Effects of high field electric pulses on the activity of selected enzymes

Journal of Food Engineering 31 (1997) 69-84 Copyright 0 1997 Elsevier Science Limited

PlI:SO260-8774(96)00052-O

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ELSEVIER

Effects of High Field Electric Pulses on the Activity of Selected Enzymes S. Y. Ho,” G. S. Mittal”” & J. D. Crosd’ “School of Engineering, “Department of Electrical

(Received

University of Guelph, Guelph, Ontario, Canada, NlG 2Wl and Computer Engineering, University of Waterloo, Waterloo, Ontario, Canada, N2L 3Gl

14 February

1995; revised 3 May 1996; accepted

15 June 1996)

ABSTRACT A compact and low cost bench-top, pulsed electric field treatment system was designed and developed. The unit consisted of a high-voltage pulse generator ( 530 kV) and a treatment chamber with I 148 ml capacity. Over the set-up voltage range of 4-26 kV 30 pulses (with instant charge reversal) were applied to eight different enzyme solutions using a 0.3-cm electrode distance, a 13-87 kVlcm field, 0.5Hz pulse frequency, 2-p pulse width and 20°C process temperature. For some enzymes, activities were reduced after the pulse treatments: lipase, glucose oxidase and heat-stable r-amylase exhibited a vast reduction of 70-85%; peroxidase and polyphenol oxidase showed a moderate 30-40% reduction whereas alkaline phosphatase only displayed a slight 5% reduction under the conditions employed. On the other hand, the enzyme activities of lysozyme and pepsin were increased under a certain range of voltages. Electric pulse profile (instant charge reversal) played a very important role in reducing the activities of various enzymes. 0 1997 Elsevier Science Limited. All rights reserved.

INTRODUCTION Food spoilage can be caused by enzymes naturally present in the food, or by the enzymes produced from certain microorganisms. Many food preservation techand freezing, are aimed at niques, such as thermal processing, dehydrating controlling or preventing the activity of indigenous or microbial enzymes. On the other hand, some enzymes bring desirable changes, such as flavour and odour, to the foods. Moreover, some exogenous enzymes are used to develop new food products or used as alternative methods to produce products such as aspartame and cyclodextrins (Gross, 1991; Nielsen, 1991). *To whom correspondence

should be addressed. 69

70

S. Y Ho et al.

Conventionally, enzymes in foods are inactivated by thermal processing. In the last 8 years, pulsed electric fields have been investigated as a potential non-thermal technique for food preservation (Ho et al., 1995). Enzyme activity, product yield on carrot mesh juice and water retention in wheat dough and bread firmness were also shown to be positively affected by pulsed electric fields (Aibara et al., 1992; Geulen et al., 1993). Results from these and other studies have indicated sufficient microbial control, product yield and shelf-life improvements, with negligible product quality changes. The study of enzymatic activity under pulsed electric field treatment was prompted by the very limited information available on the issue (Vega-Mercado et al., 1995a,b; Castro et al., 1994), and the frequent use of various enzymes in the food industry. Earlier, Gilliland & Speck (1967) reported inactivation of enzymes using electrohydraulic shock, which is quite different from pulsed electric field. The enzymes that were inactivated included lactic dehydrogenase, trypsin and proteinases of Bacillus subtilis. Some toxicity was caused by the liberation of copper from the electrodes during electrohydraulic discharge. The 15 kV was applied across a l-6-6.4 mm electrode gap. Vega-Mercado et al. (1995b) treated protease from Pseudomonas fluorescens with a pulsed electric field of 20 and 35 kV/cm on a pilot plant pulser. The reduction in proteolytic activity was a function of electric field and number of pulses as: 25% reduction at 10 pulses of 20 kV/cm, 50% at 20 pulses of 20 kV/cm, 60% at 10 pulses of 35 kV/cm, and 70% at 20 pulses of 35 kV/cm. Castro et al. (1994) reported the inactivation of alkaline phosphatase in simulated milk ultrafiltrate by applying a field of 22 kV/cm in pulses of 0.7-0.8 ms duration using a Gene electroporator (GeneZapper by Kodak). The high pulse time might have generated electrolysis in the treated sample. Similarly Vega-Mercado et al. (1995a) reported 90% reduction in the activity of plasmin after applying 50 pulses of 30 or 50 kV/cm at 15°C. Eight enzymes were selected for study based on their functional properties in major food groups. Table 1 describes the beneficial and detrimental functions of selected enzymes which are frequently encountered in food processing. The effects of a pulsed electric field on relative enzyme activity of these eight enzymes in aqueous solutions were determined using a pilot scale pulser, generating pulses with instant charge reversal.

MATERIALS

AND METHODS

Equipment (pulser and treatment chamber) The pulsed electric field treatment system consisted of a 130 kV d.c. high voltage pulse generator and a circular treatment chamber of I 148 ml capacity. Figure 1 shows a block diagram of the design unit. The 110 V a.c. was raised in voltage through a high voltage transformer, and then rectified. The d.c. high voltage supply then charged up the 0.12 ,uF capacitor through a series of 6 MQ resistors. The power source can provide up to 30 kV. The resistors were immersed in oil to prevent corona and arcing. The generation of high electric field pulses relied on the discharge of the 0.12 PF capacitor through the thyratron. This produced a pulsed electrical field (exponential decay pulse) between the electrodes in the treatment chamber.

71

High field electric pulses and enzyme activity

The pulse generator emitted a train of 5-V pulses, and the trigger circuit served to convert that to 500-V pulses using a silicon control rectifier (SCR). Terminologies for a pulse waveform are shown in Fig. 2. For this research, high electric field pulses with instant charge reversal’ were used. These were earlier found to be very effective in inactivating microorganisms (Ho et al., 1995) The circular treatment chamber (250-cm diameter) had two circular and parallel stainless-steel electrodes (16*5-cm diameter), as illustrated in Figure 3. The insulation, Delrin, was constructed to have close physical contact with the electrodes. The distance between the electrodes could be adjusted by inserting Delrin circular plates (14.5-cm diameter) with thickness 0.3, 0.6 or 0.9 cm. Thus, the process volume could be varied between 49.5, 99-l and 148.6 ml, respectively. To eliminate air bubbles in the treatment chamber during electrical treatment, the sample could go into the cavity from the bottom through a channel in the chamber under 51-kPa vacuum. The cell was held in place on a hinge-jointed movable frame, so the sample could be treated either vertically or horizontally. All samples were treated using a horizontally positioned chamber to avoid any chance of electrical sparking. The use of a vertically oriented chamber could increase the possibility of

Functions

TABLE 1 of Selected Enzymes in the Food Industry

Enzyme

Food industty

Peroxidase

vegetables milk

Alkaline phosphatase

milk

r-Amylase (heat stable)

starch, syrup, cereal, distillery, brewing, baking

Lipase

cheese milk oil cereal

Lysozyme Glucose oxidase

cheese beer, wine, fruit juices, eggs

Polyphenol oxidase

tea, coffee fruits, vegetables

Pepsin

meat, cheese

(deMan,

Beneficial functions

detects blanching efficiency preservative detects pasteurization efficiency converts/removes starch to dextrin (corn syrups), maltose, glucose, and oligosaccharides character build up, develops cured flavour, converts lipids to fatty acids and glycerol preservative antioxidant (oxygen removal), anti-browning (glucose removal) develops browning

tenderization ripening, coagulation

1982; Fox, 1991) Detrimental functions

off flavours browning action

heat stable

hydrolytic rancidity hydrolytic rancidity, over browning

browning, off flavour, loss of vitamins

S. Y Ho et al.

72 A.C. I 1OV + Transformer + Rectifier

I D.C. High voltage

Capacitor 0.12 FF

Resistor P 6Mohms

z

Ground

Fig. 1. Block diagram of the pulsed electric field treatment

system.

d

b

Time

Fig. 2. Pulse waveform profiles. a: pulse period(s), b: pulse width or duration(s), c: pulse rise time(s), d: spike or negative pulse width or duration(s), e: pulse peak voltage(V), f: spike peak voltage(V).

High field electric pulses and enzyme activity

73

3mm. Outlet tube

Front electrode

Rear platten Food cavity 3mm. Inlet tube ~____ Front platten Valve screw Electrode

~-

terminals

Fig. 3. Schematics of the treatment chamber.

gas pocket formation at the insulation-fluid interface by capillary action or gravitational force, and thereby increase the chance of sparking as compared with a horizontal orientation. A digital storage oscilloscope was connected to the system so that each pulse voltage across the chamber could be monitored. Enzyme solutions and experimental design Eight enzymes were selected based on their functional properties in major food groups, as shown in Table 1. Table 2 shows the experimental matrix with 35 tests designed. All tests were repeated at least twice (separately), in the reverse experimental order as the previous set. Only the enzyme solutions (prepared from pure enzyme as indicated in Table 2) were subjected to pulse tests, and the measurements were taken immediately before and after each treatment. The pulse period, pulse width and electrode distance were fixed to be 2 s (05Hz pulse frequency), 2 ,LLSand 0.3 cm, respectively. The sample temperature was 20°C and the set-up voltage range was 4-26 kV giving a maximum electric field of 13-87 kV/cm. The electrical conductivity values of the enzyme solutions varied from 400 to 16000 p.S/ cm. Due to the changes in electrical conductivity, there were changes in the durations for the rise and decay of positive and negative sections of the pulses. Details are given in Ho et aE. (1995).

74

S. Y Ho et al.

TABLE 2 Selection of Assays and Electrical Conditions for Pulsed 0.3-cm Electrode Distance, 2-s Pulse Period, 2-~LS Pulse Orientation Assay method

EnZyme”

Enzyme concentration (uniislmL)h

Electric Field Treatments, Using Width and Horizontal Chamber

Buffer

Voltage

Number of pUlSKS applied

WPLY PV)

continuous spectrophotometric rate continuous spectropholometric rate

0.674

100 mM potassium phosphate

12, 18, 22

30, 100

0.197

12, 18, 24

30

1-Amylase

calorimetric

1.27

titrimetric

903

Lysozyme

turbidimetric

218

continuous spectrophotometric rate continuous spectrophotometric rate spectrophotometric stop rate Test procedure

0.019

6, 9, 15, 18, 24 6, 14, 17, 24, 26 4, 12, 15, 18.22 5, 12, 15, 19

30

Lipase

1000 mM diethanolamine, 0.5 mM magnesium chloride de-ionized water de-ionized water 66 mM potassium phosphate SO mM sodium acetate

2007

SO mM potassium phosphate

6, 15, 24

30

40.5

6, 12, 18, 24 Structure

30

Molecular weight

10 mM hydrochloric Buffer pH

Peroxidase

EC 1.11.1.7

Pa) 44 000

6.0

monomer

Alkaline phosphatase

EC 3.1.3.1

160000

9.8

dimer

a-Amylase

EC 3.2.1.1

62 650

7.0

monvmer

Lipase

EC 3.1.1.3

62000

7.0

monomer

Peroxidase

Alkaline phosphatase

Glucose

oxidase

Polyphenol oxidase Pepsin Enzyme

Lysozyme

EC 3.2.1.17

14300

6.2

acid

monomer

Glucose oxidase

EC 1.1.3.4

186000

5.1

dimer

Polyphenol oxidase Pepsin

EC 1.14.18.1

128000

6.5

tetramer

EC 3.4.23.1

35 000

2.0

monomer

30 30 30

Enzymf origin and product number

soybean, P-1432 bovine intestinal mucosa, type VII-S, P-5521 Bacillus lichenijbtmis, A-4551 wheat germ, type 1, L-3001 chicken egg white, type 1, L-6876 Aspergillus niger, type VII-S, G-7016 mushroom, T-7755

porcine stomach mucosa, type A, P-6887

“All enzymes were obtained from Sigma Chemical Co., Saint Louis, MO. “Enzyme concentration: data in table show the average concentration used in two separate

trials

High field electric pulses and enzyme uctivity

7s

Sample preparation and loading Each enzyme solution of the required concentration was prepared immediately before test. Enzyme concentration was based on the activity of the pure enzyme in units/mg which was given by the supplier. The enzyme concentrations used in the tests (Table 2) were the recommended concentration for the respective enzyme assay. This recommended enzyme concentration facilitated the activity measurement without sample dilution. The sample was fed at 20°C into the treatment chamber from the bottom with a vacuum pump (Waters Millipore, Milford, MA, Model DOA-V152-AA) at 51-kPa vacuum to remove air bubbles. The vacuum was released after 30 s to eliminate any chance of sparking. A high electric field pulse test was then performed on a horizontally oriented chamber, and the pulse waveform (shape, peak voltage, pulse width) was recorded using a digital storage oscilloscope and a high voltage probe (Tektronix Inc, Beaverton, OR, Model 2230 for oscilloscope and Model P6015A for probe) at loo-MHz sampling rate. All the enzyme was recovered in the treated sample as there was no deposition on the electrodes at the end of the treatment. The fouling of the electrodes was completely eliminated due to negligible temperature increase and instant charge reversal pulses. The treatment chamber and the sample feeding tubes were then rinsed thoroughly with sterile distilled water. Temperature (Fisher Scientific, Whitby, Ontario, Digi-thermal Model 15-077) pH (Fisher Scientific, Whitby, Ontario, Accumet pH meter Model 925) and enzyme activity of each solution before and after each pulse treatment were also measured. Enzyme assays Standard enzymatic assay procedures (Sigma Chemical Co., Saint Louis, MO) were followed to prepare the reagents and enzyme solutions. Enzyme activity for both the pulse treated samples and the untreated solution (control) were determined using the appropriate apparatus (Table 2). The major apparatus used was: spectrophotometer (visible) (Spectronic 20 D, Milton Roy Co., Rochester, NY), and spectrophotometer (UV) (UV-260, Kyoto, Japan, supplied by Mandel Scientific Corp., Ltd., Guelph, Ontario). A light path of 1 cm was used in spectrophotometric determination for the activities of all the enzymes. The activity of peroxidase was based on the fact that one unit of enzyme will form 1 mg of purpurogallin from pyrogallol in 20 s at 20°C pH 6 and A420 “,,, (Sigma procedure EC 1.11.1.7). Assay of alkaline phosphatase (Sigma procedure EC 3.1.3.1) was based on the definition that one unit of enzyme will hydrolyse 1 ktmol of p-nitrophenol phosphate per min at 37°C pH 9.8 and A40s ,,,,,. Similarly, r-amylase activity (Sigma procedure EC 3.2.1.1) was based on the reaction that one unit of enzyme will liberate 1 mg of maltose from starch in 3 min at 20°C pH 6.9 and As4() “,,,. Assay for lipase (Sigma procedure EC 3.1.1.3) was based on the reaction that one unit of enzyme will hydrolyse 1 microequivalent of fatty acid from a triglyceride in 1 h at 37°C and pH 7.7. Lysozyme assay (Sigma procedure EC 3.2.1.17) was based on the definition that one unit of enzyme will produce a M 450 “,,, of 0.001 per min at 2°C pH 6.24 and 450 nm wavelength using a suspension of Micrococcus lysodeikticus as substrate in a 2.6 ml reaction mixture. Similarly, glucose oxidase assay (Sigma procedure EC 1.1.3.4) was based on the definition that one unit of enzyme will oxidize 1 pmole of B-D-glucose to D-

76

S. Y Ho et al.

glucuronic acid and H202 per min at 35”C, pH 5.1 and A500 “,,,. Polyphenol oxidase assay (Sigma procedure EC 1.14.18.1) was based on the definition that one unit of enzyme is equal to a hA 265nm of 0.001 per min at 25°C pH 65 andAzc5 nm in 3 ml reaction mix containing L-DOPA and L-ascorbic acid. Finally, pepsin assay (Sigma procedure EC 3.4.23.1) was based on the definition that one unit of enzyme is equal to a AAze5,,,,, of 0.001 per min at 37°C pH 2 and Az8,, nm in 3 ml, reaction mix containing L-DOPA and L-ascorbic acid. Detailed procedures are available from Sigma Corporation, including the reagents, method and calculations required. The enzyme activity calculations were based on the maximum linear rate principle (as recommended by Sigma Chemical Co.), meaning that the substrate concentration should not be the limiting factor during reaction. RESULTS AND DISCUSSION Figures 4-11 illustrate the relative enzyme activity as a function of supplied voltage at O-3-cm electrode gap. The data in the figures show the results of two separate trials. In general, with the exception of alkaline phosphatase, results from the two trials of each enzyme are in good agreement with each other (at 5% confidence level). A maximum variation of &4*8% points was observed in replications of the

Volti\ge supplied (kV)

Fig. 4. Changes in activity of enzyme peroxidase due to electric pulse treatment using 0.3-cm electrode distance and horizontal chamber for 30 and 100 pulses at 2-s pulse period and 2-~LS pulse width.

77

High field electric pulses and enzyme activity

activity of alkaline phosphatase (Fig. 5). Since variation was not high and change in activity was relatively small, no additional replication was conducted. No change in temperature or pH was recorded before and after the experiments. Thus, any change detected before and after the tests should be due to an effect on the enzymes by the high electric field pulses. Analysis of variance (ANOVA) results indicated no effect on enzyme activity of replication (except alkaline phosphatase) and number of pulses for peroxidase at 5% level. Pulse field had significant effect on the activity. Besides lysozyme (Fig. 8), and pepsin (Fig. ll), all other enzymes exhibited a similar activity profile. Enzyme activity decreased with increase in applied voltages or field intensity. Lipase, glucose oxidase and heat-stable r-amylase exhibited a vast reduction of 70-85%; peroxidase and polyphenol oxidase showed a moderate 30-40% reduction; whereas alkaline phosphatase only displayed a slight 5% reduction under the conditions employed. To prevent denaturation or degradation, an enzyme has to maintain its native structure. Enzymes are stabilized by weak noncovalent forces, such as hydrogen bonds, electrostatic forces, van der Waals forces and hydrophobic interactions; internal salt bridges; and, in some cases, disulphide bonds (Price & Stevens, 1991). A change in the magnitude of any of these could cause denaturation. The applica-

‘“1 n

. n

1st Trial

A 2nd Trial

Voltage supplied (kV)

Fig. 5. Changes in activity of enzyme alkaline phosphatase due to electric pulse treatment using 0.3-cm electrode distance and horizontal chamber orientation for 30 pulses at 2-s pulse period and 2-ps pulse width.

78

S. Z: Ho et al.

tion of high electric field pulses might have affected the forces involved in maintaining the three-dimensional structure (secondary, tertiary and quaternary structure) or conformation of the globular protein. Secondary structure refers to local interactions between amino acids in close proximity in the sequence to generate regular structural features, such as helices and turns. Tertiary structure refers to the overall folding of the polypeptide chain so as to cause interactions between amino acids remote from one another in the sequence. Quaternary structure refers to the arrangement of polypeptide chains of an enzyme which contains more than one such chain (Price & Stevens, 1991). As seen from Table 2, the eight enzymes tested had a very wide range of molecular weight, 14.3-186 kDa, and quaternary structure, monomers to dimers to tetramers (Sigma Chemical product specification sheets). However, the results seem to suggest that they are of minor importance to the reduction of enzymatic activity under pulse treatment. Since the degree of denaturation varied from enzyme to enzyme, secondary structure, or more likely, tertiary structure of the enzyme may play a more important role. In the case of lysozyme and pepsin, high electric field pulses appeared to have a stimulatory effect. Pepsin showed a bell-shaped activity-voltage profile, whereas lysozyme exhibited a wave-like contour. The activation might be due to the creation

.

1st Trial

A 2nd Trial

IO

1 0

I

I

I

I

5

IO

15

20

.

,

25

Voltage supplied (kV)

Fig. 6. Changes

in activity of enzyme a-amylase (heat stable) due to electric pulse treatment using 0.3-cm electrode distance and horizontal chamber orientation for 30 pulses at 2-s pulse period and 2-~LSpulse width.

High field electric pulses and enzyme activity

19

of more active sites or by increasing the size of the existing sites, and thereby increasing the reaction rate. However, the creation of active sites would indicate that the existing native form is not the most active form. This can be verified by doing pulse tests using various different enzymatic assays, different initial substrate concentrations, and examining the enzyme structure using X-ray crystallography or other methods. Comparison of the V,,.JK,,, ratios using the Michaelis-Menten equation is also beneficial. V,,,,, is the maximum reaction velocity at infinite substrate concentration and K,,, is the Michaelis constant, which corresponds to the substrate concentration at half of the maximum reaction velocity. The higher the ratio, the greater the preference of the enzyme towards that substrate. Furthermore, the presence of spikes (negative field pulses due to instant charge reversal) in the output pulses indicate oscillatory motion (high mobility effect) in the solutions. The high field pulses might also have decreased the free energy of activation of the enzymatic reaction by proximity and orientation effects, that is, the bringing together of reactants in the correct relative orientation for reaction at a faster rate. In both cases, pepsin and lysozyme exhibited an inhibitory effect once a particular field was reached. Using the same treatment chamber configuration, a pulse electric field strength of 10 kV/cm at 2-ps pulse width and 2-s pulse interval for 10 pulses (with instant charge reversal) was proved to be more than enough to achieve significant microbial

IO

1 0

I

I

I

I

5

10

IS

20

I 2s

I 30

Voltage supplied (kV)

Fig. 7. Changes electrode

distance

in activity of enzyme lipase due to electric pulse treatment using 0.3-cm and horizontal chamber orientation for 30 pulses at 2-s pulse period and 2-ps pulse width.

S. I: Ho et al.

80

reduction (6 log cycles) of Pseudomonas fluorescens (Ho et al., 1995). Using the same electrical parameters, only < 10% inactivation was observed in lipase (Fig. 7), glucose oxidase (Fig. 9) and polyphenol oxidase (Fig. 10). Peroxidase, alkaline phosphatase and or-amylase required 20 kV/cm for 10% reduction (Figs 4-6). Only lysozyme exhibited moderate reduction (50%) under low electric field strength. On the other hand, 10 kV/cm imposed a 10% stimulatory effect on pepsin (Fig. 11). Knowing that some enzymes are positively used in the industry, the results indicated that optimum conditions can be established to obtain sufficient microbial reduction but not at the expense of enzymatic inactivation. Vega-Mercado et al. (1995a) achieved a 90% inactivation of plasmin using 50 pulses at both 30 and 45 kV/cm and a processing temperature of 15°C. Inactivation of enzyme depended on the intensity of field and number of pulses applied. In current research, about 90% inactivation of a-amylase and lipase was achieved by applying 30 pulses (instant charge reversal) of 80 and 88 kV/cm, respectively, at 20°C. Inactivation of enzyme is also dependent on the type of enzyme, hence optimum conditions to inactivate the enzyme at a certain level will be different for different enzymes. Castro et al. (1994) achieved a 44% reduction of alkaline phosphatase using 20 pulses at 22 kV/cm. On the other hand, alkaline phosphatase was only slightly

.

1st Trial

A 2nd Trial

30 1 0

I 5

I IO

I IS

I 20

I 2s

Voltage supplied (kV)

Fig. 8. Changes in activity of enzyme lysozyme due to electric pulse treatment using 0.3-cm electrode distance and horizontal chamber orientation for 30 pulses at 2-s pulse period and 2-ps pulse width.

High field electric pulses and enzyme activity

81

affected (5%) in the current research. Comparison of the findings indicates that pulse width and pulse waveform may have a more important role than the electric field strength in the reduction of enzymatic activity. Castro et al. (1994) employed a commercial electroporator (Gene-zapper, Kodak) with a much wider pulse width, 0.7-0.8 ms, as compared to the 2 ps with instant charge reversal pulse employed in the current research. They concluded that the pulsed electric field preferentially increases accessibility of aliphatic hydrophobic regions in the phosphatase, and that it does not cause proteolysis. More understanding of the kinetics, structures and functions of the enzymes under high electric fields will be beneficial. CONCLUSIONS Enzymatic activity was proved to be affected by high electric field pulses on the eight enzymes tested. The degree of denaturation varied from enzyme to enzyme. Lipase, glucose oxidase and heat-stable cl-amylase exhibited a vast reduction of 70-85%; peroxidase and polyphenol oxidase showed a moderate 30-40% reduction; whereas alkaline phosphatase only displayed a slight 5% reduction under the conditions employed. Knowing that enzymes are stabilized by weak non-covalent forces, such as hydrogen bonds and hydrophobic interactions, the application of high elec-

Voltage supplied (kV)

Fig. 9. Changes in activity of enzyme glucose oxidase due to electric pulse treatment using 0.3-cm electrode distance and horizontal chamber orientation for 30 pulses at 2-s pulse period and 2-ps pulse width.

82

S. Y.Ho et aL

tric field pulses might have affected the three-dimensional structure of the globular protein. Comparisons between the results with previous findings from other researchers indicated that electric field strength, pulse width, and pulse waveform. all played a very important role. Lysozyme and pepsin showed a stimulator-y profile under a certain range of voltages. Several hypotheses on active site configuration were proposed. Repeating the experiments with different assays and initial substrate concentrations will increase the confidence level. More work is needed to understand the kinetics and structural changes. Comparing the results between microbial depletion and enzyme inactivation under pulsed electric field treatments indicated that the latter demanded a higher voltage for significant reduction. Knowing that some enzymes are positively used in the industry, this provides opportunities for achieving sufficient microbial control and yet minimizing damage to the enzymes. ACKNOWLEDGEMENTS This research was supported by the Ontario Ministry of Agriculture through their special Ontario Food Processing Research Program.

.

100 .-

and Food

1st Trial

A 2nd Trial

65

-

60

-

.

A 55 0

I

I

I

I

I

5

IO

15

20

25

Voltage supplied (kV)

Fig. 10.Changes in activity of enzyme polyphenol oxidase due to electric pulse treatment using 0.3-cm electrode distance and horizontal chamber orientation for 30 pulses at 2-s pulse period and 2-,us pulse width.

High jield electric pulses and enzyme activity

.

260 .-

I

2

240

-

220

-

200

~

1x0

-

83

.

1st Trial

A 2nd Trial

g 0 ;i 3 x .z

100

v

0

I

I

I

I

I

5

IO

15

20

25

Voltage supplied (kV)

Fig. 11. Changes in activity of enzyme pepsin due to electric pulse treatment using 0.3-cm electrode distance and horizontal chamber orientation for 30 pulses at 2-s pulse period and ? ..,. -..I_.. .. ..Arl.

REFERENCES Aibara, S., Hisaki, K. & Watanabe, K. (1992). Effects of high voltage electric field treatment on wheat dough and bread making properties. Cereal Chem., 69,465-467. Castro, A. J., Swanson, B. G. & Barbosa-Canovas, G. V. (1994). Protein denaturation by strong electric fields. IFT 1994 Annual Meeting: Book of Abstracts, paper no. 47B-36. I31 de#&

J. M. (1982). Principles of Food Chemistry. AVI

348-384.

Publishing,

Westport,

CT, pp.

Fox, P. F. (1991). Food Enzymology, Volume 1 and II. Elsevier Applied Science, New York. Gross, A. (1991). Enzymatic catalysis in the production of novel food ingredients. Food Zkhnol.,

45(l),

96-97.

Geulen, M., Teichgraber, P. & Knorr, D. (1993). Effects of high electric field pulse treatment on carrot juice yield. IFT 1993 Annual Meeting, Book of Abstracts, paper no. 198, 53 pp. Gilliland, S. E. & Speck, M. L. (1967). Mechanism of the bactericidal action produced by electrohydraulic shock. Appl. Microbial., 15, 1038-1044. Ho, S. Y., Mittal, G. S.. Cross, J. D. & Griffiiths, M. W. (1995). Inactivation of Pseudomonas puorescens by high voltage electric pulses. J. Food Sci., 60, 1337-1343. Nielsen, H. K. (1991). Novel bacteriolytic enzymes and cyclodextrin glycol transferase for the food industry. Food Technol., 45(l), 102-104. Price, N. C. & Stevens, L. (1991). Enzymes: structure and function. In Food Enzymology, Volume I and II, ed. P. F. Fox. Elsevier Applied Science, New York, pp. l-25.

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Vega-Mercado, H., Powers, J. R., Barbosa-Canovas, G. V. & Swanson, B. G. (1995a). Plasmin inactivation with pulsed electric fields. J. Food Sci, 60, 1143-1146. Vega-Mercado, H., Powers, J. R., Barbosa-Canovas, G. V., Swanson, B. G. & Luedecke, L. (1995b). Inactivation of a protease from Pseudomonas j7uorescens M3/6 using high voltage pulsed electric fields. IFT 1995 Annual Meeting, Book of Abstracts, paper no. 89-3, 267 PP.