Plasmin Inactivation with Pulsed Electric Fields HUMBERTO VEGA-MERCADO, JOSEPH R. POWERS, GUSTAV0 V. BARBOSA-CANOVAS, and BARRY G. SWANSON ABSTRACT Plasmin(fibrinolysin E.C.3.4.21.7), an indigenous enzymein bovine milk, added to simulated milk ultrafiltrate (SMUF) at 100 pg/mL (pH 6.11 and ionic strength 0.056 M) was treated at 10°Cand 15°Cwith pulsed electric fields (HVPEF)of 15, 30 and 45 kV/cm andnumber of pulses10, 20, 30, 40 and 50. The plasminactivity measured using a commercial assay, was reduced 90% after 50 pulses at both 30 and 45 kV/cm andat a processing temperature of 15°C. Similar inactivation was obtained whenplasmin (100 pg/mL) in SMUF was heated at 40°C for 15 min. Inactivation of the enzyme depended on the number of pulses appliedduring treatment, intensityof the appliedfield, and processing temperature. Key Words:plasmin, milk, inactivation, pulsed electric fields INTRODUCTION PLASMIN (fibrinolysin E.C.3.4.21.7) or milk alkaline protease is an indigenous enzyme in bovine milk (Kaminogawa et al., 1972; Eigel, 1977; Grufferty and Fox, 1988). Plasmin and plasmino- gen are components of the proteolytic enzyme system in bovine blood. They pass from the blood to the milk via the mammary glands. Activation of plasminogen results in formation of plas- min. Plasminogen activators and plasmin inhibitors also appear in the milk. The activators of plasminogen are associatedwith the casein micelles in milk while the plasmin inhibitors are found only in the serum phase (Grufferty and Fox, 1988). Plas- min activity results in production of y-caseins and proteose- peptones from p-caseins (Bastian et al., 1991; Eigel, 1977; Kohlmann et al., 1991). The concentration of plasmin in milk is related to several factors including the stageof lactation, breed of cows, age of cows, mastitis and storage temperature (Rich- ardson, 1983b; Grufferty and Fox, 1988; Politis et al., 1989a, b). The proteolytic activity of plasmin promotes several changes in milk. These include a decreaseof viscosity of casein disper- sions prepared from milk (Grufferty and Fox, 1988) and an in- crease in the amount of soluble protein due to the formation of peptides (Grufferty and Fox, 1988). An increase in rennet co- agulation time also occurs with an increase in p-casein hydrol- ysis during cheese ripening (Humbert and Alais, 1979; Richardson and Pearce, 1981) and gelation of UHT milk (Gruf- ferty and Fox, 1988; Kohlmann et al., 1991). Pasteurization de- creases initial plasmin activity in milk (Korycka-Dahl et al., 1983; Richardson, 1983a), but the proteolytic activity increases during storage of processed milk (Humbert and Alais, 1979; Korycka-Dahl et al., 1983; Richardson, 1983a). The increase in proteolytic+.activity can be explained by the destruction of in- hibitors of plasminogen activators during heating (Richardson, 1983a). The use of P-lactoglobulin as an inhibitor of plasmin has been reported by Bastian et al. (1993). Factors which affect activity of the enzyme are temperature, reaction time, enzyme concentration, nature of substrate, and thermostability of the enzyme and inhibitors (Visser, 1981). In- activation of plasmin in milk is a function of pH during heating, Authors Vega-Mercado and Barbosa-C&novas are with the Bio- logical Systems Engineering Dept. and Authors Powers and Swanson are with the Food Science & Human Nutrition Dept., Washington State Univ., Pullman, WA 99164-6120. Address in- quiries to Dr. G.V. Barbosa-Crinovas. decreasingplasmin stability with an increasein pH (Richardson, 1983a; Grufferty and Fox, 1988). Kaminogawa et al. (1972) reported a rapid decrease of plasmin activity after heating a buf- fer solution (PH 7.0) containing the enzyme at 40 to 80°C for 10 min. In addition, the stability of plasmin is lower in a non- micelle system than in a micellar casein dispersion (Grufferty and Fox, 1988). In the 1920s and 193Os, the use of electric treatments to pas- teurize milk was widely studied (Beattie and Lewis, 1925; Fet- terman, 1928; Moses, 1938). The earlier objective was to heat the milk by passing current through the product (ohmic heating). Sale and Hamilton (1967) demonstrated the nonthermal lethal effect of electric fields on bacteria such as Escherichia coli, Staphylococcus aureus, Micrococcus lysodeikticus, Sarcina lu- tea, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, and Clostridium welchii, and yeasts such as Saccharomycescerevi- siae and Candida utilis. Similar results were reported by Sato et al. (1988), Mizuno and Hori (1988), Zhang et al. (1995) and Pothakamury et al. (1995). The main effect of an electric field on microorganisms is an increase in membrane permeability becauseof compression and poration. Cell breakdown or inactivation is achieved becauseof osmotic imbalance across the cell membrane induced by pora- tion. Reduction of up to 9 log cycles (9D) for Escherichia coli (E. coli) was obtained with pulsed electric fields of 50 kV/cm (Zhang et al., 1995). Gilliland and Speck (1967) reported the inactivation of trypsin and protease from B. subtilis using an electric field of 3 1.5 kV/cm. Castro (1994) reported the inactivation of alkaline phos- phatase in simulated milk ultrafiltrate (SMUF) by applying an electric field of 20 kV/cm. The inactivation mechanism for the enzymes proposed by Gilliland and Speck (1967) was an oxi- dative reaction of key components induced by electric fields as a function of treatment time. Our objective was to examine the feasibility of using pulsed electric fields (PEF) in the inactivation of plasmin in simulated milk ultrafiltrate basedupon limited information available on the effects of PEF on enzymatic activity. The variables included number of pulses, electric field strength and processing temper- ature. MATERIALS & METHODS Plasmin solution and simulated milk ultrafiltrate Plasmin E.C.3.4.21.7 from bovine plasma (Sigma Chemical, St.Louis, MO, 80 mg freeze-dried sample) wasreconstituted with 10mL of 1 mM HCl and frozenin l-mL vials at -35°C until used.Simulated milk ul- trafiltrate (SMUF) provided a casein micelle-free medium with electrical and chemical properties similar to milk. SMUF consists of lactose (50 g), potassium phosphate monobasic (1.58 g), &i-potassium citrate(0.98 g), tri-sodium citrate (1.79 g), calcium chloride (1.32 g), magnesium citrate (0.38 g), potassium carbonate (0.30 g), and potassium chloride (1.08 g) in 1L deionized water. SMUF was diluted with deionized water to modify electrical properties in the proportion 1:2 (v/v). The solution was filtered through a 0.22~km sterile filter and stored in sterile bottles. The pH of SMUF was 6.11 with an ionic strength of 0.056 M. An experimental SMUF/plasmin solution was prepared by mixing a portion of the reconstituted plasmin solution and diluted SMUF to a final con- centration of 100 pg/mL plasmin. The solution was stored at 4°C while inactivation experiments were conducted. Pulsed electric fields (PEF) A continuous flow chamber (Fig. 1) consisting of two parallel stainless steel electrodes and a polysulfone spacer was used to apply the PEF Volume 60, No. 5, 1995-JOLJRNAL OF FOOD SCIENCE-l 143