Effect of High-Pressure Treatment on Lipoxygenase Activity Ratchada Tangwongchai, Dave A. Ledward,* and Jennifer M. Ames Department of Food Science and Technology, The University of Reading, P.O. Box 226, Whiteknights, Reading RG6 6AP, U.K. Solutions of commercial soybean lipoxygenase (100 μg/mL) in 0.2 M citrate-phosphate and 0.2 M Tris buffer were subjected to pressures of 0.1, 200, 400, and 600 MPa for 20 min. The enzyme was stable at atmospheric pressure (0.1 MPa) over a wide pH range (5-9). In citrate-phosphate buffer, the enzyme had maximum stability over the pH range 5-8 in untreated samples and after treatment at 200 MPa, but with increasing pressure, the pH stability range become narrower and centered around pH 7-8. The enzyme was more sensitive to acid than alkali, and at pH 9, it lost virtually all activity after pressurization at 600 MPa for 20 min in both buffers. The activity of the crude enzyme extracted from tomatoes treated at 200 and 300 MPa for 10 min was not significantly different from that of the untreated tomatoes, while a pressure of 400 MPa for 10 min caused a significant decrease in activity and treatment at 600 MPa led to complete and irreversible activity loss. Compared to unpressurized tomatoes, treatment at 600 MPa gave significantly reduced levels of hexanal, cis-3-hexenal, and trans-2-hexenal, which are important contributors to ‘fresh‘ tomato flavor, and this was attributed to the inactivation of lipoxygenase. Keywords: High-pressure treatment; tomato; lipoxygenase; pH; flavor INTRODUCTION Consumer demand for fresh or minimally processed food of high nutritional and organoleptic quality has stimulated research into novel nonthermal or combined processes (Hoover et al., 1989). High-pressure treatment provides an alternative method of food processing since it can decrease microbial load and enzyme activity while retaining sensory and nutritional quality (Cheftel, 1992; Hoover et al., 1993; Galazka and Ledward, 1995). High- pressure treatment can increase or decrease the kinetics of enzyme-mediated reactions, depending on whether their reaction volumes are negative or positive, and may destroy enzyme activity entirely by modifying the enzyme structure. Inhibition of enzyme activity by high- pressure treatment depends on the properties of the medium (including its pH), temperature, and time of treatment (Knorr et al., 1992). There are several reports on the effect of high-pressure treatment on enzyme activity (e.g., Gomes and Ledward, 1996; Herna ´ ndez and Cano, 1998; Ludikhuyze et al., 1998). Seyderhelm et al. (1996) classified enzymes, with respect to their sensitiv- ity to high pressure (up to 900 MPa), as pressure- sensitive or pressure-tolerant. They classified lipoxyge- nase as pressure-sensitive, the activity decreasing noticeably after 2 min at 600 MPa in both pH 7 Tris buffer and soymilk at 25 °C and complete inactivation occurring in Tris buffer after 10 min at 600 MPa at 25 °C. Ludikhuyze et al. (1998) reported that pressure- induced, as well as thermal, inactivation of lipoxygenase could be explained by first-order kinetics. They also reported that, in the temperature range 10-64 °C, the enzyme was most resistant to pressure at temperatures slightly above room temperature. Lipoxygenase (EC 1.13.11.12) plays an important role in the genesis of volatile flavor aroma compounds in many plant foods, including tomato, cucumber, and banana (Eskin et al., 1977). The enzyme degrades linoleic and linolenic acids to volatiles such as hexanal and cis-3-hexenal. The latter compound transforms to trans-2-hexenal, which is more stable. These compounds are thought to be the major volatile compounds that contribute to the ‘fresh‘ flavor of blended tomatoes. (Kazeniac and Hall, 1970). The purpose of the present work was to obtain fundamental information regarding the effect of high pressure on the stability of lipoxyge- nase and to investigate how these effects modified the generation of volatile flavor compounds in tomatoes. MATERIALS AND METHODS Lipoxygenase type I and linoleic acid (∼99% free acid) were obtained from Sigma Chemical Co. (Gillingham, U.K.). Other reagents were obtained from BDH (Lutterworth, U.K.). All chemicals used were analytical grade. High-Pressure Treatment. A prototype Stansted Food- lab model high-pressure rig (Stansted Fluid Power Ltd., Stansted, U.K.) was used to pressure-treat the samples (Cheah and Ledward, 1996). A mixture of castor oil and ethanol (20: 80) was used as the pressure transmitting medium, and all treatments were carried out at room temperature (∼20 °C). Temperature changes in the pressure transferring medium were measured by a thermocouple, and during pressurization the temperature of the medium increased to a maximum of 38 °C at 200 MPa and 45 °C at 600 MPa within 1-2.5 min and returned to ambient within 4 min from the start of processing. Pressurization of Samples. Soybean Lipoxygenase. Soy- bean lipoxygenase type I at a concentration of 100 μg/mL was prepared in 0.2 M citrate-phosphate buffer at pH 4, 5, 6, 7, 8, and 9 and 0.2 M Tris buffer at pH 6, 7, 8, and 9. A few samples were prepared in 0.2 M Tris buffer containing sodium chloride to give solutions having the same ionic strength as the citrate-phosphate ones. The enzyme solutions were sealed * To whom correspondence should be addressed. Tel: +44 118 931 8715. Fax: +44 118 931 0080. E-mail: D.A.Ledward@ reading.ac.uk. 2896 J. Agric. Food Chem. 2000, 48, 2896-2902 10.1021/jf9913460 CCC: $19.00 © 2000 American Chemical Society Published on Web 06/06/2000