Nonthermal Inactivation of Soy (Glycine Max Sp.

)
Lipoxygenase by Pulsed Ultraviolet Light
Bhaskar A. Janve, Wade Yang, Maurice R. Marshall, José I. Reyes-De-Corcuera, and Taha M. Rababah
C: Food Chemistry

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)

Practical Application: 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 phylls (King and Klein 1987). Hence, inactivation of deleterious

Lipoxygenase (linoleate: oxygen oxidoreductase, EC
1.13.11.12; LOX) is one of the most studied groups of en-
zymes. 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 (Boy-
ington 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 Demirdöven
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 as-
sociated with quality deterioration by producing off-flavor, odor
production, and loss of pigments such as carotenes and chloro-
MS 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: wade.yang@ufl.edu).
enzymes is considered a critical processing step for food preserva-
tion. Blanching is one of the most common unit operations for
inactivating enzymes, particularly peroxidase. Blanching temper-
ature and time are adjusted for inactivating peroxidase because its
assay is simple and rapid. However, complete inactivation of perox-
idase often leads to overblanching. A residual activity (RA) of 3%
to 10% peroxidase in blanched and frozen-food products is recom-
mended (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; In-
drawati 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 LOX-
related oxidation. The detrimental effects of thermal treatment
on organoleptic quality, heat liable nutrients, and volatile com-
ponents 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 heat-
induced degradation on organoleptic and nutritive properties has
resulted in developing nonthermal processes (Mertens and Knorr
1992).



C 2013 Institute of Food Technologists
R

C8 Journal of Food Science r Vol. 79, Nr. 1, 2014 doi: 10.1111/1750-3841.12317

Further reproduction without permission is prohibited
Nonthermal inactivation of soyLOX by PUV . . .
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), miti-
gating allergens (Chung and others 2008; Yang and others 2010),
and decontaminating food surfaces and packaging materials (Guil-
lou and others 2007) are among the emerging applications of PUV
in foods. PUV is comprised of high-frequency pulses of broad-
spectrum 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-Cánovas
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 re-
gions, 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 mod-
ifications, 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 absorp-
tion at 280 nm (Hollosy 2002). Peptide bonds [–C(O)–NH–] in
proteins exhibit a strong and weak absorption in the Far UV re-
gion 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 pheny-
lalanine 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 ab-
sorbed 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 in-
activate enzymes. Dunn and others (1989) showed that 2 to 5
flashes of light with approximately 3 J cm−2 were enough to in-
hibit 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 parame-
C: Food Chemistry
ters affect enzyme properties. Specific propertiesevaluated include
enzyme inactivation kinetics, LOX protein, and sample tempera-
ture profile. PUV parameters examined include duration of illu-
mination (Hiramoto 1984) and the distance (Gómez-López and
others 2005) from the light source.
Materials and Methods
Materials
Soybean LOX type 1 B (EC 1.13.11.12, Lot # 050M1910V),
linoleic acid, and Tween 20 were obtained from Sigma Chemi-
cal 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 electrophore-
sis (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, LH-
840 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 ran-
domly 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 pro-
vided 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 dur-
ing treatment was measured using a handheld noncontact infrared
thermometer (Omega OS423-LS, Omega Technologies, Stam-
ford, Conn., U.S.A.). The bulk temperature profile of the sample
during treatment was recorded by K-type thermocouple using a
PicoR 8-channel thermocouple data logging interface (PC-08) at-
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
 Nonthermal inactivation of soyLOX by PUV . . .
Lipoxygenase activity
The substrate was prepared fresh according to a modified
method of Axelrod (1981). Linoleic acid (C18 H32 O2 , cis-9, cis-
12-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 as-
say was used to measure LOX activity at 25◦ C. The assay mix-
C: Food Chemistry
ture 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 mix-
ture. 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
Figure 2–Schematic representation of PUV treatment and temperature data acquisition.
C10 Journal of Food Science r Vol. 79, Nr. 1, 2014
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 heat-
ing 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 Stan-
dards; 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 densito-
metry performed using ImageJ (NIH, Bethesda, Md., U.S.A.) soft-
ware (Girish and Vijayalakshmi 2004). Quantitative densitometry
Figure 1–Experimental workflow for analyzing
PUV illumination parameters on the activity
and protein profile of LOX.
Nonthermal inactivation of soyLOX by PUV . . .

measurements of gels was subjected to statistical analysis (Paulo The above equations are simplified to form (Eq.) 6, as other factors
and others 2010). remain constant during the experiment. The RA after treatment
depends directly on PUV illumination time and inversely to the
Protein fragment profile by RP-HPLC proximity from the PUV light source. A similar relation was used
SDS-PAGE provided protein profile information within the by Gómez-López and others (2005) to explain microbial inacti-
range 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

C: Food Chemistry
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
300 μL (a 6× concentration). B is a constant specific to each specimen (s−1 ).
Chromatography of the <10 kDa fractions (50 μL) was carried t is PUV illumination time (s).
out on a Perkin Elmer HPLC system consisting of a series 200 d is distance from the quartz window (cm).−1
autosampler, series 200 LC pump, and series 235C diode array C is a constant specific to each sample (cm ).
detector using an Agilent Zorbax SB-C18 (5.0 μm, 4.6 × 250
mm) HPLC column; solvent: acetonitrile:water: trifluoroacetic Statistical analysis
acid (TFA) at (60:39.9:0.1) under isocratic conditions; flow rate: All experiments in this study followed RBD. Data were ana-
1 mL/min; and detection: 280 nm. lyzed using factorial analysis of variance (ANOVA), considering
main effects and two-way interactions. The effect of treatment
Inactivation modeling of PUV-treated LOX distances from the quartz window on k and D-value was ana-
The RA of LOX obtained after each PUV treatment was de- lyzed using one-way ANOVA. Fisher’s least significant difference
fined as: (LSD) was used for multiple mean comparisons. Inactivation ki-
netic data were analyzed using regression models on seminatural
RA = A/A0 (1) logarithmic plots. Linear and nonlinear model fitting of inac-
tivation data and temperature rise was achieved by a least square
where A is LOX activity after treatment, and A0 is initial LOX method with model parameter estimation (SAS Model Procedure).
activity before treatment. Experimental data were fitted to the The prediction performance and total variation explained by the
first-order inactivation model (Eq.) 2. Inactivation rate constant k models were evaluated using the coefficient of determination
was computed from the slope of the regression curve for ln (RA) (R2 value). Data analysis was performed using the statistical analy-
with respect to PUV illumination time (t). sis system (SAS) software, version 9.1 (SAS Inst. Inc., Cary, N.C.,
U.S.A.).
l n (RA) = −kt (2)

Decimal reduction times (D) were calculated using (Eq.) 3, where Results and Discussion
k is the first-order inactivation rate constant (s−1 ).
D = 2.303/k (3)
A mathematical relationship between RA of LOX to that of PUV
time and distance was developed by considering the nonthermal
photochemical inactivation occurring for the ultraviolet region of
the PUV spectrum. The relationship between RA to illumination
time and distance from lamp source to sample by photo-chemical
inactivation can be expressed mathematically by Hiramoto (1984)
equations (Eq.) 4 and (Eq.) 5, where No and N are the number of
organisms present before and after PUV irradiation, respectively. P
is a specific-constant for the organism (W.cm−2 .s), e is the base of
the natural logarithm, I is the intensity of the pulsed ultraviolet rays
in the wavelength range effective for inactivating the organisms
(W.cm−2 ), Io is the PUV intensity applied to the surface layer
of the organisms (W.cm−2 ), t is the irradiation time (s), α is the
ultraviolet absorption factor of the organism (cm−1 ), β is constant,
and d is distance of lamp to surface upper layer of the organisms
(cm).
N −I.t
ln ( )= (4)
N0 p (P > 0.05) difference between treatments at distances 8.5 and 11
I = I0 · e −α·β·N0 ·d (5)
Lipoxygenase activity
Inactivation of LOX activity was achieved by PUV processing
with little rise in sample temperature. PUV treatment of 16 s at a
proximity of 11 to 6 cm from the quartz window resulted in 1.5
and 3.5 log (95% to 99.95%) reductions in LOX initial activity
and temperature rise of 6 and 17◦ C, respectively.
LOX inactivation data (Table 1) followed first-order reaction
kinetics with PUV illumination time (Figure 3). Kinetic param-
eters, that is, reaction rate constant (k) and D-value for LOX in-
activation, were significantly (P < 0.05) influenced by treatment
distance from the quartz window and PUV illumination. How-
ever, no significant difference (P > 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
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

 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
the quartz window. ln(A/A0 ) = −1.15 · t · e −0.15·d (6)
Distance∗∗∗ Overall LOX inactivation by PUV followed a first-order kinetic
Time (s)∗∗ 6 cm 8.5 cm 11 cm model. The half-life for conversion of native LOX to inactive
C: Food Chemistry

0 100 ± 10.14 aA state was independent of the initial activity of LOX and inversely
1 66.67 ± 16.60 aB 74.07 ± 26.20 aC 85.19 ± 10.10 aC proportional to the first-order rate-constant (k). The constant (k)
2 37.04 ± 13.10 abB 48.15 ± 10.10 abC 59.26 ± 24.20 abC was dependent on PUV treatment time and distance from the
4 18.52 ± 4.40 bB 22.22 ± 8.30 bC 33.33 ± 8.30 bC light source keeping other extrinsic (exposed sample surface area,
8 3.03 ± 2.00 cB 7.81 ± 2.00 cC 16.71 ± 12.80 cC
16 1.12 ± 1.70 dB 1.6 ± 1.30 dC 2.23 ± 0.40 dC
thickness of the treated sample, initial temperature, and so on)
k (s−1 ) 0.482 ± 0.190 A 0.299 ± 0.050 B 0.245 ± 0.030 B and intrinsic factors (initial enzyme activity, composition, and so
D-value (s) 5.6 ± 2.5 A 7.9 ± 1.2 AB 9.5 ± 1.2 B on) unchanged. LOX inactivation may involve a number of re-
R2 0.998 0.984 0.994 versible (decomposition and denaturation) as well as irreversible
∗
Values obtained for n = 5, and expressed as mean ± standard deviation. (decomposition, aggregation, and coagulation) reactions (Lencki
∗∗
Values having different smaller case alphabets in the same column are significantly (P < and others 1992). Inactivation of LOX followed similar first-order
0.05) different.
∗∗∗
Values having different capital case alphabets in the same row are significantly (P < inactivation as other processing technologies either independently
0.05) different. or as combinations. Processing technologies studied for LOX inac-
tivation 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
of PUV on the enzyme protein (Gómez-López and others 2007). and Burgos 1995). First-order reaction rate constants for different
Inactivation of LOX complies with the assumption of the photo- processing technologies having similar LOX assay conditions are
chemical inactivation mechanism by the UV region of the PUV shown in Table 2. The rate-constant (k), that is, 0.245 ± 0.014
spectrum. The log RA was directly proportional to PUV illu- and 0.482 ± 0.084 s−1 for PUV illumination at treatment distances
mination time from 1 to 16 s and exponentially decreased with 11 and 6 cm, respectively, was found to be higher than reported
treatment distance in the range of 6 to 11 cm from the quartz values for other processes without a considerable rise in sample
window. The relationship for (Eq.) 5 resulted in an adequate coef- temperature (33 to 36◦ C for apex treatment at 6 cm, 16 s). The
ficient of determination (R2 = 0.987). The estimated parameters exception was the reaction constant (k) of 1.128 s−1 at 90◦ C with
and their standard errors are: B = –1.15 ± 0.11 (s−1 ), C = 0.15 ± microwave treatment obtained based on 2 points by Kermasha and
0.01(cm−1 ). Substituting these values gives the following modified 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.

C12 Journal of Food Science r Vol. 79, Nr. 1, 2014

Nonthermal inactivation of soyLOX by PUV . . .

Table 2–First-order reaction rate constants for lipoxygenase in- the combined area of the peaks after the void volume. The total-
activation by different processing methods using similar sub- response area of fragments (≤10 kDa) was affected (P ≤ 0.05)
strate, activity assay, and sample preparation.
by PUV illumination time. The control and treatment sample at
Method Condition Rate constant (s−1 ) (R2 ) 1 s PUV illumination time were significantly (P < 0.05) differ-
Conventional water-bath 60◦ C 0.00120 0.99
ent from treatments at 4 and 16 s (Figure 7). The LOX protein
heatinga (≤10 kDa) samples treated at 6 cm distances from the quartz
70◦ C 0.00443 0.88 window during PUV treatment (Figure 7) showed a significant

C: Food Chemistry
80◦ C 0.01795 0.93 (P ≤ 0.05) change in total-response area from control, unlike
90◦ C 0.03842 0.94 samples treated at 8.5 and 11 cm. However, considerable varia-
Microwave heatinga 70◦ C 0.03458 0.82
80◦ C 0.16161 0.87 tions were observed among the treated samples (≤10 kDa) total
Mixed modeb (water bath 60◦ C 0.00062 0.98
in microwave)
70◦ C 0.00176 0.97
80◦ C 0.00975 0.93
90◦ C 0.01464 0.97
Manothermoinactivationc (0.1 MPa, 68◦ C) 0.00305 >0.95
(250 MPa, 68◦ C) 0.00298 >0.95
(625 MPa, 25◦ C) 0.00282 >0.95
a
Kermasha and others (1993).
b
(Water bath in microwave) Kermasha and others (1993).
c
Indrawati and others (1999).

Lipoxygenase SDS-PAGE and RP-HPLC LOX (≤10 kDa)

protein profile
The SDS-PAGE for control and treated LOX samples is illus-
trated 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, re-
spectively. 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) re-
duction 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 indi-
cation 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, neg-
ative 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

 Nonthermal inactivation of soyLOX by PUV . . .
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 decreas-
ing LOX activity provided sufficient evidence of enzyme inactiva-
tion due to protein degradation into smaller fragments (≤10 kDa)
when exposed to PUV illumination. Similar results of PUV on
C: Food Chemistry
the marked reduction in protein bands intensity on SDS-PAGE
were seen for tropomyosin (36 kDa) in shrimp (Shriver and oth-
ers 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 ag-
glomeration. 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 aro-
matic residues in the PUV-treated LOX protein fragment sam-
ples. 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 pro-
cessing. For the majority of PUV treatments (≤8 s), the rise in
temperature was in the range of 5 to 7◦ C. The combined photo-
thermal and photochemical effects might occur for PUV treat-
ments 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. Inactiva-
tion 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 frag-
mentation. SDS-PAGE revealed the loss in the LOX band with
C14 Journal of Food Science r Vol. 79, Nr. 1, 2014
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 vul-
nerable 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 photo-
sensitive amino acids, that is, cysteine (Vliegenthart and Veldink
1982), tryptophan (Shibata and others 1987), tyrosine, and pheny-
lalanine (Park and others 2005). Therefore, photolysis and struc-
tural 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, wave-
length 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 en-
ergy 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 (7)
where, m = mass of the sample (g), cp = specific heat capacity
[W.s.(g.◦ C) –1 ], T = observed temperature in◦ C, T0 = initial tem-
perature 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, ori-
entation 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 mathemati-
cal 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.
Nonthermal inactivation of soyLOX by PUV . . .
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 de-
pending 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
depends upon absorption factor and initial concentration of the
treated sample.
T = (T − T0 ) = ε · t · e γ d (8)
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 )
C: Food Chemistry
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.
Vol. 79, Nr. 1, 2014 r Journal of Food Science C15
 Nonthermal inactivation of soyLOX by PUV . . .
values, respectively. These values were substituted in 8 to de-
velop 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.
T = 2.11 · t · e −0.13·d (9)
C: Food Chemistry
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 treat-
ment at different distances from the quartz window. The temper-
C16 Journal of Food Science r Vol. 79, Nr. 1, 2014
ature rise profile with illumination time obtained by the infrared
and K-type thermocouple is demonstrated in Figure 8. The dis-
tance 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 K-
type thermocouple. The overall surface temperature rise profile
was observed by the infrared-thermometer. However, no signifi-
cant (P > 0.05) difference was observed between the 2 temperature
rise profiles at experimental data points obtained by thermocou-
ple 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.
Nonthermal inactivation of soyLOX by PUV . . .
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 prox-
imity 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).
Conclusions
PUV illumination time and treatment distance from the quartz
window successfully modeled LOX inactivation. First-order ki-
netic 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
0.05) affected inactivation of LOX. The overall loss in activity of
LOX by PUV was accounted for by LOX protein fragmentation. Anthon
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
illumination time. Information from this research would be use- Bank
ful for using PUV illumination in enzyme inactivation, LOX, in
particular.
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
ε constant specific to the sample properties (◦ C.s−1 )
γ constant specific to sample radiation absorption properties
(cm−1 )
Acronyms
FDA Food and Drug Administration
C: Food Chemistry
LOX lipoxygenase
MWCO molecular weight cut off
PAGE polyacrylamide gel electrophoresis
PUV pulse ultraviolet light
RBD randomized block design
RP-HPLC reverse phase high-performance liquid chromatogra-
phy
SDS sodium dodecyl sulfate
handbook. Berlin: Springer. p 3–6.
GE, Barrett DM. 2003. Thermal inactivation of lipoxygenase and hydroperoxytrienoic
acid lyase in tomatoes. Food Chem 81(2):275–9.
Linoleate: oxygen oxidoreductase. Methods Enzymol 71:441–51.
H, John J, Schmehl M, Dratch R. 1990. Bactericidal effectiveness of modulated UV light.
Appl Environ Microbiol 56(12):3888.
Barbosa-Cánovas GV, Pothakamury UR, Palou E, Swanson BG. 1998. Nonthermal preservation
of foods. New York: Marcel Dekker. p 139–61
Barrett DM, Theerakulkait C. 1995. Quality indicators in blanched, frozen, stored vegetables. J
Food Technol 49(1):62–5.
Baysal T, Demirdöven A. 2007. Lipoxygenase in fruits and vegetables: a review. Enzyme Microb
Technol 40(4):491–6.
Boyington JC, Amzel LM. 1993. Structure of soybean lipoxygenase-1. Biochem Soc Trans
21:744–8.
Chasteen ND, Grady JK, Skorey KI, Neden KJ, Riendeau D, Percival MD. 1993. Charac-
terization of the non-heme iron center of human 5-lipoxygenase by electron paramagnetic
resonance, fluorescence, and ultraviolet-visible spectroscopy: redox cycling between ferrous
and ferric states. J Biochem 32(37):9763–71.
Chung SY, Yang W, Krishnamurthy K. 2008. Effects of pulsed UV-light on peanut allergens in
extracts and liquid peanut butter. J Food Sci 73(5):C400–4.
Cui W, Thompson MS, Reilly JP. 2005. Pathways of peptide ion fragmentation induced by
vacuum ultraviolet light. J Am Soc Mass Spectrom 16(8):1384–98.
Curley K, Lawrence DS. 1999. Light-activated proteins. Curr Opin Chem Biol 3(1):84–8.
Davies MJ, Truscott RJ. 2001. Photo-oxidation of proteins and its role in cataractogenesis.
J Photochem Photobiol B 63(1):114–25.
De Corcuera JIR, Cavalieri R, Powers J. 2003. Prototype instruments for laboratory and on-line
measurement of lipoxygenase activity. Food Sci Technol Int 9(1):5–9.
Dunn JE, Clark RW, Asmus JF, Pearlman JS, Boyer K, Painchaud F, Hofmann GA, inventors.
1989. Methods for preservation of foodstuffs. U.S. Patent 4,871,559.
Dunn J, Ott T, Clark W. 1995. Pulsed-light treatment of food and packaging. J Food Technol
49(9):95–8
Elmnasser NEN, Guillou SGS, Leroi FLF, Orange NON, Bakhrouf ABA, Federighi MFM.
2007. Pulsed-light system as a novel food decontamination technology: a review. Can J
Microbiol 53(7):813–21.
Eskin NAM, Grossman S, Pinsky A, Whitaker JR. 1977. Biochemistry of lipoxygenase in relation
to food quality. Crit Rev Food Sci Nutr 9(1):1–40.
Friso G, Barbato R, Giacometti G, Barber J. 1994. Degradation of D2 protein due to UV-B
irradiation of the reaction centre of photosystem II. FEBS Lett 339(3):217–21.
Giese N, Darby J. 2000. Sensitivity of microorganisms to different wavelengths of UV
light: implications on modeling of medium pressure UV systems. Water Res 34(16):4007–
13.
Girish V, Vijayalakshmi A. 2004. Affordable image analysis using NIH Image/ImageJ. Indian J.
Cancer 41(1):47.
Gómez-López V, Devlieghere F, Bonduelle V, Debevere J. 2005. Factors affecting the inactiva-
tion of micro-organisms by intense light pulses. J Appl Microbiol 99(3):460–70.
Gómez-López VM, Ragaert P, Debevere J, Devlieghere F. 2007. Pulsed light for food decon-
tamination: a review. Trends Food Sci Technol 18(9):464–73.
Green S, Baskaran N, Swanson BG. 2003. High-intensity light. In: Zeuthan P, Sorenson LB,
editors. Food preservation techniques. Boca Raton, Fla.: CRC Press. p 284–302.
Greenberg JR. 1979. Ultraviolet light-induced crosslinking of mRNA to proteins. Nucleic Acids
Res 6(2):715–32.
Guillou S, Leroi F, Orange N, Bakhrouf A, Federighi M, Elmnasser N. 2007. Pulsed-light
system as a novel food decontamination technology: a review. Can J Microbiol 53(7):813–21.
Gunes B, Bayindirli A. 1993. Peroxidase and lipoxygenase inactivation during blanching of green
beans, green peas and carrots. Lebensm Wiss Technol 26(5):406–10.
Hillegas SL, Demirci A. 2003. Inactivation of Clostridium sporogenes in clover honey by pulsed
UV-light treatment. CIGR J AE Sci Res Dev 5:6–7.
Hiramoto T, inventor. 1984. Method of sterilization. Tokyo, Japan: Ushio Denki
Kabushikikaisha: U.S. Patent 4,464,336.
Hollosy F. 2002. Effects of ultraviolet radiation on plant cells. Micron 33(2):179–97.
Indrawati, Van Loey AM, Ludikhuyze LR, Hendrickx ME. 1999. Soybean lipoxygenase inacti-
vation by pressure at subzero and elevated temperatures. J Agric Food Chem 47(6):2468–74.
Vol. 79, Nr. 1, 2014 r Journal of Food Science C17
 Nonthermal inactivation of soyLOX by PUV . . .
Jun S, Irudayaraj J, Demirci A, Geiser D. 2003. Pulsed UV-light treatment of corn meal for
inactivation of Aspergillus niger spores. Int J Food Sci Technol 38(8):883–8.
Kaviany M. (2011). Essentials of heat transfer: principles, materials, and applications. Cambridge,
United Kingdom: Cambridge Univ. Press. p 328.
Keklik NM, Demirci A, Patterson PH, Puri VM. 2010. Pulsed UV light inactivation of
Salmonella enteritidis on eggshells and its effects on egg quality. J Food Prot 73(8):1408–
15.
Kermasha S, Bisakowski B, Ramaswamy H, Van de Voort F. 1993. Thermal and microwave
inactivation of soybean lipoxygenase. Lebensm Wiss Technol 26(3):215–9.
C: Food Chemistry
King DL, Klein BP. 1987. Effect of flavonoids and related compounds on soybean lipoxygenase
1 activity. J Food Sci 52(1):220–1.
Krishnamurthy K, Demirci A, Irudayaraj J. 2007. Inactivation of Staphylococcus aureus in milk
using flow-through pulsed UV-light treatment system. J Food Sci 72(7):M233–9.
Krishnamurthy K, Tewari JC, Irudayaraj J, Demirci A. 2010. Microscopic and spectroscopic
evaluation of inactivation of Staphylococcus aureus by pulsed UV light and infrared heating.
Food Bioprocess Technol 3(1):93–104.
Lencki RW, Arul J, Neufeld RJ. 1992. Effect of subunit dissociation, denaturation, aggregation,
coagulation, and decomposition on enzyme inactivation kinetics: I. First-order behaviour.
Biotechnol Bioeng 40(11):1421–6.
Lopez P, Burgos J. 1995. Lipoxygenase inactivation by manothermosonication: effects of soni-
cation on physical parameters, pH, KCl, sugars, glycerol, and enzyme concentration. J Agric
Food Chem 43(3):620–5.
Luse R, McLaren A. 1963. Mechanism of enzyme inactivation by ultraviolet light
and the photochemistry of amino acids (AT 2537 Å)∗. Photochem Photobiol 2(3):
343–60.
Mach H, Middaugh CR, Lewis RV. 1992. Statistical determination of the average values of the
extinction coefficients of tryptophan and tyrosine in native proteins. Anal Biochem 200(1):74–
80.
McLaren A, Luse R. 1961. Mechanism of inactivation of enzyme proteins by ultraviolet light.
Sci 134(3482):836–7.
Mertens B, Knorr D. 1992. Developments of nonthermal processes for food preservation. J Food
Technol 46(5):124–33.
Min S, Min SK, Zhang Q. 2003. Inactivation kinetics of tomato juice lipoxygenase by pulsed
electric fields. J Food Sci 68(6):1995–2001.
Oms-Oliu G, Martı́n-Belloso O, Soliva-Fortuny R. 2010. Pulsed light treatments for food
preservation. A review. Food Bioprocess Technol 3(1):13–23.
Ozer NP, Demirci A. 2006. Inactivation of Escherichia coli O157: H7 and Listeria monocyto-
genes inoculated on raw salmon fillets by pulsed UV-light treatment. Int J Food Sci Technol
41(4):354–60.
Park HJ, Adsit FG, Boyington JC. 2005. The 1.4 Å crystal structure of the human oxidized low
density lipoprotein receptor Lox-1. J Biol Chem 280(14):13593–9.
C18 Journal of Food Science r Vol. 79, Nr. 1, 2014
Paulo JA, Lee LS, Wu B, Repas K, Banks PA, Conwell DL, Steen H. 2010. Optimized sample
preparation of endoscopic collected pancreatic fluid for SDS-PAGE analysis. Electrophoresis
31(14):2377–87.
Regulations CoF. 2000. Irradiation in the production, processing, and handling of food. Office
of the Federal Register. Washington, D.C.: U.S. Government Printing Office. Title 21(Part
179).
Ridolfi M, Terenziani S, Patumi M, Fontanazza G. 2002. Characterization of the lipoxygenases
in some olive cultivars and determination of their role in volatile compounds formation. J
Agric Food Chem 50(4):835–9.
Sheu S, Chen A. 1991. Lipoxygenase as blanching index for frozen vegetable soybeans. J Food
Sci 56(2):448–51.
Shibata D, Steczko J, Dixon J, Hermodson M, Yazdanparast R, Axelrod B. 1987. Primary
structure of soybean lipoxygenase-1. J Biol Chem 262(21):10080–5.
Shriver SK, Yang WW. 2011. Thermal and nonthermal methods for food allergen control. Food
Eng Rev 3(1):26–43.
Shriver S, Yang W, Chung SY, Percival S. 2011. Pulsed ultraviolet light reduces immunoglobulin
E binding to Atlantic white shrimp (Litopenaeus setiferus) extract. Int J Environ Res Public
Health 8(7):2569–83.
Slowinski EJ, Wolsey WC, Rossi RC. 2011. Chemical principles in the laboratory. 10th ed.
Independence, KY: Cengage Learning. p 97.
Steczko J, Donoho GP, Clemens JC, Dixon JE, Axelrod B. 1992. Conserved histidine residues in
soybean lipoxygenase: functional consequences of their replacement. J Biochem 31(16):4053–
7.
Takeshita K, Shibato J, Sameshima T, Fukunaga S, Isobe S, Arihara K, Itoh M. 2003. Damage
of yeast cells induced by pulsed light irradiation. Int J Food Microbiol 85(1):151–8.
Todd JF, Paliyath G, Thompson JE. 1990. Characteristics of a membrane-associated lipoxygenase
in tomato fruit. Plant Physiol 94(3):1225–32.
Vladimirov YA, Roshchupkin D, Fesenko E. 1970. Photochemical reactions in amino acid
residues and inactivation of enzymes during UV-irradiation. A Review. Photochem Photobiol
11(4):227–46.
Vliegenthart J, Veldink G. 1982. Lipoxygenases. In: Pryor W, editor. Free radicals in biology.
Vol. 5. New York: Academic Press. p 29–63.
Wekhof A. 2000. Disinfection with flash lamps. PDA J Pharm Sci Technol 54(3):264–76.
Wetlaufer DB. 1962. Ultraviolet spectra of proteins and amino acids. Adv Protein Chem 17:303–
90.
Williams D, Lim M, Chen A, Pangborn R, Whitaker J. 1986. Blanching of vegetables prior to
freezing: which indicator enzyme to choose. J Food Technol 40:130–40.
Yang WW, Chung SY, Ajayi O, Krishnamurthy K, Konan K, Goodrich-Schneider R. 2010.
Use of pulsed ultraviolet light to reduce the allergenic potency of soybean extracts. Int J Food
Eng 6, no. 3, doi: 10.2202/1556-3758.1876.