Prototype Instruments for Laboratory and On-line Measurement of Lipoxygenase Activity J.I. Reyes De Corcuera,1 R.P. Cavalieri2,* and J.R. Powers3 1
Department of Biological Systems Engineering, Washington State University, Pullman WA 99164-6120 USA 2 Agricultural Research Center, Washington State University, Pullman WA 99164-6240 USA 3 Department of Food Science and Human Nutrition, Washington State University, Pullman WA 99164-6376 USA A laboratory bench-top and a continuous prototype for measurement of lipoxygenase activity was developed. Lipoxygenase activity determination was based on the measurement of the rate of oxygen consumption by the enzyme using a polarographic sensor for the bench-top prototype and fibre optic sensors for the continuous prototype. The bench-top and the continuous sensors were compared against the standard spectrophotometric method. The range of LOX activity according to the spectrophotometric assay was from 0 to 0.163 mmol/L s. Accuracy and precision were 0.009 and 0.005 mmol/L s respectively for the bench top prototype and 0.007 and 0.006 mmol/L s for the on-line prototype. Correlation coefficients (R2) were 0.99 for both prototypes with respect to the spectrophotometric method. Key Words: lipoxygenase activity, blanching, bench-top prototype, on-line sensor prototype
INTRODUCTION Currently in the frozen vegetable industry, blanching control is based on temperature measurements and is often done manually. A semi-quantitative peroxidase test is the most common quality assurance test performed to confirm the effectiveness of blanching. However, lipoxygenase (LOX), which is less thermally stable than peroxidase, was found responsible for the deterioration of many vegetables such as corn, green peas, green beans and soy bean during frozen storage (Williams et al., 1986; Velasco et al., 1989; Sheu and Chen, 1991; Barrett and Theerakulkait, 1995). LOX catalyses the oxidation of unsaturated fatty acids containing 1,4-cis,cis-pentadiene system, resulting in the production of hydroperoxides (Axelrod et al., 1981) that are precursors of aldehydes and alcohols that are responsible for off-flavours or offaroma. Therefore, using lipoxygenase instead of peroxidase as the indicator of the extent of blanching and implementing continuous measurement and control of lipoxygenase activity while blanching would result in considerable energy savings due to the shorter blanching times. Also, shorter blanching may result in increased retention of fresh-like quality. Determination of LOX activity is important in other foods such as peanuts (Pattee and Singleton, 1981), avocado (Marcus et al., 1988), asparagus (Ganthavorn and Powers, 1989) canola *To whom correspondence should be sent (e-mail:
[email protected]). Received 11 September 2001; revised 25 November 2002. Food Sci Tech Int 2003;9(1):0005–5 ß 2003 Sage Publications ISSN: 1082-0132 DOI: 10.1177/108201303033014
seeds (Khalifa et al., 1990) and pumpkin and melon seeds (Al-Khalifa, 1996). Determination of lipoxygenase activity by the standard spectrophotometric technique requires optically clear solutions of the enzyme (Grossman and Zakut, 1979). To obtain such clear solutions, time-consuming extraction and separation of the enzyme from the vegetable tissue is required. Some other colorimetric methods are simpler but are mainly visual and remain semi-quantitative (Villafuerte-Romero and Barrett, 1997; Anthon and Barrett, 2001; Garrote et al., 2001). Based on a previous prototype (Zhang et al., 1991) Reyes de Corcuera et al. (2002) proposed a benchtop instrument for the rapid determination of residual LOX activity using the amperometric measurement of the rate of oxygen consumption by the enzyme catalysed oxidation of linoleic acid. This method is rapid (< 2 min) requires very small quantities of vegetable tissue crude homogenate and no purification step. The ability to rapidly and quantitatively determine LOX activity without need of purification creates the possibility of continuous measurement and therefore of on-line instrumentation for process control. The objective of this study was to build an on-line prototype instrument for the continuous measurement of lipoxygenase activity and compare its performance to the bench-top instrument developed by Reyes De Corcuera et al. (2002). and to the standard spectrophotometric assay.
MATERIAL AND METHODS Materials Linoleic acid 99%, Trizma-Base (Tris[hydroxymethyl] amino methane) buffer, -cyclodextrin and soybean 5
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lipoxygenase (oxygen oxidoreductase EC 1.13.11.12) were purchased from Sigma Chemicals Co. Sodium hydroxide pellets and hydrochloric acid 36.5–38.0% were purchased from Fisher Chemical Co. Ultrafiltered, deionised water was used for the preparation of all solutions. Methods Lipoxygenase solutions (0.1180, 0.0885, 0.0590 and 0.0295 g/L) were prepared 0.1 M Trizma-Base buffer. Three sub-samples from each of the four LOX solutions and from Trizma-Base buffer used for the enzyme blank were taken for each of the three methods used in this study. The mean of the three sub-samples measured by the spectrophotometric method was considered as the reference value. One sub-sample of each level of enzyme concentration was used to correlate the spectrophotometric assay with our prototypes. Readings of two subsamples using our prototypes were corrected with the calibration from the previous correlation and compared to reference values determined by the spectrophotometric assay. All experiments were carried out at room temperature (20 C). Spectrophotometric Method Two mL of 1 g/L linoleic acid solution and one mL of LOX solution were rapidly mixed in a quartz cuvette. Absorbance data at 234 nm was collected every two seconds for three minutes. LOX activity was measured by determining the highest rate of change in absorbance with respect to time. LOX activity is ultimately converted into the rate of linoleic acid oxidation with units of mmol/L s using molar absorptivity equal to 2.5 104 1/Mcm for the produced dienehydroperoxide (Axelrod et al., 1981).
ET AL.
Bench-top Sensor Prototype Lipoxygenase activity was determined using the bench-top prototype (Figure 1) by injecting 7.5 mL of LOX solution and 15 mL of 1 g/L linoleic acid solution into the reaction cell. The oxygen probe, an amperometric miniature electrode (Diamond General Cat. No. 730) was calibrated using a 2% sodium sulphite solution for 0% O2 saturation level, and water in equilibrium with air for the 100% O2 saturation. Data of percent oxygen saturation and reaction time were collected every second for two minutes and processed using a data acquisition board and a computer programme written in WorkBench for Windows from Strawberry Tree Company. LOX activity is computed at the end of each run as the rate of oxygen consumption with units of mmol/L s. On-line Prototype Lipoxygenase activity was determined using the online sensor prototype (Figure 2) that consists of a tubular reactor in which the rate of oxygen depletion is measured by means of two fluorescence oxygen probes (Ocean Optics Cat. No. FOXY). The double head diaphragm pump model 071135-40 and the static mixer cat no. A-04667-08 and other fittings were from Cole Parmer Instrument Co. Two static mixture elements were inserted in 12.7 mm ID polypropylene tube. The total length of the reactor was 50 cm. Flow rates were set to 7.21 and 13.60 mL/min for LOX solution and linoleic acid solutions respectively to maintain the same concentration of LOX and LA as in the bench-top prototype while keeping very low product consumption (approximately 5 g/min of tissue homogenate; that is, 1–4 g/min of raw vegetable would be required to obtain similar response). Each run was started with the system
Figure 1. Laboratory bench-top instrument prototype.
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Prototype Instruments
Table 1. Mean LOX activity determined by the spectrophotometric method.
Concentration of LOX Powder (g/L)
empty. Oxygen probes were calibrated using a 2% sodium sulphite solution for 0% O2 saturation level and water in equilibrium with air for the 100% O2 saturation. Data of percent of oxygen saturation at each probe, LOX activity expressed as the rate of oxygen consumption in mmol/L s and reaction time were collected using an Ocean Optics data acquisition board and a LabVIEW (National Instruments) computer programme. Data were collected every 2 s for 8 min to observe the transient and steady state reaction phases.
Zone I
1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
Even though purified LOX was used, it was necessary to determine the actual activity using a reference spectrophotometric method because our experimental conditions and the method used by Sigma Chemicals Co. are different. LOX activity was higher than predicted (according to Sigma specification) from the weight of enzyme powder as shown in Table 1 Columns 2 and 3 for different concentrations of LOX powder (Column 1). Column 4 in Table 1 lists LOX activity converted to the rate of linoleic acid oxidation in mmol/ L s. The increase of one unit of absorbance at 234 nm corresponds to the oxidation of 0.12 mmol of linoleic acid in the reaction cuvette. The increase in absorbance for the solution containing 0.0197 g/L in the reaction cuvette recorded by the spectrophotometer (Figure 3) displayed 3 zones. Zone I represented a lag phase. In Zone II the rate of oxidation of linoleic acid was maximum. Zone III showed a decrease in the rate of oxidation. The curve levels due to the limitation of dissolved oxygen in the reaction mixture at this stage of the reaction. Enzyme activity is determined from Zone II data. Lipoxygenase activity can also be defined as the rate of oxidation of linoleic acid.
0 3850 7700 11500 15400
0.002 0.024 0.074 0.117 0.163
Zone II
0
Zone III
20
40
60
80
Time
Figure 3. Absorbance vs. time for the solution that contains 7.5 units /L1 of LOX using the spectrophotometer.
% O2 saturation
Spectrophotometric Method
725 11900 37100 58500 81400
Replicate Replicate Replicate
120
RESULTS AND DISCUSION
Rate of Linoleic acid Oxidation (mmol/L s)
0 0.010 0.020 0.030 0.039
Absorbanc
Figure 2. On-line instrument prototype. TA-01: enzyme solution; TA-02: linoleic acid solution; PD-01: dual head diaphragm pump; O2-01 and 02: fibre optic oxygen probes; SM-01: static mixer.
Spectrophotometric Mean Measured LOX Activity (units)
LOX Activity Estimated from the Weight of Enzyme Powder (units)
Zone I
Zone II
Zone III
100 80 Replicate 1 60
Replicate 2
40
Replicate 3
20 0 0
20
40
60
80
100
120
Time (s)
Figure 4. Change in percent oxygen saturation with respect to time in the bench-top reaction cell.
Bench-top Sensor Prototype Lipoxygenase activity, as measured by the bench-top sensor, was defined as the rate of change of oxygen concentration with respect to time in the reaction cell. Figure 4 shows the depletion of oxygen as recorded by the data acquisition system for three replicates using the bench-top prototype. In this figure we can identify three zones. Zone I represents a lag phase. In Zone II the rate of oxygen consumption is maximum. In Zone III a decrease in the rate of oxygen consumption is caused by the deficiency in oxygen concentration. In other words, in Zone III oxygen became the limiting reagent. Enzyme activity is determined from Zone II. The oxygen
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J. I. REYES DE CORCUERA 120
Zone I
%O2 Saturation
100
Zone II
Zone III
80 Probe 1 Probe 2
60 40 20 0 0
100
200
300
400
500
600
Time
Figure 5. Determination of percent oxygen saturation at the inlet (probe 1) and at the outlet (probe 2) of the on-line LOX sensor prototype. concentration in water at 20 C and 101.325 kPa is 258 mmol/L. This concentration corresponds to 100% saturation. On-line Sensor Prototype Lipoxygenase activity measured by the on-line sensor was defined as the difference in oxygen concentration between oxygen probes 1 and 2 with respect to the residence time of the reaction mixture. Figure 5 illustrates the measurement of percent of oxygen saturation at both of the oxygen probes of the on-line prototype for one replicate of the reaction mixture containing approximately 31.3 mg/L1 of LOX powder. Three zones can be identified. Zone I is delimited by the time at which the reaction mixture reaches oxygen probe 1 and the time the reaction mixture reaches oxygen probe 2. Zone I included the first 102 s and corresponded to the residence time of the reaction mixture in the tubular reactor. Zone II corresponded to the transient phase for probe 2. Zone III corresponded to the steady state phase for probe 2. Probe 1 reached steady state before probe 2. However, enzyme activity is determined once steady state is reached at both probes, in this case for time > 400 s. The difference in percent oxygen saturation between probe 1 and probe 2 over the steady state period is averaged. Then, as for the benchtop sensor, these values are converted to the rate of oxygen consumption with units of mmol/L s. Comparison Among Methods Lipoxygenase activity data obtained from the benchtop and the on-line prototype were different but linearly related to data obtained with the spectrophotometric assay. LOX activities measured by the bench-top and the on-line prototypes were linearly correlated to the mean of the three replicates of enzyme activity determined by the spectrophotometric method. These linear regressions were used for calibration of the benchtop (y ¼ 0.985x þ 0.002, R2 ¼ 0.99) and the on-line
ET AL.
sensors (y ¼ 0.992x þ 0.001, R2 ¼ 0.99) with respect to the spectrophotometric method. The high correlation coefficients confirmed the good correlation existing between the spectrophotometric methods and the methods based on the uptake of oxygen. Lipoxygenase activities determined with the benchtop sensor were significantly higher than those determined by the on-line sensor for all samples containing LOX. This can be explained because in the bench-top sensor, the reaction mixture is well mixed by a stirrer while in the on-line sensor the flow of reagents is laminar and the mixing of substrate and enzyme takes place throughout the tubular reactor. Accuracy and precision were 0.009 and 0.005 mmol/L s respectively for the bench-top prototype and 0.007 and 0.006 mmol/L s for the on-line prototype. Accuracy was defined here as the maximum measured error with respect to the reference value. Precision was defined here as the maximum measured error with respect to the mean of the measured values. Since the spectrophotometric assay is considered the reference value, the accuracy could not be determined. However, the precision was 0.027 mmol/L s. This indicated that both of the proposed prototypes are at least four times more precise that the standard spectrophotometric assay. Relative mean errors for the bench-top and the on-line prototypes with respect to the spectrophotometric method (Table 2) were calculated as the difference between the mean LOX activity measured by the spectrophotometric method and the mean of the linearly calibrated LOX activity determined by another method, divided by the mean LOX activity of the solution with the highest LOX concentration (determined by the spectrophotometric method). Other recently proposed rapid methods are based on the visual assessment of colour change (Villafuerte-Romero and Barrett, 1997; Anthon and Barrett, 2001) for corn and green beans and further applied to carrots, broccoli, Brussels sprouts and potatoes (Garrote et al., 2001). However, in all cases the results remain semi-quantitative and can only give relative results and cannot be used for on-line measurement.
Table 2. Estimation of the mean error for the benchtop prototype and the on-line prototype with respect to the spectrophotometric method. Spectrophotometer Mean LOX Activity (mmol/L s) 0.002 0.024 0.074 0.117 0.163
Bench-top Prototype % Error
On-line Prototype % Error
0.289 0.564 1.856 0.858 0.146
1.494 1.838 0.025 0.373 –
Prototype Instruments
ACKNOWLEDGEMENTS The authors gratefully acknowledge the support of Key Technology, Inc., The International Marketing Program for Agricultural Commodities and Trade (IMPACT) at WSU, Fulbright-CONACyT (National Council for Science and Technology, Me´xico), U.S. Department of Energy and the Washington State University Agricultural Research Center.
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Methods of Biochemical Analysis. New York: Wiley and Sons. pp. 303–327. Khalifa A., Kermasha S. and Alli I. (1990). Partial purification and characterization of lipoxygenase of canola seed (Brassica napus var. westar). Journal of Agricultural and Food Chemistry 38: 2003–2008. Marcus L., Prusky D. and Jacoby B. (1988). Purification and characterization of avocado lipoxygenase. Phytochemistry 27(2): 323–327. Pattee H.E. and Singleton J.A. (1981). Peanut quality: its relationship to volatile compounds – a review. In: Teranishi R. and Barrera-Benitez H. (eds.), Quality of Selected Fruits and Vegetables of North America. Washington DC: American Chemical Society. pp. 147–161. Reyes-De-Corcuera J.I., Cavalieri R.P. and Powers J.R. (2002). Improved amperometric method for the rapid quantitative measurement of lipoxygenase activity in vegetable tissue crude homogenates. Journal of Agricultural and Food Chemistry 50(5): 997–1001. Sheu S.C. and Chen A.O. (1991). Lipoxygenase as blanching indicator for frozen vegetable soybeans. Journal of Food Science 56(2): 448–451. Velasco P.J., Lim M.H., Pangborn R.M. and Whitaker J.R. (1989). Enzymes responsible for off-flavor and offaroma in blanched and frozen vegetables. Biotechnology and Applied Biochemistry 11: 118–127. Villafuerte-Romero M. and Barrett D.M. (1997). Rapid methods for lipoxygenase assay in sweet corn. Journal of Food Science 62(4): 696–700. Williams D.C., Lim M.H., Chen A.O., Pangborn R.M. and Whitaker J.R. (1986). Blanching of vegetables for freezing- which indicator enzyme to choose. Food Technology 40: 130–140. Zhang Q., Cavalieri R.P., Powers J.R. and Wu J. (1991). Measurement of lipoxygenase activity in homogenized green bean tissue. Journal of Food Science 56(3): 719–721,742.
