Zhu et al., 1995). Transglutaminase can be used to modify the functional proper- ties of food proteins, including altering solubility and functional prop- erties (Ikura et al., 1985; Matheis and Whitaker, 1987). Cross-link- ing -casein with -casein, -lactoglobulin, and soybean 11S and 7S globulins has been reported to enhance functional properties, in- cluding gelation and hydration (Motoki et al., 1984; Nio et al., 1985). The formation of covalent bonding between protein molecules by TG catalysis was very effective in forming a self-supporting net- work (Chanyongvorakul et al., 1994). Edible coatings and films can prevent quality changes in foods by acting as barriers to control transfer of moisture, oxygen and car- bon dioxide, lipid oxidation and loss of volatile flavors and aromas. Most edible coatings and films contain high-molecular-weight poly- mers, including polysaccharides, lipids or proteins (Kester and Fen- nema, 1986).  s1 -Casein film prepared by TG catalyzed cross-link- ing reaction was water insoluble, but digestible by proteolytic en- zymes. Tensile strength of this film was two times higher than that of the control (Motoki et al., 1987). Mahmoud and Savello (1992; 1993) produced whey protein films using TG activity that were di- gestible by proteolytic enzymes and showed moderate solubility in buffered solvents and in protein denaturants. Whey is a coproduct of the cheese and casein industry, and in the U.S. 60% of total whey is used for food applications and animal feed (Kinsella and Whitehead, 1989). The rest is waste effluent, resulting in a loss of food energy and an economic burden. Only a small quan- tity of soy protein in the U.S., less than 5%, is used directly in hu- man foods (Hettiarachchy and Kalapathy, 1997). Soybean proteins are composed of two major components 11S (glycinin) and 7S (- conglycinin) globulin. The 11S globulin (11S) can be purified more easily than the 7S globulin. The 11S has better nutritional value and functional properties than 7S globulin (Saio and Watanabe, 1978; Utsumi et al., 1994). Furthermore, cross-linking of 11S is faster than that of 7S globulin since the proximity of glutamyl or lysyl residues in 7S may not be easily accessible for TG activity (Ikura et al., 1980). Transglutaminase-catalyzed reactions to cross-link soybean 11S and whey protein isolate (WPI) gave biopolymers with improved functionality (Yildirim et al., 1996; Yildirim and Hettiarachchy, 1997). There is no published report on films produced by cross-linking whey proteins and soybean 11S globulin using TG catalysis. Therefore, the objectives of this research were to produce films by cross-link- ing whey protein isolate and 11S globulin using TG and to investi- gate mechanical properties, solubility and hydrolyzability of the re- sulting films. MATERIALS & METHODS Materials Transglutaminase (E.C. 2.3.2.13), -chymotrypsin and trypsin were obtained from Sigma Chemical Co. (St. Louis, MO). Soybeans (Glycine max, var. Walters) were obtained from the Dept. of Agron- omy, Univ. of Arkansas. Whey protein isolate (WPI) was purchased from Davisco International Inc. (LeSueur, MN). Sepharose 6B-CL was purchased from Pharmacia (Uppsala, Sweden). The reagents were analytical reagent grade and purchased from Fisher Scientific (Pitts- burgh, PA) and Sigma Chemical Co. (St. Louis, MO). Soybean 11S globulin preparation and purification Ground soybean flour was defatted three times at room tempera- ture using hexane (1:4 soybean flour:hexane ratio). Soybean 11S was prepared from hexane-defatted soybean flour by acid and salt pre- cipitation and purified using a Sepharose 6B-CL column as described by Thanh and Shibasaki (1976) and Iwabuchi and Yamauchi (1987). Preparation of transglutaminase catalyzed 11S globulin and whey protein isolate films In the presence of CaCl 2 , concentrated 11S solution was almost insoluble; therefore, 2.5-5.0% 11S solution was heated at 75°C for 30 s to increase its solubility by modifying its three dimensional structure. Six films, control WPI (CWF), TG cross-linked WPI (TWF), control 11S (C11SF), TG cross-linked 11S (T11SF), control WPI/11S (CMIXF), and TG cross-linked WPI/11S (TMIXF) were produced. WPI (5.0%), 11S (5.0%) or the mixture of these two (2.5% WPI and 2.5% 11S) were dispersed in 0.1 M Tris-HCl buffer (pH 7.8), containing 5 mM CaCl 2 and 20 mM dithiothreitol. Glycerol (1.25%) and TG (0.02 units/mg protein) were added. One enzyme unit catalyzes the formation of 1.0 μ mole of hydroxamate in 1 min from N--carbobenzyloxy-Gln-Gly and hydroxylamine at pH 6.0, at 37°C (Folk and Cole, 1966). This film forming solution was poured on plastic plates and incubated at 37°C for 5 h followed by over- night drying at room temperature (22-25°C). Control films (CWF, CHEMISTRY/BIOCHEMISTRY The authors are affiliated with the Dept. of Food Science, Univ. of Arkansas, Fayette- ville, AR 72704.