Innovative Food Science and Emerging Technologies 56 (2019) 102186

Contents lists available at ScienceDirect

Innovative Food Science and Emerging Technologies

journal homepage: www.elsevier.com/locate/ifset

Enhanced bacterial inactivation in apple juice by synergistic interactions T

between phenolic acids and mild food processing technologies
⁎
Erick F. de Oliveiraa,b, Cuong H. Nguyena, Kayla Stepaniana, Andrea Cossua, Nitin Nitina,c,
a
Department of Food Science and Technology, University of California, Davis, CA, USA
b
CAPES Foundation, Ministry of Education of Brazil, Brasilia, DF, Brazil
c
Department of Biological and Agricultural Engineering, University of California, Davis, CA, USA

A B S T R A C T

This study demonstrated an innovative processing approach based on synergistic antimicrobial activity of two phenolic acids with mild thermal and non-thermal
processing technologies. The two selected model phenolic acids were gallic acid (GA; 10 mM) and ferulic acid (FA; 1 mM). The processing technologies evaluated for
processing of a model clarified apple juice were UV-A light, mild heat (55 °C) and moderate pressure (250 MPa), with processing times ranging from 1 to 30 min. The
results demonstrated that combinations of selected phenolic acids and a mild physical processing were able to lower E. coli O157:H7 and Listeria innocua counts from
6-log CFU mL−1 to below the detection limit of 1-log CFU mL−1. Bacterial inactivation was significantly enhanced by the combination of UV-A light processing and
FA, where 10 min of treatment enhanced bacterial inactivation by 5-log as compared to light processing alone, which presented no bacterial inactivation. In contrast,
the combination of GA and mild-temperature thermal processing (55 °C) or mild-levels of high-pressure processing (250 MPa), enhanced bacterial inactivation by 4-
log as compared to the physical treatments alone, which presented only 1-log of inactivation. The influence of these synergistic combinations on bacterial membrane
damage was assessed by selective plating technique under osmotic pressure. Furthermore, the total intracellular thiol content was also measured to assess for thiol
oxidation. Overall, the results demonstrated enhanced bacterial inactivation based on synergistic interactions of selected phenolic acids with both mild-thermal and
non-thermal technologies in a model food system and illustrate potential to create diversity of novel antimicrobial strategies for food processing.
Industrial relevance: This study showed that the presence of naturally-based compounds can significantly reduce intensity levels of physical processing required to
inactivate model gram-positive and gram-negative bacteria in apple juice. In this study two phenolic acids, ferulic acid and gallic acids were selected as these
compounds are naturally present in many fruits and other food products including grains. Furthermore, the levels of these natural phenolic acids used in this study
are comparable to the levels naturally present in some of the food materials.

1. Introduction
Physical processes, mainly thermal processing, have been tradi-
tionally used in the food industry to ensure microbial safety and extend
the shelf-life of processed food (Petruzzi et al., 2017). However, in-
tensive thermal processing conditions are often required to pasteurize
food, which can lead to negative effects on the sensorial and nutritional
properties of the processed food. To overcome the impact of heat on
food quality, non-thermal pasteurization methods have been developed
as an alternative to thermal processing, including high pressure pro-
cessing (HPP), ultraviolet-light treatment (UV-C), pulsed electric field
(PEF), and ultrasound (Huang, Wu, Lu, Shyu, & Wang, 2017; Jermann,
Koutchma, Margas, Leadley, & Ros-Polski, 2015). Despite significant
potential, many of these technologies are not broadly used for com-
mercial food processing and present significant limitations compared to
thermal processing such as low antimicrobial efficacy (e.g. ultrasound),
low compatibility for broader food applications (e.g. PEF, UV-C). Al-
though some of these novel technologies such as HHP are gaining
significant market acceptance, the cost associated with these technol-
ogies can be higher compared to thermal processing due to intense
processing requirements.
In addition to physical processes, some chemical agents such as
phenolics and organic acids are also commonly applied during food
processing (Jung & Matthews, 2015). Despite their low impact on food
quality, these chemical compounds are often less efficient in achieving
microbial reduction as compared to thermal processing. Due to this
limitation, chemical treatments have been traditionally applied in food
processing as an additional hurdle following an initial physical treat-
ment mainly with an overall aim to preserve and extend shelf-life of
these products (Chan, 2015; Gould, 1996). Among diverse chemical
agents, natural-based antimicrobials, such as phenolic compounds
found in plant extracts, have significant potential for antimicrobial
activity in food systems (Juneja, Dwivedi, & Yan, 2012). In addition to
their strong antimicrobial activity, other advantages of using natural-
based compounds could include: (I) adherence to the existing reg-
ulatory requirements – many of these compounds are classified as food

⁎
Corresponding author at: Robert Mondavi Institute South, Hilgard Ln, Davis, CA 95616, USA.
E-mail address: nnitin@ucdavis.edu (N. Nitin).

https://doi.org/10.1016/j.ifset.2019.102186
Received 11 April 2019; Received in revised form 25 June 2019; Accepted 25 June 2019
Available online 26 June 2019
1466-8564/ © 2019 Elsevier Ltd. All rights reserved.
E.F. de Oliveira, et al.
grade, and (II) increased consumer acceptance – meet the growing
consumer demand for replacing synthetic preservatives with natural
ingredients in food.
Recently, the simultaneous combination of chemical treatments and
physical processing has emerged as an alternative food processing ap-
proach to inactivate microbes in food and water systems (Berdejo,
Pagán, García-Gonzalo, & Pagán, 2018). In contrast to conventional
hurdle technology, which combines physical and chemical processes in
sequential treatments to enhance microbial safety, this alternative ap-
proach is based on a synergistic antimicrobial activity observed when
some physical processes, such as heat and light, and some chemical
treatments are simultaneously combined. For example, some studies
have shown that the combination of mild-temperature thermal pro-
cessing and essential oils can significantly enhance microbial load in-
activation in fruit juices as compared to mild-temperature thermal
treatment alone or essential oil treatment alone (Espina, Condón,
Pagán, & García-Gonzalo, 2014; Espina et al., 2012). Similarly, other
studies have highlighted that the combination of sub-lethal concentra-
tions of phenolic compounds and sub-lethal levels of light processing
could lead to synergistic inactivation of various microbes both in water
and food systems (de Oliveira, Tosati, Tikekar, Monteiro, & Nitin, 2018;
Ghate, Kumar, Zhou, & Yuk, 2015; Penha et al., 2017). Overall, the
main advantage of these emerging technologies is enhancement of
microbial inactivation in food while applying low intensity levels of
physical processing and, consequently, reducing the overall impact of
processing on food quality.
This present study aims to develop a broader concept of simulta-
neously combining chemical and diverse physical treatments to en-
hance bacterial inactivation in food systems. To achieve that goal, we
evaluated if combinations of low concentrations of two phenolic com-
pounds (i.e. gallic acid and ferulic acid) and mild levels of three dif-
ferent physical processing (i.e. heat, pressure and light) could lead to
rapid and synergistic inactivation of model pathogenic bacteria (i.e.
Escherichia coli O157:H7) in apple juice. The choice of mild levels of
physical and low concentrations of phenolic acids is ideal for observing
possible synergisms between these treatments as well as for lowering
the impact of processing on the quality of treated apple juice.
Furthermore, the biological damages to cellular membranes and in-
tracellular thiol content were measured in treated bacterial cells to
assess the synergistic antimicrobial activity of these treatments.
Overall, this study evaluates the synergistic combination of phenolic
compounds and mild physical processing technologies to enhance in-
activation of bacteria in a model juice system. It is expected that the
synergistic combination of phenolic compounds and mild processing
methods can also reduce the energy inputs required for processing and
also improve food quality while addressing the food safety require-
ments.
2. Materials and methods
2.1. Reagents
Clarified apple juice (Safeway Kitchens™; Pleasanton, CA, USA) was
purchased from a local retail store and kept at 4 °C until further use for
up to one month. Gallic acid (GA), ferulic acid (FA), reduced L-glu-
tathione (GSH), sodium chloride (NaCl), sodium dodecyl sulfate (SDS),
ethylenediaminetetraacetic acid (EDTA) and Triton X-100 were all
obtained from Sigma-Aldrich (St. Louis, MO, USA). Measure-iT™ Thiol
Assay Kit, a thiol-reactive fluorescence probe, was purchased from
Molecular Probes (Eugene, OR, USA). Zirconia-silica beads (0.1-mm
diameter) were acquired from Biospec Products (Bartlesville, OK, USA).
Tryptic soy broth (TSB), tryptic soy agar (TSA), phosphate-buffered
saline (PBS) and tris-hydrochloride (1 M; Tris-HCl) were purchased
from Fisher BioReagents (Pittsburgh, PA, USA). Ultrapure water was
obtained using a Milli-Q filtration system (EDM Millipore; Billerica,
MA, USA).
2
Innovative Food Science and Emerging Technologies 56 (2019) 102186
2.2. Bacterial culture
Escherichia coli O157:H7 (ATCC 700728, Manassas, VA, USA) and
Listeria innocua (ATCC 33090, Manassas, VA, USA) were provided by
Dr. Linda Harris, from the Department of Food Science and Technology
at University of California, Davis. These bacterial strains have been
modified with a rifampicin-resistant plasmid. Bacteria were grown
overnight in TSB broth containing Rifampicin 50 μg mL−1 at 37 °C and
150 rpm. Fresh bacterial suspensions were prepared before each ex-
periment by diluting overnight-grown bacteria with sterile water to a
final bacterial concentration of approximately 1.0 × 108 CFU mL−1.
2.3. Inoculation of apple juice
Before inoculation, apple juice supplemented with GA or FA were
prepared by weighing and dissolving appropriate amounts of GA and
FA in clarified apple juice to a final concentration of 10 mM of GA and
1 mM of FA. This concentration levels were selected based on the so-
lubility limits for these phenolic acids in water and also the con-
centration levels at which the compounds do not result in significant
microbial inactivation. Then, bacterial inoculation was carried out by
adding 0.02 mL of freshly prepared E. coli O157:H7 bacterial suspen-
sions to 2 mL of apple juice containing GA or FA giving a final bacterial
concentration in apple juice of 1.0 × 106 CFU mL−1. Bacteria-in-
oculated apple juice without the addition of GA and FA were also
carried out as controls. Inoculated apple juice samples were freshly
prepared before each experiment and discarded afterwards. Apple juice
samples were also inoculated with L. innocua following the above-
mentioned procedure, and the results were included in the
Supplementary material.
2.4. UV-A light processing of apple juice
UV-A light processing of bacteria-inoculated apple juice were per-
formed as previously reported (Oliveira, Cossu, Tikekar, & Nitin, 2017).
Briefly, two milliliters of freshly prepared apple juice samples (Control,
GA 10 mM and FA 1 mM) containing 1.0 × 106 CFU mL−1 of E. coli
were placed in an individual well of a sterile 12-well flat bottom
polystyrene plate. The plate was then positioned at the center of a
plastic chamber equipped with four UV-A lamps (320–400 nm; 18 W;
Actinic BL, Philips, Holland) located at the inside-top of the chamber.
The distance between the UV-A lamps and the samples was 8 cm. The
average UV-A light intensity at the center of the chamber was
3.2 ± 0.2 mW cm−2. The apple juice samples were exposed to UV-A
light for 2.5, 5, 10, 15, and 30 min. Immediately after UV-A light pro-
cessing, the samples were serially diluted in sterile PBS, spread on TSA
plates, and the plates were incubated at 37 °C for up to 2 days. After
incubation, bacterial colony-forming units (CFU) were determined by
the standard plate counting method and bacterial populations were
expressed as CFU mL−1.
2.5. Mild-thermal processing of apple juice
Mild-thermal processing of bacteria-inoculated apple juice was
performed in a water bath (Isotemp® 2150, Thermo Fisher Scientific)
set at 55 °C. Before processing, glass vials were positioned in the water
bath and pre-incubated for 10 min at 55 °C in order to achieve tem-
perature equilibrium. Then, mild-thermal processing of the juice were
conducted by adding 0.2 mL of apple juice samples (Control, GA 10 mM
and FA 1 mM) containing 1.0 × 106 CFU mL−1 of E. coli to the pre-in-
cubated glass vials at 55 °C for 1, 2, and 4 min. Immediately after
heating, the samples were serially diluted in sterile PBS, spread on TSA
plates, and the plates were incubated at 37 °C for up to 2 days. After
incubation, bacterial colony-forming units (CFU) were determined by
the standard plate counting method and bacterial populations were
expressed as CFU mL−1.
E.F. de Oliveira, et al.
2.6. High pressure processing of apple juice
High pressure processing (HPP) of bacteria-inoculated apple juice
was performed in a Isostatic Food Press (Avure Technologies Inc.,
Franklin, TN, USA) equipped with a two liter unit chamber and a
thermostatically controlled water bath to control adiabatic heating.
First, 2 mL of apple juice samples (Control, GA 10 mM and FA 1 mM)
containing 1.0 × 106 CFU mL−1 of E. coli were added to 12 cm × 4 cm
plastic bags, which were then sealed at 95% vacuum (Ultravac 250;
Koch Equipment LLC, Kansas City, MO, USA) in order to remove air
pockets from the bags prior to HPP. The sample-loaded plastic bags
were subjected to mild-levels of HPP (250 MPa) at 25 °C. Pressure
equilibrium was achieved after 10 s and treatments were performed
under 250 MPa for 2.5, 5, and 7.5 min. After HPP, the samples were
serially diluted in sterile PBS, spread on TSA plates, and the plates were
incubated at 37 °C for up to 2 days. After incubation, bacterial colony-
forming units (CFU) were determined by the standard plate counting
method and bacterial populations were expressed as CFU mL−1.
2.7. Membrane damage assay
Bacterial membrane damage induced by GA and FA in combination
with UV-A light, mild-thermal processing or HPP were evaluated
through selective medium plating technique (SMPT) as previously re-
ported (Espina, García-Gonzalo, & Pagán, 2016). TSA plates containing
3% of sodium chloride (NaCl) were used as selective medium to dis-
criminate between membrane-intact bacteria and membrane-compro-
mised bacteria, where bacteria with compromised membranes should
not be able to grow in the presence of NaCl (Espina et al., 2016).
Treatment times which had shown limited bacterial reduction were
chosen in order to differentiate between sub-lethal and lethal injuries.
First, apple juice samples containing 1.0 × 106 CFU mL−1 of E. coli
O157:H7 were exposed to UV-A light processing for 2.5 min, mild-
thermal processing for 2 min, or HPP for 2.5 min as previously de-
scribed. After processing, the samples were serially diluted in PBS,
spread on TSA plates containing 0% and 3% NaCl (w/v) and incubated
at 37 °C for up to 2 days. The number of membrane-damaged bacterial
cells were determined by subtracting the number of bacteria on TSA
plates with 3% NaCl from the number of bacteria on TSA plates without
NaCl, and were then expressed as bacterial sub-lethal injuries (log CFU
mL−1)
2.8. Intracellular thiol content quantification
The reduction in intracellular thiol content of E. coli O157:H7 cells
incubated in apple juice containing GA or FA and exposed to UV-A
light, mild-thermal processing or HPP was evaluated. The intracellular
thiol-containing compounds were extracted by lysing the bacteria cells
through bead beating. First, 1 mL of each apple juice sample (Control,
GA 10 mM and FA 1 mM) containing 1 × 109 CFU mL−1 of E. coli
O157:H7 were exposed to UV-A light, mild-temperature thermal pro-
cessing or HPP, as previously described, for 10, 2 and 5 min, respec-
tively. Following treatments, samples were centrifuged (10,000 ×g;
10 min) and the bacterial pellet was resuspended in 500 μL of a sterile
lysis buffer (Tris-HCl 50 mM, NaCl 25 mM, EDTA 25 mM, SDS 2%,
Triton X-100 1%) and transferred into a sterile 1.5 mL tube containing
400 μL of zirconia-silica beads (0.1 mm). The bacterial suspension was
vortexed for 10 min and then centrifuged (16,000 ×g; 10 min) before
recovering the supernatant to be used for the total thiol content mea-
surement. The total thiol content was quantified through fluorescence
spectroscopy using the Measure-iT™ Thiol Assay Kit from Molecular
Probes (Eugene, OR, USA). Briefly, 10 μL of the obtained lysate were
added to 100 μL of a thiol-reactive working solution and placed in a
well of a 96-well black flat bottom polystyrene plate. The plate was
incubated at room temperature in the dark for 5 min and the flores-
cence intensity was measured using a fluorescence plate reader (Tecan
3
Innovative Food Science and Emerging Technologies 56 (2019) 102186
SPECTRAFluor Plus) using an excitation filter of 488 nm and an emis-
sion filter of 520 nm. The total thiol content (μM) was determined using
a standard curve based on known concentration levels of reduced glu-
tathione (GSH). The results were expressed in terms of remaining thiol
concentration (%) compared to the untreated bacterial sample
(Control).
2.9. Statistical analysis
Statistical analysis was conducted using one-way ANOVA and the
pair-wise differences were evaluated using the Tukey's range test to
identify significant differences between each sample group (P < 0.05).
All experiments were performed in triplicates.
3. Results and discussion
3.1. Antibacterial activity of GA and FA without physical processing
The effect of GA and FA individual treatments on the survival of E.
coli O157:H7 in bacteria-inoculated apple juice was evaluated before
assessing their synergistic antimicrobial combinations with mild phy-
sical processes. The concentrations of GA and FA were selected based
on previous experiments, taking into consideration properties such as
water solubility and antimicrobial activity. As shown in Supplementary
material Fig. 1, 10 mM of GA and 1 mM of FA presented no antibacterial
activity against E. coli O157:H7 in apple juice, suggesting that the se-
lected concentration levels were in fact sub-lethal.
3.2. Low fluence UV-A light processing of GA- and FA-supplemented apple
juice
In this study, we evaluated the use of a mild form of light radiation
(UV-A light) in combination with phenolic acids (GA 10 mM and FA
1 mM) as an alternative approach to achieve E. coli O157:H7 inactiva-
tion in apple juice. As observed in Fig. 1, the combination of FA and UV-
A light (FA + UVA) exhibited strong antibacterial activity,
achieving > 5 log CFU mL−1 reduction of E. coli O157:H7 in apple juice
after 10 min of light treatment (total light fluence of 19.2 kJ m−2). In
contrast, the combination of GA and UV-A light (GA + UVA) presented
limited antibacterial activity, where < 1 log CFU mL−1 reduction in the
bacterial plate count was achieved even after 30 min of UV-A treatment
(total light fluence of 57.6 kJ m−2). Fig. 1 also illustrates that UV-A
light treatment of apple juice alone had no impact on bacterial
Fig. 1. Inactivation of Escherichia coli O157:H7 in apple juice containing either
10 mM of Gallic Acid (GA) or 1 mM of Ferulic Acid (FA) and exposed to UV-A
light processing at 25 °C. Controls consist of bacteria in apple juice without the
addition of GA and FA and exposed to UV-A light. Initial bacteria concentration
was 6 log CFU mL−1. The limit of detection of the plate counting method was 1
log CFU mL−1. See Supplementary material Fig. 1 for non-processed controls.
E.F. de Oliveira, et al.
inactivation. Similar results were also observed against Listeria innocua
in apple juice, where after 15 min the FA + UVA treatment in-
activated > 5 log CFU mL−1, and the GA + UVA treatment in-
activated < 1 log CFU mL−1 (Supplementary material Fig. 2). There-
fore, it can be inferred that the combination of UV-A light and FA can
synergistically inactivate both Gram-positive and Gram-negative pa-
thogens in a model juice environment.
In addition to bacterial inactivation, biological damages to the
bacterial membrane and to intracellular thiol content of E. coli O157:H7
cells were assessed as indicators of the synergistic interactions between
light and selected phytochemicals. As shown in Fig. 2a, GA + UVA did
not lead to an increase in the number of sub-lethal bacterial injuries
assessed based on ability of bacterial cells to form colonies in the pre-
sence of osmotic stress as compared to the controls (i.e. individual
treatments of GA and UV-A light). In contrast, treatment with
FA + UVA led to a small but significant increase in sub-lethal injuries to
E. coli O157:H7 compared to the individual controls (P < 0.05). Fur-
thermore, it was observed that GA + UVA and FA + UVA affected the
intracellular thiol content of E. coli O157:H7 in opposite ways. As ob-
served in Fig. 2b, the presence of GA alone led to a reduction in the
intracellular thiol content of the bacterial cells, which was further en-
hanced when GA was combined with UVA. In contrast, while FA alone
had no effect on intracellular thiol, the combination of FA and UVA led
to an increased on the total intracellular thiol concentration. Together,
these results suggest that FA + UVA and GA + UVA may affect E. coli
O157:H7 cells through different pathways influencing both membrane
and intracellular environment.
The synergistic antimicrobial combination of UV-A light and FA
against foodborne pathogens and spoilage microbes has been reported
by few prior studies (Shirai, Watanabe, & Matsuki, 2017; Shirai,
Kajiura, & Omasa, 2015; Shirai & Yasutomo, 2019). Many of these prior
studies were conducted using aqueous suspension of bacteria in che-
mical buffers and without the presence of a food matrix. Furthermore,
in these previous studies, FA was dissolved in DMSO for evaluating the
synergistic antimicrobial activity, which would not be appropriate for
food applications. This study demonstrates translation of this novel
antimicrobial concept using commercial apple juice as a food model
and using only food grade ingredients (i.e. FA dissolved in water). The
results demonstrate that FA + UVA could inactivate > 5-log of E. coli
O157:H7 in apple juice using low concentrations of FA (1 mM or
190 ppm) and low levels of UV-A light exposure (total light fluence of
19 kJ m−2).
It has been recently suggested in prior studies that FA may act as a
photosensitizer and thus generate reactive oxygen species (ROS) upon
exposure to light (Shirai & Yasutomo, 2019). The generation ROS can
induce multiple intracellular damages in bacteria which can further
lead to cell death. As shown in Fig. 2a, the FA + UVA treatment caused
a small but significant (P < 0.05) impact on the membrane of E. coli
O157:H7 as compared to UVA alone and FA alone, supporting the hy-
pothesis that ROS generation could lead to bacterial membrane da-
mage. In addition to membrane damage, FA + UVA was observed to
4
Innovative Food Science and Emerging Technologies 56 (2019) 102186
Fig. 2. (a) Sub-lethal injuries and (b) changes in the
total intracellular thiol content of Escherichia coli
O157:H7 after 2.5 min of UV-A light processing of
apple juice containing either 10 mM of Gallic Acid
(GA) or 1 mM of Ferulic Acid (FA). Controls consist of
bacteria in apple juice without the addition of GA or
FA. Sub-lethal injuries were evaluated by selectively
plating treated bacteria in TSA medium supplemented
with 3% NaCl. Results marked with different letters
were statistically significant (P < 0.05).
affect the total intracellular thiol content of E. coli O157:H7 (Fig. 2b).
Intracellular thiol content is commonly used as a biomarker to measure
oxidative stress in bacterial cells (Cossu, Dou, Young, & Nitin, 2017).
Interestingly, FA + UVA led to an increase in the total intracellular
thiol content of E. coli O157:H7, which, at first glance, seems to con-
tradict the idea that FA + UVA induced intracellular oxidative stress
through the generation of ROS. Similar observations were reported for
E. coli O157:H7 cells exposed to low levels of hydrogen peroxide, where
an increase in the uptake of glucose seemed to contradict the ROS-
mediated damage induced by peroxide. In that study, it was suggested
that the increase in glucose uptake at low concentration of peroxides
was triggered by the bacteria to provide protection against oxidative
stress (Cossu, Le, Young, & Nitin, 2017). Therefore, it is possible that
the observed increase in thiol content during the FA + UVA treatment
was due to increased activity of thiol-containing enzymes and redox
proteins such as glutathione reductase and thioredoxin, which are key
components of metabolic pathways activated by Gram-negative bac-
teria in response to induced oxidative stress (Netto, Oliveira, Tairum, &
Neto, 2016; Winther & Thorpe, 2014). Overall, the observed anti-
bacterial activity of FA + UVA could be attributed to photo-activity of
FA upon exposure to UV-A light. This photo-activity of FA could induce
intracellular oxidative stress, damage the bacterial membrane, among
other intracellular sites, and ultimately leading to cell death.
As opposed to FA + UVA, the GA + UVA treatment presented lim-
ited bacterial inactivation (maximum of 1 log CFU mL−1) even after
30 min of treatment. Although, despite the lower antibacterial activity,
GA alone and GA + UVA led to the reduction of intracellular thiol
content in E. coli O157:H7, indicating that GA was able to permeate into
the cells and induce intracellular stress. Similar to FA, the combination
of GA and light radiation has been suggested to generate intracellular
ROS in bacterial cells, leading to bacteria inactivation (Cossu et al.,
2016; Nakamura et al., 2012; Wang, de Oliveira, Alborzi, Bastarrachea,
& Tikekar, 2017). Furthermore, a recent study showed that GA can also
interfere with the bacterial metabolism that can further contribute to
the inactivation of bacterial cells (Oliveira et al., 2017). However, most
of these studies were performed in water or buffer systems and not in
the presence of a complex food matrix such as apple juice. Therefore,
the limited bacterial inactivation achieved by GA + UVA observed in
this study could be related to interference from various components in
apple juice, such as sugars and polyphenolic antioxidants. These com-
ponents could protect bacterial cells from the antibacterial activity of
the GA + UVA treatment. Some of these compounds could also provide
a favorable environment for bacteria to recover from the damages in-
duced by the GA + UVA treatment.
3.3. Mild-temperature thermal processing of GA- and FA-supplemented
apple juice
In this study, we also evaluated the combination of GA and FA with
mild-temperature thermal processing (55 °C) to inactivate E. coli
O157:H7 in apple juice. As shown in Fig. 3, 4 min of mild-temperature
E.F. de Oliveira, et al.
Fig. 3. Inactivation of Escherichia coli O157:H7 in apple juice containing either
10 mM of Gallic Acid (GA) or 1 mM of Ferulic Acid (FA) and exposed to mild-
temperature thermal processing at 55 °C. Controls consist of bacteria in apple
juice without the addition of GA or FA and exposed to mild-temperature
thermal treatment. Initial bacteria concentration was 6 log CFU mL−1. The
limit of detection of the plate counting method was 1 log CFU mL−1. See
Supplementary material Fig. 1 for non-processed controls.
thermal processing alone was able to reduce the initial bacterial load by
only 1 log CFU mL−1. Furthermore, when mild-temperature thermal
processing was combined with 1 mM of FA (FA + Heat), the initial
bacterial load in apple juice was reduced by 2.5 log CFU mL−1. On the
other hand, combining 10 mM of GA with the same level of mild-tem-
perature thermal processing (GA + Heat) led to the reduction of > 5
log CFU mL−1 of inoculated E. coli O157:H7. Similar results were also
observed when these combinations were applied against L. innocua in
apple juice, where GA + Heat led to significantly higher bacterial in-
activation than FA + Heat and heat energy alone (Supplementary ma-
terial Fig. 3). These results indicate that GA or FA in combination with
low levels of heat energy can lead to a synergistic antibacterial activity
against E. coli O157:H7 in apple juice.
Thermal processing is one of the most common and effective pas-
teurization approach used in the food industry to ensure microbial
safety of food but often has negative influence on the nutritional and
organoleptic properties of the processed food (Gonzalez & Barrett,
2010). In order to mitigate these negative effects, the combination of
sub-lethal levels of mild temperature and sub-lethal concentrations of
bioactive compounds to enhance microbial inactivation in food pro-
ducts have been recently investigated (Petruzzi et al., 2017). Most of
these studies have evaluated the combination of essential oils or es-
sential oils components with mild temperature processing to inactivate
microbes in juices (Ait-Ouazzou, Espina, García-Gonzalo, & Pagán,
2013; Chueca, Ramírez, Arvizu-Medrano, García-Gonzalo, & Pagán,
2016; Espina et al., 2014, 2011, 2012; Leite et al., 2016; Usaga,
Worobo, & Padilla-Zakour, 2014). One study has reported that the
addition of orange essential oil or (+)-limonene prior to mild-thermal
5
Innovative Food Science and Emerging Technologies 56 (2019) 102186
processing at 54 °C could enhance the inactivation of E. coli O157:H7 in
orange juice (Espina et al., 2014). Another study has shown that the
addition of carvacrol to apple, mango, orange and tomato juices fol-
lowed by mild-temperature processing (54 °C) could inactivate E. coli
O157:H7 up to five times faster than mild-temperature processing alone
(Ait-Ouazzou et al., 2013). In our current study, it was demonstrated
that the addition of GA, a natural and food-grade compound, to a juice
product in combination with mild-temperature thermal processing
could provide multifold enhancement inactivation of E. coli O157:H7
compared to thermal processing alone. This combination of treatments
reduces the initial inoculum level from 6 log CFU mL−1 to below the
detection limit of 1 log CFU mL−1.
Similar to the UV-A light synergistic combinations, damages to the
bacterial membrane and to the total intracellular thiol content were
assessed as indicators of heat-mediated synergistic antimicrobial com-
binations. As shown in Fig. 4, mild-temperature thermal processing
alone led to an increase in the number of sub-lethally injured bacterial
cells as compared to the control (i.e. No processing). Similar levels of
sub-lethal injuries were observed for the FA + Heat treatment, while
the GA + Heat treatment led to a significant increase in sub-lethal in-
juries of E. coli O157:H7 (P < 0.05). Furthermore, GA + Heat pre-
sented a significant decrease in the total intracellular thiol content
compared to thermal processing alone and FA + Heat (P < 0.05).
These observations indicate that the presence of GA during thermal
processing enhanced both bacterial membrane damages as well as in-
tracellular thiol oxidation in E. coli O157:H7, as compared to the other
treatments.
Thermal treatment is known to significantly affect the bacterial cell
membrane (Marcén, Ruiz, Serrano, Condón, & Mañas, 2017). In addi-
tion, some antibacterial compounds such as essential oils can also cause
damage to the bacterial cell envelop (Espina, Gelaw, de, Pagán, &
García-Gonzalo, 2013). It has been suggested that the enhanced anti-
bacterial activity of the combination of essential oils and thermal
treatment could be due to accumulative localized stresses in the bac-
terial membrane. While some studies have been performed to in-
vestigate the possible antibacterial mechanisms for the combination of
essential oils and mild-temperature processing, little have been re-
ported for the combination of mild-temperature processing and phe-
nolic acids. In this work it was shown that the combination of
GA + Heat can cause a significant damage to the membrane as well as
to the total intracellular thiol content of E. coli O157:H7 compared to
thermal treatment alone and FA + Heat (Fig. 4a). Previous studies have
shown that GA can damage membranes resulting in an increase of
membrane permeability, as well as impact cytoplasmic environment,
inducing intracellular thiols oxidation and reducing the overall meta-
bolic activity of the bacterial cells (Oliveira et al., 2017). Together,
these observations indicate that GA may not only enhance the mem-
brane damage induced by mild levels of heat but could also induce
damages to the intracellular machinery of the bacteria. Thus, this sy-
nergistic combination of membrane and intracellular damages in bac-
teria could result in the enhanced antibacterial activity observed for
Fig. 4. (a) Sub-lethal injuries and (b) changes in the
total intracellular thiol content of Escherichia coli
O157:H7 after 2 min of mild-temperature thermal
processing of apple juice containing either 10 mM of
Gallic Acid (GA) or 1 mM of Ferulic Acid (FA).
Controls consist of bacteria in apple juice without the
addition of GA or FA. Sub-lethal injuries were eval-
uated by selectively plating treated bacteria in TSA
medium supplemented with 3% NaCl. Results
marked with different letters were statistically sig-
nificant (P < 0.05).
E.F. de Oliveira, et al.
Fig. 5. Inactivation of Escherichia coli O157:H7 in apple juice containing either
10 mM of Gallic Acid (GA) or 1 mM of Ferulic Acid (FA) and exposed to high
pressure processing (HPP; 250 MPa) at 25 °C. Controls consist of bacteria in
apple juice without the addition of GA or FA and exposed to HPP. Initial bac-
teria concentration was 6 log CFU mL−1. The limit of detection of the plate
counting method was 1 log CFU mL−1. See Supplementary material Fig. 1 for
non-processed controls.
GA + Heat.
3.4. High pressure processing (HPP) of GA- and FA-supplemented apple
juice
The synergistic antibacterial activity of GA and FA in combination
with HPP was evaluated against E. coli O157:H7 in apple juice. In this
study, HPP treatment was conducted at 250 MPa, which is a sig-
nificantly lower pressure than the conventional pressures applied for
HPP pasteurization (i.e. 400–600 MPa) (Huang et al., 2017). As illu-
strated in Fig. 5, 5 min of HPP treatment without the presence of GA or
FA reduced the initial load of bacteria by around 1 log CFU mL−1.
Meanwhile, GA in combination with HPP (GA + HPP) for 5 min re-
sulted in the inactivation of approximately 5 log CFU mL−1 of E. coli
O157:H7. In contrast, the combination of FA and HPP (FA + HPP) did
not enhance bacterial inactivation as compared to HPP alone (around 1
log CFU mL−1). Furthermore, similar results were observed against L.
innocua in apple juice, where GA + HPP significantly enhanced bac-
terial inactivation as compared to HPP and FA + HPP (Supplementary
material Fig. 4). These results suggest that GA could synergistically
enhance E. coli O157:H7 inactivation in apple juice during HPP treat-
ment.
To further evaluate the indicators of bacterial damage induced by
these synergistic combinations, damages to the bacterial membrane and
changes in the total intracellular thiol content of E. coli O157:H7 cells
were evaluated after treatment with FA + HPP and GA + HPP. As
shown in Fig. 6a, after 2.5 min of treatment, both GA + HPP and
FA + HPP combinations led to a significant increase in the number of
6
Innovative Food Science and Emerging Technologies 56 (2019) 102186
sub-lethally injured E. coli O157:H7 cells as compared to HPP alone.
Although, despite the initial increase in sub-lethal injuries, FA + HPP
presented limited bacterial inactivation even after 7.5 min of treatment
(Fig. 5). These observations suggest that FA + HPP could induce bac-
terial membrane damage at early treatment stages, but the induced sub-
lethal injuries did not further translate to an increase in bacterial in-
activation as compared to HPP alone. In contrast, the sub-lethal injuries
induced by GA + HPP could be directly related to the high bacterial
lethality observed after the treatment. Characterization of the total
intracellular thiol content showed that HPP alone can affect the in-
tracellular thiol content of E. coli O157:H7 cells, but no further sig-
nificant decrease in the intracellular thiol content was observed when
HPP was combined with GA or FA (Fig. 6b).
The combination of HPP and some chemical bioactive compounds
have been suggested as an alternative to enhance microbial inactivation
in food products while reducing the impact of HPP on food quality
(Black, Kelly, & Fitzgerald, 2005; Bleoancă et al., 2016; Gayán, Torres,
& Paredes-Sabja, 2012; Li & Gänzle, 2016; Rodríguez-Calleja, Cruz-
Romero, O'Sullivan, García-López, & Kerry, 2012). The promising as-
pects of this approach lies on the fact that strong antibacterial syner-
gism could be achieved at relatively sub-lethal levels of HPP. This sy-
nergistic combination of mild or medium pressure HPP and
phytochemical compounds could reduce the energy requirements for
HPP and improve the structural properties of food products. However,
there is still a lack of understanding regarding the overall antimicrobial
mechanisms behind these synergies. It has been suggested that mem-
brane damage induced by the combination of HPP and antimicrobial
compounds, such as peptides and essential oils, could be the main factor
behind microbial inactivation (Gayán et al., 2012). Interestingly, in this
study we observed that membrane damage was not strictly related to
bacterial inactivation, since both FA + HPP and GA + HPP caused high
levels of membrane damage but only GA + HPP enhanced E. coli
O157:H7 inactivation as compared to HPP alone.
It has also been suggested that synergistic processes between HPP
and antimicrobial compounds may depend on specific reactivity of
antimicrobials in the intracellular environment of microbial cells. A
recent study highlighted that antibacterial synergism with HPP was
only achieved for thiol-reactive antimicrobial compounds, suggesting
that bacterial kill was a result of sub-lethal oxidative damages induced
by HPP and intracellular toxicity induced by antimicrobial compounds
(Feyaerts, Rogiers, Corthouts, & Michiels, 2015). In this current study,
it was shown that HPP alone could be causing significant levels of in-
tracellular oxidative stress, and that GA alone presented thiol-reactive
properties (Fig. 6b), which has been previously reported elsewhere
(Cossu et al., 2016; Oliveira et al., 2017). Therefore, the antibacterial
synergism observed for GA + HPP as opposed to FA + HPP seems to
agree with the hypothesis that bacterial inactivation may not be only
due to enhanced membrane damages but also to induced intracellular
reactions such as reaction of antimicrobial compounds with thiols.
More extensive investigations are required to better understand the
mechanism of action driving bacterial inactivation for each synergistic
Fig. 6. (a) Sub-lethal injuries and (b) changes in the
total intracellular thiol content of Escherichia coli
O157:H7 after 2.5 min of high pressure processing of
apple juice containing either 10 mM of Gallic Acid
(GA) or 1 mM of Ferulic Acid (FA). Controls consist of
bacteria in apple juice without the addition of GA or
FA. Sub-lethal injuries were evaluated by selectively
plating treated bacteria in TSA medium supple-
mented with 3% NaCl. Results marked with different
letters were statistically significant (P < 0.05).
E.F. de Oliveira, et al.
combination. The future studies may focus on partitioning of anti-
microbials into bacterial cells, changes in gene expressions, metabo-
lomics, and chemical modifications of intracellular and membrane
structure.
4. Conclusion
This manuscript demonstrated synergistic antibacterial activity of
selected phenolic acids (i.e. GA and FA) in combination with mild levels
of physical stresses in the form of light, heat, or pressure. Some of the
observed synergistic combinations, such as FA + UVA, GA + Heat, and
GA + HPP, were able to significantly enhance the inactivation of E. coli
O157:H7 in apple juice by > 4 log CFU mL−1, while individual physical
and chemical treatments combined presented limited bacterial in-
activation (up to 1 log CFU mL−1). Furthermore, the results of this
study suggested that these synergistic combinations between phenolic
acids and physical processes may present different antibacterial me-
chanisms. GA presented particularly strong antibacterial activity when
combined with heat energy and HPP, causing a significant increase in
intracellular oxidative stress and damages to the bacterial membrane.
In contrast to GA, FA was more effective against E. coli O157:H7 when
combined with UV-A light radiation, possibly due to oxidative stresses
induced by the photo-activity of FA. Overall, we believed that the sy-
nergistic approach of combining natural food-grade phenolic acids,
such as GA and FA, with physical processing can aid in reducing the
overall impact of processing technologies on food quality by either
lowering the processing conditions (e.g. temperature, pressure) and/or
decreasing the processing time without compromising microbial safety.
Further studies are required in order to evaluate these synergistic
treatments for a broader class of bioactive compounds, as well as ap-
plying these synergies against a broader range of microbial targets and
in different types of food matrices.
Funding information
This project was supported by Agriculture and Food Research
Initiative grant no. 2014-67017-21642 from the USDA National
Institute of Food and Agriculture (USDA-NIFA) Program in Improving
Food Quality (A1361) and grant no. 2015-68003-23411 from the
USDA-NIFA Program Enhancing Food Safety through Improved
Processing Technologies (A4131). CAPES/BR supported this work
through the “Science without Borders” scholarship program (recipient
n° 11897-13-9) for Erick F. de Oliveira.
Declaration of Competing Interest
The authors of this study do not have any conflicts of interests.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ifset.2019.102186.
References
Ait-Ouazzou, A., Espina, L., García-Gonzalo, D., & Pagán, R. (2013). Synergistic combi-
nation of physical treatments and carvacrol for Escherichia coli O157:H7 inactivation
in apple, mango, orange, and tomato juices. Food Control, 32(1), 159–167. https://
doi.org/10.1016/j.foodcont.2012.11.036.
Berdejo, D., Pagán, E., García-Gonzalo, D., & Pagán, R. (2018). Exploiting the synergism
among physical and chemical processes for improving food safety. Current Opinion in
Food Science. https://doi.org/10.1016/j.cofs.2018.08.004.
Black, E. P., Kelly, A. L., & Fitzgerald, G. F. (2005). The combined effect of high pressure
and nisin on inactivation of microorganisms in milk. Innovative Food Science &
Emerging Technologies, 6(3), 286–292. https://doi.org/10.1016/j.ifset.2005.04.005.
Bleoancă, I., Saje, K., Mihalcea, L., Oniciuc, E.-A., Smole-Mozina, S., Nicolau, A. I., &
Borda, D. (2016). Contribution of high pressure and thyme extract to control Listeria
monocytogenes in fresh cheese - A hurdle approach. Innovative Food Science &
Emerging Technologies, 38(Part A), 7–14. https://doi.org/10.1016/j.ifset.2016.09.
7
Innovative Food Science and Emerging Technologies 56 (2019) 102186
002.
Chan, P. N. A. (2015). Chemical properties and applications of food additives:
Preservatives, dietary ingredients, and processing aids. In P. C. K. Cheung, & B. M.
Mehta (Eds.). Handbook of food chemistry (pp. 75–100). . https://doi.org/10.1007/
978-3-642-36605-5_37.
Chueca, B., Ramírez, N., Arvizu-Medrano, S. M., García-Gonzalo, D., & Pagán, R. (2016).
Inactivation of spoiling microorganisms in apple juice by a combination of essential
oils' constituents and physical treatments. Food Science and Technology International,
22(5), 389–398. https://doi.org/10.1177/1082013215606832.
Cossu, A., Dou, F., Young, G. M., & Nitin, N. (2017). Biomarkers of oxidative damage in
bacteria for the assessment of sanitation efficacy in lettuce wash water. Applied
Microbiology and Biotechnology, 1–11. https://doi.org/10.1007/s00253-017-8314-5.
Cossu, A., Ercan, D., Wang, Q., Peer, W. A., Nitin, N., & Tikekar, R. V. (2016).
Antimicrobial effect of synergistic interaction between UV-A light and gallic acid
against Escherichia coli O157:H7 in fresh produce wash water and biofilm. Innovative
Food Science & Emerging Technologies, 37(Part A), 44–52. https://doi.org/10.1016/j.
ifset.2016.07.020.
Cossu, A., Le, P., Young, G. M., & Nitin, N. (2017). Assessment of sanitation efficacy
against Escherichia coli O157:H7 by rapid measurement of intracellular oxidative
stress, membrane damage or glucose active uptake. Food Control, 71, 293–300.
https://doi.org/10.1016/j.foodcont.2016.07.009.
de Oliveira, E. F., Tosati, J. V., Tikekar, R. V., Monteiro, A. R., & Nitin, N. (2018).
Antimicrobial activity of curcumin in combination with light against Escherichia coli
O157:H7 and Listeria innocua: Applications for fresh produce sanitation. Postharvest
Biology and Technology, 137(Supplement C), 86–94. https://doi.org/10.1016/j.
postharvbio.2017.11.014.
Espina, L., Gelaw, T. K., de, L.-C., Pagán, R., & García-Gonzalo, D. (2013). Mechanism of
bacterial inactivation by (+)-limonene and its potential use in food preservation
combined processes. PLoS One, 8(2), https://doi.org/10.1371/journal.pone.
0056769.
Espina, L., Condón, S., Pagán, R., & García-Gonzalo, D. (2014). Synergistic effect of or-
ange essential oil or (+)-limonene with heat treatments to inactivate Escherichia coli
O157:H7 in orange juice at lower intensities while maintaining hedonic acceptability.
Food and Bioprocess Technology, 7(2), 471–481. https://doi.org/10.1007/s11947-
013-1076-x.
Espina, L., García-Gonzalo, D., & Pagán, R. (2016). Detection of thermal sublethal injury
in Escherichia coli via the selective medium plating technique: Mechanisms and
improvements. Frontiers in Microbiology, 7. https://doi.org/10.3389/fmicb.2016.
01376.
Espina, L., Somolinos, M., Lorán, S., Conchello, P., García, D., & Pagán, R. (2011).
Chemical composition of commercial citrus fruit essential oils and evaluation of their
antimicrobial activity acting alone or in combined processes. Food Control, 22(6),
896–902. https://doi.org/10.1016/j.foodcont.2010.11.021.
Espina, L., Somolinos, M., Ouazzou, A. A., Condón, S., García-Gonzalo, D., & Pagán, R.
(2012). Inactivation of Escherichia coli O157:H7 in fruit juices by combined treat-
ments of citrus fruit essential oils and heat. International Journal of Food Microbiology,
159(1), 9–16. https://doi.org/10.1016/j.ijfoodmicro.2012.07.020.
Feyaerts, J., Rogiers, G., Corthouts, J., & Michiels, C. W. (2015). Thiol-reactive natural
antimicrobials and high pressure treatment synergistically enhance bacterial in-
activation. Innovative Food Science & Emerging Technologies, 27, 26–34. https://doi.
org/10.1016/j.ifset.2014.12.005.
Gayán, E., Torres, J. A., & Paredes-Sabja, D. (2012). Hurdle approach to increase the
microbial inactivation by high pressure processing: Effect of essential oils. Food
Engineering Reviews, 4(3), 141–148. https://doi.org/10.1007/s12393-012-9055-y.
Ghate, V., Kumar, A., Zhou, W., & Yuk, H.-G. (2015). Effect of organic acids on the
photodynamic inactivation of selected foodborne pathogens using 461 nm LEDs. Food
Control, 57, 333–340. https://doi.org/10.1016/j.foodcont.2015.04.029.
Gonzalez, M. E., & Barrett, D. M. (2010). Thermal, high pressure, and electric field pro-
cessing effects on plant cell membrane integrity and relevance to fruit and vegetable
quality. Journal of Food Science, 75(7), R121–R130. https://doi.org/10.1111/j.1750-
3841.2010.01763.x.
Gould, G. W. (1996). Methods for preservation and extension of shelf life. International
Journal of Food Microbiology, 33(1), 51–64. https://doi.org/10.1016/0168-1605(96)
01133-6.
Huang, H.-W., Wu, S.-J., Lu, J.-K., Shyu, Y.-T., & Wang, C.-Y. (2017). Current status and
future trends of high-pressure processing in food industry. Food Control, 72(Part A),
1–8. https://doi.org/10.1016/j.foodcont.2016.07.019.
Jermann, C., Koutchma, T., Margas, E., Leadley, C., & Ros-Polski, V. (2015). Mapping
trends in novel and emerging food processing technologies around the world.
Innovative Food Science & Emerging Technologies, 31, 14–27. https://doi.org/10.1016/
j.ifset.2015.06.007.
Juneja, V. K., Dwivedi, H. P., & Yan, X. (2012). Novel natural food antimicrobials. Annual
Review of Food Science and Technology, 3(1), 381–403. https://doi.org/10.1146/
annurev-food-022811-101241.
Jung, Y., & Matthews, K. R. (2015). Chapter 17 - Development and application of novel
antimicrobials in food and food processing. Antimicrobial resistance and food safety
(pp. 347–364). . Retrieved from http://www.sciencedirect.com/science/article/pii/
B978012801214700017X.
Leite, C. J. B., de Sousa, J. P., da Costa Medeiros, J. A., da Conceição, M. L., dos Santos
Falcão-Silva, V., & de Souza, E. L. (2016). Inactivation of Escherichia coli, Listeria
monocytogenes, and Salmonella Enteritidis by Cymbopogon citratus D.C. Stapf.
Essential oil in pineapple juice. Journal of Food Protection, 79(2), 213–219. https://
doi.org/10.4315/0362-028X.JFP-15-245.
Li, H., & Gänzle, M. (2016). Effect of hydrostatic pressure and antimicrobials on survival
of Listeria monocytogenes and enterohaemorrhagic Escherichia coli in beef.
Innovative Food Science & Emerging Technologies, 38, Part B, 321–327. doi:https://
E.F. de Oliveira, et al.
doi.org/10.1016/j.ifset.2016.05.003.
Marcén, M., Ruiz, V., Serrano, M. J., Condón, S., & Mañas, P. (2017). Oxidative stress in E.
coli cells upon exposure to heat treatments. International Journal of Food Microbiology,
241, 198–205. https://doi.org/10.1016/j.ijfoodmicro.2016.10.023.
Nakamura, K., Yamada, Y., Ikai, H., Kanno, T., Sasaki, K., & Niwano, Y. (2012).
Bactericidal action of photoirradiated gallic acid via reactive oxygen species forma-
tion. Journal of Agricultural and Food Chemistry, 60(40), 10048–10054. https://doi.
org/10.1021/jf303177p.
Netto, L. E. S., Oliveira, M. A.d., Tairum, C. A., & Neto, J. F.d. S. (2016). Conferring
specificity in redox pathways by enzymatic thiol/disulfide exchange reactions. Free
Radical Research, 50(2), 206–245. https://doi.org/10.3109/10715762.2015.
1120864.
Oliveira, E. F.d., Cossu, A., Tikekar, R. V., & Nitin, N. (2017). Enhanced antimicrobial
activity based on a synergistic combination of sub-lethal levels of stresses induced by
UV-A light and organic acids. Applied and Environmental MicrobiologyAEM.00383-17.
https://doi.org/10.1128/AEM.00383-17.
Penha, C. B., Bonin, E., da, S., Hioka, N., Zanqueta, É. B., Nakamura, T. U., … Mikcha, J.
M. G. (2017). Photodynamic inactivation of foodborne and food spoilage bacteria by
curcumin. LWT - Food Science and Technology, 76, 198–202. doi:https://doi.org/10.
1016/j.lwt.2016.07.037.
Petruzzi, L., Campaniello, D., Speranza, B., Corbo, M. R., Sinigaglia, M., & Bevilacqua, A.
(2017). Thermal treatments for fruit and vegetable juices and beverages: A literature
overview. Comprehensive Reviews in Food Science and Food Safety, 16(4), 668–691.
https://doi.org/10.1111/1541-4337.12270.
Innovative Food Science and Emerging Technologies 56 (2019) 102186
Rodríguez-Calleja, J. M., Cruz-Romero, M. C., O'Sullivan, M. G., García-López, M. L., &
Kerry, J. P. (2012). High-pressure-based hurdle strategy to extend the shelf-life of
fresh chicken breast fillets. Food Control, 25(2), 516–524. https://doi.org/10.1016/j.
foodcont.2011.11.014.
Shirai, A., Watanabe, T., & Matsuki, H. (2017). Inactivation of foodborne pathogenic and
spoilage micro-organisms using ultraviolet-A light in combination with ferulic acid.
Letters in Applied Microbiology, 64(2), 96–102. https://doi.org/10.1111/lam.12701.
Shirai, A., Kajiura, M., & Omasa, T. (2015). Synergistic photobactericidal activity based
on ultraviolet-A irradiation and ferulic acid derivatives. Photochemistry and
Photobiology, 91(6), 1422–1428. https://doi.org/10.1111/php.12507.
Shirai, A., & Yasutomo, Y. (2019). Bactericidal action of ferulic acid with ultraviolet-A
light irradiation. Journal of Photochemistry and Photobiology B: Biology, 191, 52–58.
https://doi.org/10.1016/j.jphotobiol.2018.12.003.
Usaga, J., Worobo, R. W., & Padilla-Zakour, O. I. (2014). Thermal resistance parameters
of acid-adapted and unadapted Escherichia coli O157:H7 in apple-carrot juice blends:
Effect of organic acids and pH. Journal of Food Protection, 77(4), 567–573. https://
doi.org/10.4315/0362-028X.JFP-13-371.
Wang, Q., de Oliveira, E. F., Alborzi, S., Bastarrachea, L. J., & Tikekar, R. V. (2017). On
mechanism behind UV-A light enhanced antibacterial activity of gallic acid and
propyl gallate against Escherichia coli O157:H7. Scientific Reports, 7. https://doi.org/
10.1038/s41598-017-08449-1.
Winther, J. R., & Thorpe, C. (2014). Quantification of thiols and disulfides. Biochimica et
Biophysica Acta (BBA) - General Subjects, 1840(2), 838–846. https://doi.org/10.1016/
j.bbagen.2013.03.031.