308—JOURNAL OF FOOD SCIENCE—Volume 61, No. 2, 1996 Pressure Induced Inactivation of Selected Food Enzymes ISABEL SEYDERHELM, STEFAN BOGUSLAWSKI, GU ¨ NTHER MICHAELIS and DIETRICH KNORR ABSTRACT Pectinesterase, lipase, polyphenol oxidase, lipoxygenase, peroxidase, lac- toperoxidase, phosphatase and catalase have been examined at distinct conditions within a pressure range of 0.1 to 900 MPa, temperatures from 25°C to 60°C, pH 3 to 7, and time of treatment of 2 min to 45 min. Results in model buffers made it possible to rank the enzymes according to their pressure induced inactivation in the following order: lipoxygen- ase, lactoperoxidase, pectinesterase, lipase, phosphatase, catalase, poly- phenol oxidase, peroxidase. A combination of pressure with moderate temperature increased the degree of enzyme inactivation. Pressure treat- ment of real food systems showed a protective effect of food ingredients on the pressure inactivation of most enzymes evaluated. For example sucrose protected pectinesterase from inactivation by pressure while lac- toperoxidase and lipoxigenase were as stable in milk as in buffer. Key Words: hydrostatic pressure, oxidases, hydrolases, baroprotective effects INTRODUCTION RESULTS IN HIGH PRESSURE RESEARCH emphasize that ultra high pressure (UHP) treatment shows promises for effective devel- opment of novel food processes (Hayashi, 1992). UHP treatment is independent of size and geometry of the sample treated and can be carried out at ambient temperature. Its effect on food ingredients can be related to the fact that mainly noncovalent bonds are affected (Cheftel, 1992). High pressure inactivates microorganisms, influences biopolymers and affects activities of enzymes. Food quality factors, such as nutrients or functional characteristics remain mostly unchanged (Hoover et al., 1989). For the development of high pressure processes, it is essential to know the influence of high pressure on enzyme activities. Pressure favors reactions that result in a decrease of volume and inhibits those with a volume increase (Masson, 1992). The bi- ological activity of enzymes relates to their three dimensional protein structure. Changes in the tertiary and quaternary struc- ture are associated with volume changes and may therefore be affected by pressure (Heremans, 1992; Cheftel, 1992). For ex- ample, Ogawa et al. (1992) demonstrated that pectinesterase was reduced to 20% residual activity after high pressure treatment at 600 MPa, 23°C, 10 min but peroxidase retained 70% of its activity under comparable conditions. The activity of polyphenol oxidase from peaches increased after pressure treatment at 400 MPa, 25°C, 10 min (Asaka and Hayashi, 1991). Our objective was to investigate pressure effects on several types of food re- lated enzymes and to identify the importance of additional fac- tors such as temperature, medium composition and time of treatment. MATERIALS & METHODS A HYDROSTATIC PRESSURE UNIT (National Forge, St. Niklaes, Belgium) with a maximum pressure of 600 MPa, generated by an air driven pump, and a volume of 0.59 L with water as pressure transferring medium was used. Temperature range from 5°C to 95°C was monitored by a ther- mocouple. Trials with pressure 600 MPa were performed in a mobile high pressure unit (ABB, Va ˚sterhus, Sweden). In this case the pressure was generated by a pressure transducer and castor oil used as pressure The authors are affiliated with the Dept. of Food Technology, Ber- lin Univ. of Technology, Ko ¨ nigin-Luise Str. 22, 14195 Berlin, Ger- many. transferring medium. Enzymes and substrates were purchased at Merck (Darmstadt, Germany), Sigma (Deisenhofen, Germany) and Serva (Hei- delberg, Germany). Pectinesterase and polyphenol oxidase were desalted before treatment. For high pressure treatment, samples were sealed in polyethylene bags free of air bubbles, treated and stored in ice water until analysis. Cross- linking of trypsin was carried out by incubating 11.7 mg with 10 mg bovine serum albumin and 50 μL glutardialdehyde in 3.5 mL 5 mM Na 2 HPO 4 /100 mM NaCl buffer at 4°C. After 3 hr the reaction was stopped by addition of 50 mg glycine followed by 24 hr of dialysis against tris buffer (weight cut off 14kDa). V max and K M values of poly- phenoloxidase were determined by oxygen consumption in phosphate buffer at pH 7 and 25°C using a reaction volume of 3mL and 10 μL enzyme. Determination of enzyme activity For the photometric determination of enzyme activity, the linear in- crease or decrease of absorbance per time unit was used. Wavelengths and substrates were as follows. Polyphenol oxidase activity with 0.1M catechol in phosphate buffer pH 7 at :420 nm (Cano et al., 1990). Lipoxygenase activity with 99% linoleic acid in phosphate buffer pH 6.25 at :234 nm (Al-Obaidy and Siddiqi, 1981). Peroxidase activity according to Sigma with pyrogallol in phosphate buffer pH 6 at :420 nm (Stellmach, 1988). Lactoperoxidase activity with ABTS in sodium citrate buffer pH 5 at :412 nm (FINA). Phosphatase activity with ni- trophenylphosphate at pH 10.5 at :405 nm (Bernt, 1970). Catalase ac- tivity according to Beers and Sizers (1952, modified) with perhydrol in phosphate buffer DE°76 pH 7 at :240 nm. Pectinesterase activity was determined by a modified titrimetric method: Substrate was a 1% (w/w) apple pectin solution pH 8 and 30°C. The reaction was started by ad- dition of 75 μL pectinesterase in 50 mL apple pectin solution. The pH was held constant at 8.0 by addition of 0.05N NaOH. The consumption of NaOH/min in a linear range was used for calculation of enzyme ac- tivity (Korner et al., 1980). Lipase activity determination according to Worthington (1988). Substrate conversion of trypsin and -chymotrypsin catalyzed hy- drolysis of Bz-Arg-OEt rsp. Bz-Tyr-EtOH was determined by HPLC as follows: Stationary phase: RP-8, 5μm, mobile phase: 1/15M KH 2 PO 4 buffer, flow 1.2 mL/min, detection wavelength :256 nm. Limited hydrolysis: 600 μL crosslinked trypsin in tris buffer, pH 7.5 was mixed with polyphenol oxidase in tris buffer and incubated at am- bient temperature at 0.1 or 600 MPa. After distinct intervals, samples were taken and the enzyme activity determined. Each experiment was performed 3 times. T-tests were carried out to determine significant dif- ferences between samples at p ≤ 0.05 (Sachs, 1978). RESULTS HYDROLASES AND OXIDOREDUCTASES which notably affect food quality were chosen as model enzyme systems. While the hy- drolases revealed similar barosensitivity when treated in buffer, oxidoreductases varied considerably in barotolerance. For in- activation of pectinesterase, a minimum pressure of 800 MPa at 45°C was needed. An increase of pressure from 800 to 900 MPa reduced the inactivation time from 5 to 2 min (Fig. 1). When the pressure treatment of pectinesterase was carried out in media with different sucrose contents, its inactivation was strongly af- fected. Results of hydrostatic pressure on activity of pectines- terase were compared (Table 1) in orange juice, in 30% sucrose solution, in orange juice concentrate and in tris buffer pH 7. A high sucrose concentration increased the barostability of the en- zyme. The activity of lipase could be reduced with 600 MPa at ambient temperature. A 10 min treatment resulted in a 40% reduction of initial activity (data not shown). An increase to