Nonthermal Inactivation of Soy (Glycine Max Sp.) Lipoxygenase by Pulsed Ultraviolet Light Bhaskar A. Janve, Wade Yang, Maurice R. Marshall, Jos´e I. Reyes-De-Corcuera, and Taha M. Rababah
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Abstract: This study investigated pulsed ultraviolet (PUV) illumination at different distances from the PUV source on soybean lipoxygenase (LOX) (0.4 mg/mL in 0.01 M Tris-HCl buffer, pH 9) activity. Samples (5 mL) were illuminated for 1, 2, 4, 8, and 16 s at 3 distances 6, 8.5, and 11 cm from the PUV lamp’s quartz window. The temperature of 33.5 ± 1.8◦ C was observed for the highest treatment time of 16 s at the shortest distance of 6 cm, and resulted in a 3.5 log reduction (99.95%) in initial LOX activity. Illumination time and distance from the lamp significantly (P ≤ 0.05) affected LOX inactivation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on treated LOX samples and further protein profile for treated LOX filtrate (≤10 kDa), was analyzed by reverse phase high-performance liquid chromatography (RP-HPLC). The protein profile analysis revealed that LOX protein degradation was influenced significantly (P ≤ 0.05) by PUV illumination time. Keywords: enzyme inactivation, lipoxygenase, nonthermal, pulsed ultraviolet (PUV), sodium dodecyl sulfate polyacry-
lamide gel electrophoresis (SDS-PAGE) An investigation of pulsed ultraviolet light (PUV) on lipoxygenase (LOX) activity was performed for its application and enhancement of the current literature. This study shows that PUV illumination inactivates LOX, an enzyme responsible for stimulating off-flavor generation, loss of pigments and oxidative destruction of essential fatty acids in food products and raw materials. The sample treatment distance from the quartz window and illumination time were major parameters influencing the enzyme activity. The overall inactivation of LOX under PUV was accounted by the enzyme protein fragmentation with out considerable rise in the temperature of the sample. This shows PUV can successfully act as a nonthermal process for inactivation of LOX enzymes.
Introduction Lipoxygenase (linoleate: oxygen oxidoreductase, EC 184.108.40.206; LOX) is one of the most studied groups of enzymes. Lipoxygenases are ubiquitously present in biological organs and tissues, most abundant in grain legume seeds and potato tubers (Eskin and others 1977). Soybean LOX is well characterized among plants by a defined molecular structure (Boyington and Amzel 1993). The typical structure of LOX contains nonheme iron (Chasteen and others 1993) with a large protein structure consisting of 839 amino acids (Shibata and others 1987; Steczko and others 1992). Soybean has 3 isozymes with LOX-1 the most abundant. The LOX-1 acts on free polyunsaturated fatty acids forming 9- and 13-hydroperoxides in the ratio of 1:9 at room temperature and optimal pH of 9.0 (Baysal and Demird¨oven 2007). Catalytic hydroperoxidation of polyunsaturated fatty acids by LOX results in oxidation of essential fatty acids and produces several reactive molecules like free radicals. These radicals are associated with quality deterioration by producing off-flavor, odor production, and loss of pigments such as carotenes and chloroMS 20130598 Submitted 5/3/2013, Accepted 10/21/2013. Authors Janve, Yang, and Marshall are with Dept. of Food Science and Human Nutrition, Univ. of Florida, Gainesville, FL, 32611, U.S.A. Author Reyes-De-Corcuera is with Citrus Research and Education Center, Univ. of Florida, 700 Experiment Station Rd, Lake Alfred, FL, 33850, U.S.A. Author Rababah is with Dept. of Nutrition and Food Technology, Jordan Univ. of Science and Technology, Irbid, 22110 Jordan. Direct inquiries to author Yang (E-mail: [email protected]
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phylls (King and Klein 1987). Hence, inactivation of deleterious enzymes is considered a critical processing step for food preservation. Blanching is one of the most common unit operations for inactivating enzymes, particularly peroxidase. Blanching temperature and time are adjusted for inactivating peroxidase because its assay is simple and rapid. However, complete inactivation of peroxidase often leads to overblanching. A residual activity (RA) of 3% to 10% peroxidase in blanched and frozen-food products is recommended (Gunes and Bayindirli 1993). Using LOX as an indicator of proper blanching has been endorsed, as it is more significant in determining storage stability of frozen vegetables (Williams and others 1986; Sheu and Chen 1991). Lipoxygenase over peroxidase activity as a blanching index (Barrett and Theerakulkait 1995; Indrawati and others 1999) can also be implemented because it can be measured by rapid online methods (De Corcuera and others 2003). Thermal inactivation of LOX at elevated temperatures above 60◦ C improves the quality and shelf life of foods (Anthon and Barrett 2003). However, heating at such temperatures also increases nonenzymatic oxidations, which may surpass the impact by LOXrelated oxidation. The detrimental effects of thermal treatment on organoleptic quality, heat liable nutrients, and volatile components can surface later in foods. Hence, an alternative method to conventional thermal technology is desired (Lopez and Burgos 1995). Also, the demand for food products with minimal heatinduced degradation on organoleptic and nutritive properties has resulted in developing nonthermal processes (Mertens and Knorr 1992). R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12317 Further reproduction without permission is prohibited
As a nonthermal technology, PUV inactivates bacteria, fungi, and viruses more rapidly and effectively than continuous UV treatment (Dunn and others 1995). Bank and others (1990) were among the first to report inactivation of microorganisms by PUV. Inactivating microorganisms (Oms-Oliu and others 2010), mitigating allergens (Chung and others 2008; Yang and others 2010), and decontaminating food surfaces and packaging materials (Guillou and others 2007) are among the emerging applications of PUV in foods. PUV is comprised of high-frequency pulses of broadspectrum radiation containing wide wavelength distribution in the ranges from ultraviolet (100 to 400 nm), visible light (400 to 700 nm), and infrared (700 to 1100 nm) (Krishnamurthy and others 2010). Pulses of PUV light used for food-processing applications are typically at a rate of 1 to 20 flashes per second at an energy density from 0.01 to 50 J cm−2 at the surface (Barbosa-C´anovas and others 1998). The application of PUV on food products has been considered to be a relatively safe and nontoxic treatment (Green and others 2003). The PUV light fall into the nonionizing portion of the electromagnetic spectrum as it contains a significant component of longer wavelengths (Dunn and others 1995). The Food and Drug Administration (FDA) has approved PUV treatment of foods under Title 21 of the Code of Federal Regulation section 179.41 for surface microorganism control. The total cumulative PUV treatment approved should not exceed 12.0 J cm−2 and the pulse duration not longer than 2 milliseconds for treating food for surface microorganism control (Regulations 2000). A significant portion of PUV contains the ultraviolet region. It has been established that microbial inhibition is not achieved when the PUV wavelength region below 320 nm is removed by filters (Takeshita and others 2003). The visible and infrared regions, combined with the high peak power of PUV, are considered for lethal action (Elmnasser and others 2007). The photochemical mechanism of PUV involves nucleic acids, that is, chemical modifications, DNA cleavage, and transformation of pyrimidine bases (Giese and Darby 2000). Structural disruption during temporary overheating resulting from absorption of PUV light from a flash lamp exceeding 0.5 J. cm−2 of energy (Wekhof 2000) contributes to the photo-thermal effect. The thermal effect of PUV arises due to differences in UV light absorption between the species and that of the surrounding medium. Little information exists on enzyme inactivation by PUV. PUV radiation has an exceptionally broad spectrum (100 to 1100 nm) which may affect enzymes, as proteins have a strong UV absorption at 280 nm (Hollosy 2002). Peptide bonds [–C(O)–NH–] in proteins exhibit a strong and weak absorption in the Far UV region at of 190 nm (Aitken and Learmonth 1996) and 210 to 220 nm (Davies and Truscott 2001), respectively. Typical absorption spectrum for proteins is in the range of 250 to 300 nm. This originates from tryptophan and tyrosine residues, while phenylalanine absorbs weakly below 275 nm and cystine has a broad absorbance in the near-UV region (Wetlaufer 1962; Mach and others 1992). Ultraviolet light can inactivate enzymes as it is absorbed by amino acids in the proteins. The absorbed light induces a chemical change in protein resulting from the quantum yield of UV radiation on the protein and its residues (Luse and McLaren 1963). Broad spectrum PUV light with 54% as UV light (Shriver and others 2011; Shriver and Yang 2011) may be useful to inactivate enzymes. Dunn and others (1989) showed that 2 to 5 flashes of light with approximately 3 J cm−2 were enough to inhibit potato slice browning. Polyphenol oxidase extracted from treated slices exhibited less activity when compared to untreated
slices. In establishing how PUV enzyme inactivation occurs, an understanding of PUV’s properties and mechanism of action is desired. Additionally, PUV application parameters and how they influence enzyme inactivation is essential for designing processing applications. Thus, the objective of this research was to study the outcome of PUV on LOX activity and establish how PUV operating parameters affect enzyme properties. Specific propertiesevaluated include enzyme inactivation kinetics, LOX protein, and sample temperature profile. PUV parameters examined include duration of illumination (Hiramoto 1984) and the distance (G´omez-L´opez and others 2005) from the light source.
Materials and Methods Materials Soybean LOX type 1 B (EC 220.127.116.11, Lot # 050M1910V), linoleic acid, and Tween 20 were obtained from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). All reagents were of analytical grade. Electrophoresis equipment and reagents, including precast Tris-HCl sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) minigels (4% to 20%), Mini-PROTEAN Tetra cell tanks, Laemmli sample buffer, and Tris-glycine-SDS running buffer were purchased from Bio-Rad Laboratories, Inc. (Hercules, Calif., U.S.A.). The protein bands were scanned with a Canon Pixma MP160 scanner (Canon Inc., Melville, N.Y., U.S.A.). Experimental design An overview of experimental procedures used in this study is illustrated in Figure 1. Lipoxygenase was prepared fresh (0.4 mg/mL LOX in Tris-HCl buffer 0.01M; pH 9) every time. LOX (5 mL) was pipetted in aluminum dishes (5 cm dia) (Fisher Scientific, Pittsburg, Pa., U.S.A.). The samples (5.0 ± 0.1 mm thick) were illuminated with broad spectrum (100 to 1000 nm) PUV light comprising approximately 54% of the UV component in each pulse using a Xenon PUV system (Model RC-847, LH840 LMP-HGS, Xenon Corp., Wilmington, Mass., U.S.A.) at 3 pulses per second with a pulse width of 360 μs. Samples were illuminated for 1, 2, 4, 8, and 16 s at distances of 6, 8.5, and 11 cm from the quartz window. Experiments were conducted randomly according to a completely randomized block design (RBD) in stationary mode. The samples were cooled on ice following treatment. During treatment, temperature profiles were recorded. Spectrophotometric assay of LOX activity and SDS-PAGE provided analysis for RA and protein profile, respectively. The treated samples were further investigated after ultrafiltration through a centrifugal filtration cartridge (≤10 kDa MWCO) by reverse phase high-performance liquid chromatography (RP-HPLC). Temperature profile measurement The initial and final surface temperature of each sample during treatment was measured using a handheld noncontact infrared thermometer (Omega OS423-LS, Omega Technologies, Stamford, Conn., U.S.A.). The bulk temperature profile of the sample during treatment was recorded by K-type thermocouple using a R 8-channel thermocouple data logging interface (PC-08) atPico tached to a laptop running Pico-software (Figure 2). The K-type (Omegaette HH306, Omega Engineering Inc., Stamford, Conn., U.S.A.) thermocouple was placed at the geometrical center of the aluminum dish (1 mm tip) for every sample. The Pico software recorded the data every 300 ms. Data recording was started and stopped 30 s before and after the actual experiment. Vol. 79, Nr. 1, 2014 r Journal of Food Science C9
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Nonthermal inactivation of soyLOX by PUV . . .
Nonthermal inactivation of soyLOX by PUV . . .
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Lipoxygenase activity The substrate was prepared fresh according to a modified method of Axelrod (1981). Linoleic acid (C18 H32 O2 , cis-9, cis12-octadecodienoic acid) (50 μL) and an equal part Tween 20 (polyoxyethylenesorbitanmonolaurat) were homogenized with 0.2 M borate buffer (500 μL), pH 9.0. A spectrophotometric assay was used to measure LOX activity at 25◦ C. The assay mixture contained 0.95 mL borate buffer (0.2 M, pH 9), 2.0 mL substrate solution, and 0.05 mL LOX. The substrate solution (0.017% v/v) remained fixed in the total (3 mL) reaction mixture. For low residual LOX activity after treatment, the sample volume was increased to a maximum of 0.3 mL by reducing buffer volume. LOX activity was determined using a spectrophotometer (Beckman Coulter, DU 730, Life Sciences UV/VIS, Lawrence, Kans., U.S.A.) over a 3-min reaction time. One unit of activity is defined as a change of 0.001 A234nm /min at pH 9 and 25◦ C and
obtained from the greatest initial linear slope (Ridolfi and others 2002).
Protein profile analysis by SDS-PAGE SDS-PAGE (50 μL, 10 wells, 10 to 125 kDa range) was used to run LOX after treatment. Sample (50 μL) was denatured by heating with an equal amount of XT buffer 2X in a hot-water bath at 100◦ C for 10 min. LOX (8 μg) was loaded in each well along with marker proteins (kDa; Precision Plus Protein All Blue Standards; Bio-Rad) . Electrophoresis was carried out for 1 h at 160 V using a high-current power supply (PowerPac HC, Bio-Rad). Gel Code Blue Safe Protein Stain (Pierce Biotechnology Inc., Rockford, Ill., U.S.A.) was used in staining the gels for 45 min. Gels were destained overnight using deionized water and densitometry performed using ImageJ (NIH, Bethesda, Md., U.S.A.) software (Girish and Vijayalakshmi 2004). Quantitative densitometry
Figure 1–Experimental workflow for analyzing PUV illumination parameters on the activity and protein profile of LOX.
Figure 2–Schematic representation of PUV treatment and temperature data acquisition.
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measurements of gels was subjected to statistical analysis (Paulo The above equations are simplified to form (Eq.) 6, as other factors remain constant during the experiment. The RA after treatment and others 2010). depends directly on PUV illumination time and inversely to the proximity from the PUV light source. A similar relation was used Protein fragment profile by RP-HPLC SDS-PAGE provided protein profile information within the by G´omez-L´opez and others (2005) to explain microbial inactirange of 10 to 250 kDa. To elucidate protein degradation below vation and can be extended by first-order inactivation kinetics. 10 kDa, treated samples (2 mL) were loaded onto centrifugal filter concentrators (Millipore, Bedford, Mass., U.S.A.; 10 kDa cutoff) l n (RA) = B · t · e −C·d (6) and centrifuged (30 min, 2000 × g, 5◦ C). Filtered samples were freeze-dried using a VIRTIS freeze-dryer model ES-53 (VIRTIS Co. Inc., Gardiner, N.Y., U.S.A.). Prefrozen filtrate samples were where dried (0◦ C, 350 mTorr) and reconstituted with distilled water to B is a constant specific to each specimen (s−1 ). 300 μL (a 6× concentration). Chromatography of the 0.05) was observed between k and D value for treatment distances of 8.5 cm and 11 cm from the quartz window, respectively. LOX RA with respect to PUV illumination time and distance from the quartz window is presented in Table 1. There were no significant (P > 0.05) changes in LOX activity for initial PUV illumination between control and treatment up to 2 s. Treatment distance from the quartz window, illumination time, and their mutual interaction (time∗distance) significantly (P < 0.05) affected overall inactivation of LOX. However, there was no statistical (P > 0.05) difference between treatments at distances 8.5 and 11 cm from the quartz window. Photo-thermal and/or photochemical reactions taking place during treatment mainly contribute to deactivation mechanisms Vol. 79, Nr. 1, 2014 r Journal of Food Science C11
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Nonthermal inactivation of soyLOX by PUV . . .
Nonthermal inactivation of soyLOX by PUV . . . Table 1–Percentage residual activity (A/A0 )∗ of LOX illuminated Eq. 6 with respect to illumination time t (s) and distance d (cm) with PUV at 3 and 5 different distances and times. The first- from the quartz window: order reaction coefficient of regression (R2 ), reaction constant (k), and D value was calculated for each treatment distance from (6) ln(A/A0 ) = −1.15 · t · e −0.15·d the quartz window. Distance∗∗∗
C: Food Chemistry
Time (s)∗∗ 0 1 2 4 8 16 k (s−1 ) D-value (s) R2
6 cm 66.67 37.04 18.52 3.03 1.12 0.482 5.6
± 16.60 aB ± 13.10 abB ± 4.40 bB ± 2.00 cB ± 1.70 dB ± 0.190 A ± 2.5 A 0.998
8.5 cm 100 ± 10.14 aA 74.07 ± 26.20 aC 48.15 ± 10.10 abC 22.22 ± 8.30 bC 7.81 ± 2.00 cC 1.6 ± 1.30 dC 0.299 ± 0.050 B 7.9 ± 1.2 AB 0.984
11 cm 85.19 59.26 33.33 16.71 2.23 0.245 9.5
± 10.10 aC ± 24.20 abC ± 8.30 bC ± 12.80 cC ± 0.40 dC ± 0.030 B ± 1.2 B 0.994
∗ Values ∗∗
obtained for n = 5, and expressed as mean ± standard deviation. Values having different smaller case alphabets in the same column are significantly (P < 0.05) different. ∗∗∗ Values having different capital case alphabets in the same row are significantly (P < 0.05) different.
of PUV on the enzyme protein (G´omez-L´opez and others 2007). Inactivation of LOX complies with the assumption of the photochemical inactivation mechanism by the UV region of the PUV spectrum. The log RA was directly proportional to PUV illumination time from 1 to 16 s and exponentially decreased with treatment distance in the range of 6 to 11 cm from the quartz window. The relationship for (Eq.) 5 resulted in an adequate coefficient of determination (R2 = 0.987). The estimated parameters and their standard errors are: B = –1.15 ± 0.11 (s−1 ), C = 0.15 ± 0.01(cm−1 ). Substituting these values gives the following modified
Overall LOX inactivation by PUV followed a first-order kinetic model. The half-life for conversion of native LOX to inactive state was independent of the initial activity of LOX and inversely proportional to the first-order rate-constant (k). The constant (k) was dependent on PUV treatment time and distance from the light source keeping other extrinsic (exposed sample surface area, thickness of the treated sample, initial temperature, and so on) and intrinsic factors (initial enzyme activity, composition, and so on) unchanged. LOX inactivation may involve a number of reversible (decomposition and denaturation) as well as irreversible (decomposition, aggregation, and coagulation) reactions (Lencki and others 1992). Inactivation of LOX followed similar first-order inactivation as other processing technologies either independently or as combinations. Processing technologies studied for LOX inactivation involved, microwaves (Kermasha and others 1993), pulse electric field (Min and others 2003), mano-thermal inactivation (Indrawati and others 1999), and mano-thermo-sonication (Lopez and Burgos 1995). First-order reaction rate constants for different processing technologies having similar LOX assay conditions are shown in Table 2. The rate-constant (k), that is, 0.245 ± 0.014 and 0.482 ± 0.084 s−1 for PUV illumination at treatment distances 11 and 6 cm, respectively, was found to be higher than reported values for other processes without a considerable rise in sample temperature (33 to 36◦ C for apex treatment at 6 cm, 16 s). The exception was the reaction constant (k) of 1.128 s−1 at 90◦ C with microwave treatment obtained based on 2 points by Kermasha and others (1993).
Figure 3–Seminatural logarithmic plot of LOX (residual activity) inactivation by PUV operating at 6, 8.5, and 11 cm from the quartz window with respect to illumination time consisting of 3 pulses per second. Error bars at each data point represent standard error, n = 5.
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Nonthermal inactivation of soyLOX by PUV . . .
Rate constant (s−1 )
Conventional water-bath heatinga
70◦ C 80◦ C 90◦ C 70◦ C 80◦ C 60◦ C
0.00443 0.01795 0.03842 0.03458 0.16161 0.00062
0.88 0.93 0.94 0.82 0.87 0.98
70◦ C 80◦ C 90◦ C (0.1 MPa, 68◦ C) (250 MPa, 68◦ C) (625 MPa, 25◦ C)
0.00176 0.00975 0.01464 0.00305 0.00298 0.00282
0.97 0.93 0.97 >0.95 >0.95 >0.95
Microwave heatinga Mixed modeb (water bath in microwave)
by PUV illumination time. The control and treatment sample at 1 s PUV illumination time were significantly (P < 0.05) different from treatments at 4 and 16 s (Figure 7). The LOX protein (≤10 kDa) samples treated at 6 cm distances from the quartz window during PUV treatment (Figure 7) showed a significant (P ≤ 0.05) change in total-response area from control, unlike samples treated at 8.5 and 11 cm. However, considerable variations were observed among the treated samples (≤10 kDa) total
Kermasha and others (1993). bath in microwave) Kermasha and others (1993). Indrawati and others (1999).
b (Water c
Lipoxygenase SDS-PAGE and RP-HPLC LOX (≤10 kDa) protein profile The SDS-PAGE for control and treated LOX samples is illustrated in Figure 4. The LOX protein band (approximately 100 kDa) (Todd and others 1990) appeared in samples treated at 6 cm (4a), 8.5 cm (4b), and 11 cm (4c) from the quartz window, respectively. No other bands appeared throughout the gel for treated and control samples. The treated LOX protein was subjected to size fractionation during SDS electrophoresis. Quantification of stained bands (75 to 125 kDa) was achieved by densitometry. Relative molecular mass of LOX after treatment ruled out the possibilities of cross-linking, agglomeration, and polymerization (Greenberg 1979; Curley and Lawrence 1999) of protein due to UV radiation. The SDS gel densitometry disclosed the pattern of degradation for LOX protein (Figure 5). Significant (P < 0.05) reduction in band density compared with controls was observed for treatment times greater or equal to 2 s of PUV illumination. Band degradation patterns for treatment distances 6 and 8.5 cm were found to be similar but significantly (P < 0.05) different from the 11-cm distance from the quartz window due to reduction in PUV intensity. The increase in PUV source distance from the samples was inversely proportional to inactivation of microorganisms and spores during treatment (Jun and others 2003; Krishnamurthy and others 2007). A decline in gel lane densities at all distances with an increase in PUV illumination time indicated photo degradation of LOX, and further RP-HPLC analysis was obtained to support this findings. Protein profiles by SDS-PAGE indicated photo degradation of the soy LOX band for PUV-treated samples, without any indication of protein fragmentation or aggregation. The possibility of LOX protein degradation to smaller fragments by the action of PUV was postulated. RP-HPLC was employed to analyze the ≤10 kDa protein fractions after treatment. The different PUV treatment samples revealed changes in the chromatogram patterns with respect to the control. The PUV-treated samples at 6 cm from the quartz window are illustrated in Figure 6. The elution profiles from 6a to 6c were different, as new peaks occurred when PUV time increased. The peak before void volume (that is, negative baseline at RT = 4.0 min) did not affect the quatificaiton Figure 4–Protein profile for SDS-PAGE control and PUV illuminated LOX at of LOX peaks (RT > 4.2 min) . The complete response area different times (1, 2, 4, 8, 16 s) and 3 distances from quartz window 6 (A), on the LOX protein (≤10 kDa) samples were analyzed by taking 8.5 (B), and 11 (C) cm, respectively. Vol. 79, Nr. 1, 2014 r Journal of Food Science C13
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Table 2–First-order reaction rate constants for lipoxygenase in- the combined area of the peaks after the void volume. The totalactivation by different processing methods using similar sub- response area of fragments (≤10 kDa) was affected (P ≤ 0.05) strate, activity assay, and sample preparation.
Nonthermal inactivation of soyLOX by PUV . . .
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response area. The random nature of protein fragmentation under ultraviolet radiation has been proposed (Friso and others 1994; Cui and others 2005). RP-HPLC profile patterns and gel electrophoresis of decreasing LOX activity provided sufficient evidence of enzyme inactivation due to protein degradation into smaller fragments (≤10 kDa) when exposed to PUV illumination. Similar results of PUV on the marked reduction in protein bands intensity on SDS-PAGE were seen for tropomyosin (36 kDa) in shrimp (Shriver and others 2011) as well as the soy allergen proteins glycinin (14 to 34 kDa) and β-conglycinin (50 kDa) (Yang and others 2010). PUV treatment showed the disappearance of distinct protein bands for LOX from the gel confirming protein distruction with out agglomeration. The distinct change in chromatograms after 4.2 min compared to control resulting in an increase in total-response area might have been due to the presence of UV-absorbing aromatic residues in the PUV-treated LOX protein fragment samples. Hence, involvement and participation of UV photosensitive amino acids might have occurred during PUV treatment of LOX samples by photochemical reactions resulting in photolysis of the protein.
Influence of PUV on LOX protein The action of ultraviolet light responsible for LOX inactivation can be considered as the dominating mechanism in PUV processing. For the majority of PUV treatments (≤8 s), the rise in temperature was in the range of 5 to 7◦ C. The combined photothermal and photochemical effects might occur for PUV treatments involving longer illumination time. However, to identify the specific mechanism(s) of LOX inactivation by PUV, a detailed study on protein structure for treated LOX is required. Inactivation of enzymes by ultraviolet light involves chemical reactions and further photolysis of disulfide and aromatic residues (McLaren and Luse 1961). The cause of UV inactivation of enzymes might be the selective photochemical destruction of certain amino acids, that is, cysteine, tryptophan, tyrosine, and phenylalanine (Vladimirov and others 1970). The exclusive absorption of light energy by these amino acids causes photolysis and structural destruction of proteins. PUV inactivation of LOX was observed due to protein fragmentation. SDS-PAGE revealed the loss in the LOX band with
treatment while RP-HPLC did show an increase in fragments as treatment time increased. It is speculated the PUV inactivation of LOX is slightly different from UV radiation. PUV inactivation might involve histidine and other light-sensitive amino acids vulnerable to visible light as well as ultraviolet light, and breakage of hydrogen bonds for inactivation-denaturation of enzymes (Luse and McLaren 1963). Native LOX contains all the major photosensitive amino acids, that is, cysteine (Vliegenthart and Veldink 1982), tryptophan (Shibata and others 1987), tyrosine, and phenylalanine (Park and others 2005). Therefore, photolysis and structural destruction of proteins under intense UV portion of the PUV spectrum is feasible.
Temperature profile Initial temperature of the enzyme samples began at 20 ± 1.5◦ C and rose to 27 ± 1.3, 28.5 ± 1.5, and 33.5 ± 1.8◦ C for treatment distances 11, 8.5, and 6 cm from the quartz window, respectively. The emission intensity (I) is radiation energy per unit time, wavelength interval, surface area, and solid angle (Kaviany 2011). The PUV radiation energy was solely responsible for the heat gained in the system during treatment. It can be equated to the heat energy estimated by mass, specific heat, and increase in temperature (Slowinski and others 2011) assuming the treatment parameters (wavelength, surface area, and solid angle) were constant during treatment and radiation time was variable. The relation can be simplified to the following empirical equation (Eq. 6) m · c p · (T − T0 )α I · t
where, m = mass of the sample (g), cp = specific heat capacity [W.s.(g.◦ C) –1 ], T = observed temperature in◦ C, T0 = initial temperature in◦ C, I = intensity of PUV spectra range effective for heating (W.cm−2 ), and t = PUV illumination time (s). Since most of the reaction and treatment parameters (composition, mass, orientation during treatment, and specific heat of system) remained fixed, using (Eq.) 5 and 7 with the assumption that the parameters (treatment medium, wavelength spectra of each pulse, physical, and chemical properties of the system) remain constant during the experiment, (Eq.) 7 resulted. Thus, a simplified mathematical form was obtained 7 explaining the temperature rise during
Figure 5–Quantitative gel densitometry measurements of gel lanes (between the 125 and 75 kDa markers) as determined using ImageJ software. Data points obtained in triplicates, normalized with respect to the control value. Each axis point containing the different alphabet after underscore sign was significantly different (P < 0.05). Error bars at each data point represent standard error, n = 3.
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Nonthermal inactivation of soyLOX by PUV . . . depends upon absorption factor and initial concentration of the treated sample. T = (T − T0 ) = ε · t · e γ d
Estimations were obtained for ε, γ parameters using a fitted model and resulted in 2.11 ± 0.13 (◦ C.s−1 ) and –0.13 ± 0.01 (cm−1 )
Figure 6–RP-HPLC chromatograms of control (A) and PUV-treated LOX samples for 4 s (B) and 16 s (C) at 6 cm from the quartz window. Samples were filtered through a centrifugal filter concentrator (≤10 kDa), freeze-dried, and reconstituted to 300 μL by DI water.
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PUV treatment. Hiramoto (Eq.) (1984) radiation intensity (I) was substituted with addition of the proportionality constant “ε” and combined exponent constant “γ ” for balancing the equation (Eq.) 7 dimentionally to get Eq. 8. The “ε” is a numerical-constant depending upon the mass (m), surface-area, specific heat (cp ), and radiation intensity (I0 ) applied to the treated sample (◦ C.s−1 ); γ is a constant (cm−1 ) related to radiation absorption factor and
Nonthermal inactivation of soyLOX by PUV . . . values, respectively. These values were substituted in 8 to develop the relation between rise in temperature profile (T = T-T0 ) with respect to illumination time t (s) and distance d (cm) from the quartz window 9 with a coefficient of determination of R2 = 0.95.
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T = 2.11 · t · e −0.13·d
PUV illumination caused a rise in sample temperature (T). The difference between the initial and final temperature was used to measure the rise in temperature between the samples during treatment at different distances from the quartz window. The temper-
ature rise profile with illumination time obtained by the infrared and K-type thermocouple is demonstrated in Figure 8. The distance from the quartz window, duration of PUV illumination, and mutual interaction of both variables (time∗distance) significantly (P < 0.05) affected the temperature profile (T-T0 ) obtained by Ktype thermocouple. The overall surface temperature rise profile was observed by the infrared-thermometer. However, no significant (P > 0.05) difference was observed between the 2 temperature rise profiles at experimental data points obtained by thermocouple and infrared thermometer. The treatment time ranging from 1 to 16 s resulted in similar temperature rises in samples for both temperature data-acquisition methods. Uniform distribution of
Figure 7–RP-HPLC quantification of chromatographic peaks, LOX protein ≤ 10 kDa (RT 4.5 to 4.7 min), and total response area comprising of LOX protein and earlier peak eluted (RT 3.3 to 3.7 min). The error bar represents the standard error for the data point, n = 3.
Figure 8–Temperature rise profile for samples with respect to initial temperature (20 ± 1.5◦ C). Results obtained using different PUV illumination time at 6, 8.5, and 11 cm from the quartz window by K-type thermocouple using data logger and infrared thermometer (IR). Each error bar represents the standard deviation from a mean, n = 3.
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Nonthermal inactivation of soyLOX by PUV . . .
ε constant specific to the sample properties (◦ C.s−1 ) γ constant specific to sample radiation absorption properties (cm−1 ) Acronyms FDA LOX MWCO PAGE PUV RBD RP-HPLC
Food and Drug Administration lipoxygenase molecular weight cut off polyacrylamide gel electrophoresis pulse ultraviolet light randomized block design reverse phase high-performance liquid chromatography SDS sodium dodecyl sulfate
PUV illumination time and treatment distance from the quartz window successfully modeled LOX inactivation. First-order kinetic models adequately described PUV inactivation of LOX. Treatment distance from the quartz window, illumination time, References and their mutual interaction (time∗distance) significantly (P < Aitken A, Learmonth M. 1996. Protein determination by UV absorption. The protein protocols handbook. Berlin: Springer. p 3–6. 0.05) affected inactivation of LOX. The overall loss in activity of GE, Barrett DM. 2003. Thermal inactivation of lipoxygenase and hydroperoxytrienoic LOX by PUV was accounted for by LOX protein fragmentation. Anthon acid lyase in tomatoes. Food Chem 81(2):275–9. PUV distruction of LOX protein was governed primarily by PUV Axelrod B, Cheesbrough TM, Laakso S. 1981. Lipoxygenase from soybeans: EC 1.13. 11.12 Linoleate: oxygen oxidoreductase. Methods Enzymol 71:441–51. illumination time. Information from this research would be use- Bank H, John J, Schmehl M, Dratch R. 1990. Bactericidal effectiveness of modulated UV light. ful for using PUV illumination in enzyme inactivation, LOX, in Appl Environ Microbiol 56(12):3888. Barbosa-C´anovas GV, Pothakamury UR, Palou E, Swanson BG. 1998. Nonthermal preservation particular. of foods. New York: Marcel Dekker. p 139–61
Acknowledgments The authors gratefully acknowledge Dr. Yagiz Yavuz, Univ. of Florida, Dept. of Food Science and Human Nutrition, for his intellectual input concerning protein analysis in this work.
Nomenclature Symbols A observed enzyme activity at the particular time (units/mL) A0 initial enzyme activity (units/mL) B constant specific to each specimen (s−1 ) C constant specific to each sample (cm−1 ) cp specific heat capacity [W.s.(g.◦ C)−1 ] D decimal reduction time (s) d distance from the quartz window (cm) e exponential function I intensity of the radiation (W.cm−2 ) I0 intensity of the radiation applied to the surface (W.cm−2 ) k inactivation rate (s−1 ) m mass of the sample (g) kDa kilodalton MPa mega pascal (psi) N the number of organisms presented after treatment No the number of organisms presented before treatment P constant specific to the organism (W.s.cm−2 ) RA remaining residual enzyme activity relative to initial activity RT retention time (min) t PUV illumination (s) T temperature (◦ C) T0 initial temperature of the sample (◦ C) Subscripts 0 initial conditions Greek Letters α the ultraviolet absorption factor of the organism (cm−1 ) β dimentionless constant
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temperature during illumination might result from the shallow thickness 2 to 3 mm of the treated samples. The lower rise in temperature occurred with a decrease in proximity of enzyme samples from PUV source. The observations were similar to findings of PUV temperature rise profiles of 10 and 7.6◦ C corresponding to 9.5 and 14.5 cm from source on eggshells (Keklik and others 2010). The temperature rise profile data were also similar to PUV illumination on honey (2 mm, 8 cm) studied by Hillegas and Demirci (2003). However, during PUV treatment, the ease for thermal conduction by LOX solutions caused higher temperature rise profiles (9◦ C, 8 cm) when compared with the data (5◦ C, 8 cm) for salmon muscle (Ozer and Demirci 2006).
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