Short communication

Protein oxidation in processed cheese slices treated with pulsed light

technology
M. Fernndez
a,
, M. Ganan
a,b
, C. Guerra
a
, E. Hierro
a
a
Departamento de Nutricin, Bromatologa y Tecnologa de los Alimentos, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
b
CEI Campus Moncloa, UCM-UPM e INIA, Madrid, Spain
a r t i c l e i n f o
Article history:
Received 23 September 2013
Received in revised form 29 January 2014
Accepted 27 February 2014
Available online 15 March 2014
Keywords:
Pulsed light
Processed cheese
Protein oxidation
DNPH assay
Carbonyls
a b s t r a c t
The effect of pulsed light technology on protein oxidation was studied in sliced processed cheese by mea-
suring the protein-bound carbonyls with a spectrophotometric DNPH assay. Bovine serum albumin was
also tested as a protein standard. Fluences of 0.7, 2.1, 4.2, 8.4 and 11.9 J/cm
2
were applied to vacuum-
packaged cheese slices and to an aqueous solution of the protein. Treatments up to 4.2 J/cm
2
did not pro-
mote protein oxidation immediately after ashing either in cheese or in the standard. Samples treated
with 8.4 and 11.9 J/cm
2
showed signicantly higher carbonyl amounts than non-treated ones. Protein
oxidation increased along cheese storage at 4 C, and differences among treatments remained. Further
studies on the sensory properties will be needed to clarify the impact of pulsed light on processed cheese
quality.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
Several non-thermal technologies are being studied for control-
ling pathogens and extending the shelf-life of ready-to-eat (RTE)
foods. Among them, pulsed light (PL) is a promising one. PL is
based on the application of short length light ashes of intense
broad-spectrum light and it has been proven to be useful to decon-
taminate surfaces. Some studies have shown that PL can inactivate
pathogenic microorganisms on the surface of various high protein
RTE foods, such as cooked meats (Hierro, Barroso, Ordez, &
Fernndez, 2011) and carpaccio (Hierro, Ganan, Barroso, & Fernn-
dez, 2012). This technology has been approved for food processing
by FDA (1996), which has established that the total cumulative
treatment must not exceed 12 J/cm
2
. PL is easy to integrate at
industrial processing lines and, once implemented, could highly re-
duce energy costs and production times (Gmez-Lpez, Regaert,
Debevere, & Devlieghere, 2007). Also, from an environmental point
of view, PL could reduce the environmental footprint of industrial
processes (Pereira & Vicente, 2010).
Similarly to other RTE foods, processed cheese can be contami-
nated with microorganisms after pasteurization, particularly dur-
ing slicing and packaging. PL technology could offer an adequate
treatment for surface decontamination, once the product is
packaged. In this sense, previous studies have shown that PL can
easily penetrate packaging lms (Fernndez, Manzano, de la Hoz,
Ordez, & Hierro, 2009; Keklik, Demirci, & Puri, 2010). However,
exposure to light may promote physicochemical changes in food
components, such as oxidation. While oxidative reactions of lipids
have been deeply studied in foods, less attention has been paid to
protein oxidation, which has been shown to change functionality
(Lund, Lametsch, Hviid, Jensen, & Skibsted, 2007; Martins & Netto,
2006; Xiong & Decker, 1995), to lower digestibility (Morzel,
Gatellier, Sayd, Renerre, & Laville, 2006), and to induce the
formation of unpleasant odors (Dalsgaard et al., 2010; Mortensen,
Bertelsen, Mortensen, & Stapelfeldt, 2004).
Semi-hard, hard, and processed cheeses are considered to be
readily oxidised by light exposure (Mortensen et al., 2004), but
studies on the oxidation of proteins are scarce. Elmnasser et al.
(2008) investigated the effect of PL on the conformational structure
and amino acid composition of milk proteins. Scheidegger, Pecora,
Radic, and Kivatinitz (2010) studied protein-bound carbonyls in
milk along storage at 4 C under UV and uorescent light. The
aimof our study was to evaluate the effect of PL technology on pro-
tein oxidation in processed cheese, at the uences that could be
used for decontamination purposes.
2. Materials and methods
2.1. Samples
Cheese slices (80 80 2 mm thick) were purchased in a local
supermarket. Once in the laboratory, slices were individually vac-
uum-packaged in plastic bags (Cryovac Sealed Air, Barcelona,
http://dx.doi.org/10.1016/j.foodchem.2014.02.165
0308-8146/ 2014 Elsevier Ltd. All rights reserved.

Corresponding author. Tel.: +34 913943946.

E-mail address: manuela@vet.ucm.es (M. Fernndez).
Food Chemistry 159 (2014) 388390
Contents lists available at ScienceDirect
Food Chemistry
j our nal homepage: www. el sevi er . com/ l ocat e/ f oodchem
Spain) made of 48 lm polyamide/polyethylethylene/vinyl acetate,
and kept at 4 C until experiments were performed. This lm was
selected on the basis of previous investigations which indicated
that PL could easily penetrate it (Fernndez et al., 2009). In these
experiments, the inactivation of Listeria monocytogenes was tested
on supercially inoculated agar plates wrapped with different
packaging polymers, and the reduction achieved (5.5 log cfu/cm
2
)
was the same as that obtained in unwrapped plates using the same
uence (0.35 J/cm
2
).
2.2. Pulsed light treatment
PL treatment was applied using a desktop Steribeam SBSXeMat-
ic-2L-A device (Steribeam Systems, Kehl am Rhein, Germany). The
apparatus consists of a metal housing surrounding a treatment
chamber made of polished stainless steel (20 cm wide 14 cm
deep 12 cm high) and equipped with two (upper and lower) xe-
non lamps and a quartz table located in the centre. The spectral
output of the lamps corresponds to 30% UV light (12% UV-C, 10%
UV-B and 8% UV-A), 30% infrared radiation and 40% visible light.
Each pulse is delivered in 250 ls and corresponds to a uence of
0.7 J/cm
2
at the level of the quartz table. The uence was measured
with a Laserpoint A-2-D12-BBF energy meter coupled to a 4p read-
out unit (Laserpoint, Milano, Italy). A vacuum pump was connected
to the chamber purge to extract the ozone produced by the lamps.
Samples were placed on the quartz table and received different
uences: 0.7, 2.1, 4.2, 8.4 and 11.9 J/cm
2
. These uences were se-
lected on the basis of previous studies on microbial decontamina-
tion carried out in our laboratory. Untreated control samples were
also analysed. The temperature of the samples was monitored dur-
ing the PL treatment using a Testo720 thermocouple equipped
with a needle-type probe (Testo AG, Lenzkirch, Germany). A tem-
perature increase of approximately 2.5 C per pulse was recorded.
Pulsed and non-pulsed samples were stored at 4 C in the dark.
Analyses were performed immediately after treatment and at days
15 and 30 of cold storage. Results are the mean of two independent
experiments.
2.3. Protein oxidation
The protein-bound carbonyl content is the most commonly
used marker of protein oxidation (Dalle-Donne, Rossi, Giustarini,
Milzani, & Colombo, 2003). In this study, carbonyls were measured
by spectrophotometric quantication of the acid hydrazones
formed by reaction with 2,4-dinitrophenylhydrazine (DNPH),
according to Oliver, Ahn, Moerman, Goldstein, and Stadtman
(1987).
Previously to studies in cheese, bovine serum albumin (BSA)
was used as protein model to evaluate oxidation caused by PL in
a simple system. A standard solution of BSA 1:1 (w/v) (Sigma-Al-
drich, Saint Louis, MO) was placed on a quartz dish and PL treated.
Two aliquots of BSA per uence applied were taken and precipi-
tated with trichloroacetic acid (TCA, 10% nal concentration).
One aliquot was added with 0.2% (w/v) DNPH in 2 N HCl and the
other, used as blank, was only added with 2 N HCl. Both samples
were incubated at 30 C for 1 h and stirred every 10 min. Then,
samples were reprecipitated with TCA and centrifuged at 2000g
for 10 min. The supernatants were discarded and the pellets were
again precipitated with TCA and washed twice with ethanol:ethyl
acetate (1:1, v/v) to remove free DNPH. Pellets were then resus-
pended in 6 M guanidine HCl with 20 mM sodium phosphate buf-
fer (pH 6.5), followed by 10 min stirring. The amount of hydrazone
formed was determined by measuring absorbance at 370 nm.
Results were expressed as nmol carbonyls/mg protein, using a mo-
lar extinction coefcient of 21.0 mM
1
cm
1
. Protein concentration
of cheese was calculated by spectrophotometry at 280 nm using
BSA as standard.
For the determination of carbonyl content in processed cheese
slices, a previous step of protein extraction was followed. A cheese
homogenate, containing approximately 1.2 mg protein/mL, was
prepared by mixing 0.1 g of cheese in 10 mL of 0.15 M KCl for
3 min using a Polytron PT 1035 GT probe (Kinematica, Lucerne,
Switzerland) in a glass vessel immersed in an ice bath. TCA 10% (-
nal concentration) was added to precipitate proteins, and samples
were centrifuged at 2000g for 10 min to obtain the pellets. Two ali-
quots were taken for each PL condition, and carbonyls were deter-
mined following the procedure previously detailed for BSA.
2.4. Statistical analysis
A one-way ANOVA was conducted to compare the results of the
different batches, using Statgraphics Centurion XVI.I (Statpoint
Technologies, Warrenton, VA). Statistical signicance was identi-
ed at 95% condence level. Duncans multiple range test was used
to assess differences between means.
3. Results and discussion
The effect of PL on BSA oxidation is shown in Fig. 1. The car-
bonyl content signicantly increased (p < 0.05) when uences of
8.4 and 11.9 J/cm
2
were used. In cheese, the amount of carbonyls
detected immediately after PL treatment ranged between 3.8 and
5.4 nmol/mg protein (Fig. 2). Only the highest uences showed sig-
nicant differences (p < 0.05) with the control samples. Carbonyl
content increased during storage, and differences among treat-
ments remained.
Light-induced oxidation of proteins is mainly caused by wave-
lengths of the UV region, although other bands of the spectrum
can also be involved (Hollsy, 2002; Scheidegger et al., 2010). PL
ashes generally range between 200 and 1000 nm, and UV repre-
sents about 30% of the total radiation. Therefore, a certain degree
of protein oxidation due to PL could be expected. In this study, this
effect was observed at the highest uences tested and was more
noticeable in BSA. The higher carbonyl values recorded for the pro-
tein standard in comparison to those of cheese slices could be ex-
plained by the penetration depth of PL. While BSA was a
transparent solution, light fails in penetrating the opaque surface
of cheese and then, oxidative changes are restricted to the surface.
Protein oxidation may occur by a direct mechanism through the
absorption of UV light by amino acids, but also by an indirect via
with the involvement of photosensitizers (Mortensen et al.,
2004). The direct mechanism would be responsible for BSA
oxidation. This protein has a maximum absorbance at 280 nm
(Mortensen et al., 2004), that is also the wavelength at which
a
a
b b
b
b
0
2
4
6
8
10
12
0 0.7 2.1 4.2 8.4 11.9
n
m
o
l

c
a
r
b
o
n
y
l
s
/
m
g

B
S
A
Fluence (J/cm
2
)
Fig. 1. Protein-bound carbonyl content of a BSA solution treated with different
uences of pulsed light. Bars show standard deviation.
M. Fernndez et al. / Food Chemistry 159 (2014) 388390 389
aromatic amino acids (phenylalanine, tyrosine and tryptophan),
and also histidine and cysteine, absorb light (Hollsy, 2002).
Approximately 18% of the BSA chain corresponds to these amino
acid residues (Hirayama, Akashi, Furuya, & Fukuhara, 1990).
In relation to the results obtained in cheese, in principle, the
indirect mechanism of protein oxidation could also be relevant
due to the presence of riboavin, which is an efcient photosensi-
tizer (Mortensen et al., 2004). This compound, due to its conju-
gated double bond system, absorbs visible light apart from UV
(Choe, Huang, & Min, 2005), both components of PL ashes, as pre-
viously stated. When irradiated, riboavin can produce reactive
oxygen species, such as singlet oxygen or hydroxyl radical, in the
presence of oxygen. However, this mechanism can be balanced
by b-carotene, which also absorbs a signicant fraction of light,
protecting riboavin (Li, King, & Min, 2000; Mortensen et al.,
2004). The protein-bound carbonyl content reported in the litera-
ture ranges from 1.5 to 4.5 nmol/mg protein for different cheese
varieties (Fedele & Bergamo, 2001; Balestrieri et al., 2002).
Although in our study the PL treatment with the highest uences
signicantly increased (p < 0.05) protein oxidation, the values ob-
tained immediately after ashing can be considered as normal
for cheese, taking into account the thermal treatment applied for
the manufacture of processed cheese. On the other hand, it is pos-
sible that vacuum-packaging and the low penetration of light in
the samples had contributed to control light-induced oxidation.
4. Conclusion
In this preliminary study, PL treatment up to 4.2 J/cm
2
did not
increase protein oxidation in processed cheese slices, while higher
uences signicantly enhanced oxidation. Further studies on the
sensory attributes will be the next step to evaluate the real impact
of these changes on cheese quality.
Acknowledgements
This work has been supported by Project AGL2011-29325
(Ministerio de Educacin y Ciencia, Government of Spain) and
UCM Group 920276. Dr. Ganan was the recipient of a postdoctoral
contract from the International Program of Talent Recruitment
from the Campus of International Excellence, University Complu-
tense of Madrid.
References
Balestrieri, M., Spagnuolo, M. S., Cigliano, L., Stori, G., Ferrara, L., Abrescia, P., &
Fedele, E. (2002). Evaluation of oxidative damage in mozzarella cheese
produced from bovine or water buffalo milk. Food Chemistry, 77, 293299.
Choe, E., Huang, R., & Min, D. B. (2005). Chemical reactions and stability of riboavin
in foods. Journal of Food Science, 70, 2836.
Dalle-Donne, I., Rossi, R., Giustarini, D., Milzani, A., & Colombo, R. (2003). Protein
carbonyl groups as biomarkers of oxidative stress. Clinica Chimica Acta, 329,
2338.
Dalsgaard, T. K., Srensen, J., Bakman, M., Voghsen, L., Nebesl, C., Albrechtsen, R., &
Nielsen, J. (2010). Light-induced protein and lipid oxidation in cheese:
Dependence on fat content and packaging conditions. Dairy Science and
Technology, 90, 565577.
Elmnasser, N., Dalgalarrondo, M., Orange, N., Bakhrouf, A., Haertl, T., Federighi, M.,
& Chobert, J. M. (2008). Effect of pulsed-light treatment on milk proteins and
lipids. Journal of Agricultural and Food Chemistry, 56, 19841991.
FDA (1996). Code of Federal Regulations. 21CFR179.41. Title 21, Volume 3. Revised
as of April 1, 2003.
Fedele, E., & Bergamo, P. (2001). Protein and lipid oxidative stresses during cheese
manufacture. Journal of Food Science, 66, 932935.
Fernndez, M., Manzano, S., de la Hoz, L., Ordez, J. A., & Hierro, E. (2009). Pulsed
light inactivation of Listeria monocytogenes through different plastic lms.
Foodborne Pathogens and Disease, 6, 12651267.
Gmez-Lpez, V. M., Regaert, P., Debevere, J., & Devlieghere, F. (2007). Pulsed light
for food decontamination: a review. Trends in Food Science & Technology, 18,
464473.
Hierro, E., Barroso, E., Ordez, J. A., & Fernndez, M. (2011). Efcacy of pulsed light
for shelf-life extension and inactivation of Listeria monocytogenes on ready-to-
eat cooked meat products. Innovative Food Science & Emerging Technologies, 12,
275281.
Hierro, E., Ganan, M., Barroso, E., & Fernndez, M. (2012). Pulsed light treatment for
the inactivation of selected pathogens and the shelf-life extension of beef and
tuna carpaccio. International Journal of Food Microbiology, 158, 4248.
Hirayama, K., Akashi, S., Furuya, M., & Fukuhara, K. (1990). Rapid conrmation and
revision of the primary structure of bovine serum albumin by ESIMS and
frit-FAB LC/MS. Biochemical and Biophysical Research Communications, 173,
639646.
Hollsy, F. (2002). Effects of ultraviolet radiation on plant cells. Micron, 33, 179197.
Keklik, N. M., Demirci, A., & Puri, V. M. (2010). Decontamination of unpackaged and
vacuum-packaged boneless chicken breast with pulsed ultraviolet light. Poultry
Science, 89, 570581.
Li, T. L., King, J. M., & Min, D. B. (2000). Carotenoids in riboavin photosensitized
singlet oxygen oxidation of vitamin D
2
. Journal of Food Biochemistry, 24,
477492.
Lund, M. N., Lametsch, R., Hviid, M. S., Jensen, O. N., & Skibsted, L. H. (2007). High-
oxygen packaging atmosphere inuences protein oxidation and tenderness of
porcine longissimus dorsi during chill storage. Meat Science, 77, 295303.
Martins, V. B., & Netto, F. M. (2006). Physicochemical and functional properties of
soy protein isolate as a function of water activity and storage. Food Research
International, 39, 145153.
Mortensen, G., Bertelsen, G., Mortensen, B. K., & Stapelfeldt, H. (2004). Light-
induced changes in packaged cheeses A review. International Dairy Journal, 14,
85102.
Morzel, M., Gatellier, Ph., Sayd, T., Renerre, M., & Laville, E. (2006). Chemical
oxidation decreases proteolytic susceptibility of skeletal muscle myobrillar
proteins. Meat Science, 73, 536543.
Oliver, C. N., Ahn, B. W., Moerman, E. J., Goldstein, S., & Stadtman, E. R. (1987). Aged-
related changes in oxidized proteins. The Journal of Biological Chemistry, 262,
54885491.
Pereira, R. N., & Vicente, A. A. (2010). Environmental impact of novel thermal and
non-thermal technologies in food processing. Food Research International, 43,
19361943.
Scheidegger, D., Pecora, R. P., Radic, P. M., & Kivatinitz, S. C. (2010). Protein oxidative
changes in whole and skim milk after ultraviolet or uorescent light exposure.
Journal of Dairy Science, 93, 51015109.
Xiong, Y. L., & Decker, E. A. (1995). Alterations of muscle protein functionality by
oxidative and antioxidative processes. Journal of Muscle Foods, 6, 139160.
2
3
4
5
6
7
8
0 3 5 1 0
n
m
o
l

c
a
r
b
o
n
y
l
s
/
m
g

p
r
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t
e
i
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Time (days)
Fig. 2. Changes in the protein-bound carbonyl content of vacuum-packaged slices
of processed cheese treated with different uences of pulsed light and stored at
4 C. Fluences: () 0, (s) 0.7, (4) 2.1, (N) 4.2, () 8.4, (h) 11.9 J/cm
2
. Bars show
standard deviation.
390 M. Fernndez et al. / Food Chemistry 159 (2014) 388390