LWT - Food Science and Technology 197 (2024) 115908

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LWT
journal homepage: www.elsevier.com/locate/lwt

Inhibition of Salmonella growth in exudates drained from poultry meat by

bacteriophage cocktail-containing absorbent food pad
Inho Lee a, Jieon Lee a, b, Minsik Kim a, *
a
Laboratory of Molecular Food Microbiology, Department of Food and Nutrition, BK21 FOUR, College of Human Ecology, Yonsei University, Seoul, 03722, Republic of
Korea
b
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Absorbent food pads are commonly used to absorb exudate drained from food to maintain initial food quality
Alternative antimicrobial throughout shelf life. However, the absorbed exudate often serves as a nutrient for contaminating pathogens in
Chicken meat the pads, requiring appropriate antimicrobial strategies. Here, we developed bacteriophage cocktail-containing
Food packaging
absorbent food pads, called phage pads, by impregnating commercial absorbent food pads with a phage cocktail
Phage
Phage cocktail
consisting of two Salmonella phages, ФYMFM0293 and ФYMFM0433, followed by air drying for 14 h. The
prepared phage pad demonstrated reliable phage titers and water absorbing capacity, and its anti-Salmonella
activity was maintained for 84 days under simulated storage conditions. Consistent with the efficient inhibition
of in vitro Salmonella growth by the phage cocktail, Salmonella spiked directly on the phage pad at ~7-log CFU/
pad was significantly suppressed for 48 h at 4 ◦ C. When Salmonella-contaminated chicken meat was loaded,
Salmonella loads below the detection limit were achieved within the phage pad, in contrast to the control pad,
throughout 7 days of refrigerated storage. Considering its stable long-term activity and efficient reduction of
specific foodborne pathogens in food exudates, the phage cocktail-containing absorbent food pads could be an
effective antibacterial strategy to ensure microbial safety during food distribution to consumers.

1. Introduction
When various meat and fish are cut and stored, their water-holding
capacity (WHC) gradually decreases over time, resulting in the release
of liquid from the muscle tissue, commonly known as ‘food exudate’
(Wang, Li, Zhao, Bi, & Xie, 2022; Young, Karlsson, & Henckel, 2004).
Vegetables and fruits also often become watery during distribution and
storage due to physical maceration and/or prior washing and inade­
quate dehydration (Lufu, Ambaw, & Opara, 2020). These food exudates
and wateriness have a significant negative impact on food yield and
quality. The accumulation of food exudates on the contact surfaces of
food containers and packaging materials can cause food deterioration by
triggering undesirable enzymatic reactions in the food, including lipid
rancidity and discoloration (Ren et al., 2018). Because exudates contain
numerous nutrients from the food and therefore have the potential to be
a good growth medium for various microorganisms, they also often lead
to food spoilage and unsalability by supporting the growth of unwanted
spoilage and/or food poisoning bacteria (Orkusz, 2018; Papuc, Goran,
Predescu, Nicorescu, & Stefan, 2017; Sanches-Silva et al., 2014).
In the case of poultry meat, for instance, WHC and food exudates
have a major impact on meat quality. Several factors, including a
decrease in the pH of the avian carcass after slaughter, result in a
decrease in the WHC of fresh poultry meat and a consequent increase in
exudates (Mir, Rafiq, Kumar, Singh, & Shukla, 2017). Not only does this
cause a sensory deterioration of the meat by increasing toughness and
developing odor, but it can also raise microbial risks. Because the avian
intestine is an ideal habitat for Salmonella, poultry meat is at high risk for
S. Enteritidis and S. Typhimurium contamination (Eng et al., 2015;
Vandeplas, Dauphin, Beckers, Thonart, & Théwis, 2010), and poultry
meat exudates could serve as a carrier medium for Salmonella
cross-contamination. As the production and consumption of poultry
meat have recently increased worldwide due to its lower price compared
to other meats and increased consumer preference for white meat
(OECD & FAO, 2021), control of exudates from poultry meat is required
to provide consumers with safer and higher quality products.
An absorbent food pad is a food packaging product made primarily of
silica gel or cellulose coated with a plastic layer to absorb food exudates
rapidly and efficiently (Han, Ruiz-Garcia, Qian, & Yang, 2018; Otoni,

* Corresponding author. Department of Food and Nutrition, Yonsei University, Seoul, 03722, Republic of Korea.
E-mail address: m.kim@yonsei.ac.kr (M. Kim).

https://doi.org/10.1016/j.lwt.2024.115908
Received 31 December 2023; Received in revised form 19 February 2024; Accepted 26 February 2024
Available online 28 February 2024
0023-6438/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
I. Lee et al.
Espitia, Avena-Bustillos, & McHugh, 2016). Removing exudates from food
surfaces can significantly reduce the potential for unnecessary chemical
reactions and inhibit bacterial growth on food surfaces. Paradoxically,
however, food pads can sometimes pose a microbiological safety issue in
food storage and distribution. This is because harmful bacteria that
contaminate the pad during the manufacturing and/or food storage pro­
cess can grow within the pad by using the absorbed exudates as a nutrient
source (de Azeredo, 2013; Gouvêa, Mendonça, Lopez, & Batalha, 2016).
Although some previous studies have incorporated antimicrobial sub­
stances and chemicals such as fruit peel extracts, essential oils, and
1-chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC) into the absorbent
food pads (Maroufi, Tabibiazar, Ghorbani, & Jahanban-Esfahlan, 2021;
Oral et al., 2009; Ren et al., 2018), the storage stability or potential
toxicity of such materials could be noted as a limitation (Otoni et al.,
2016). The need for additional elaborate processes and related specialized
machinery such as freeze dryers and electric thermostatic ovens, which
ultimately lead to higher product costs, may also be another barrier to the
production of antimicrobial absorbent food pads (Zhou et al., 2020).
Bacteriophages are a type of virus that specifically infect bacteria.
Upon recognition of a specific receptor on the host bacterial cell surface,
phages inject their genome into the host cytosol. After synthesizing new
progeny using host resources and machinery, the infected host cell is
lysed by phage-encoded lysis proteins to release the progeny. Because of
this antibacterial activity, as well as their ubiquity in nature and efficient
propagation within host bacteria, phages are emerging as novel and
cost-effective alternative antimicrobials (Nagel et al., 2022). A distinct
advantage of phages over conventional antimicrobials is their ability to
selectively control host bacteria, leaving the good and beneficial
microbiota present in the treated environment minimally affected or
unaffected (Culot, Grosset, & Gautier, 2019; Stone et al., 2019). Begin­
ning with the U.S. FDA’s approval of Listeria phage P100 as GRAS
(Generally Recognized As Safe) and as a food additive in 2006 (Carlton,
Noordman, Biswas, de Meester, & Loessner, 2005), the development of
phage products to effectively control harmful bacteria in foods and food
processing steps is being actively explored.
In view of the above, the aim of the present study was to develop a
phage cocktail-containing absorbent food pad, called phage pad, by
adsorbing two Salmonella-specific phages onto a commercially available
absorbent food pad and drying it with filtered air. The water-absorbing
capacity (WAC) and long-term anti-Salmonella activity of the developed
phage pad were investigated to determine its suitability as an absorbent
food pad with reliable function. We also evaluated and discussed the
effectiveness of the phage pads in reducing Salmonella load in a model of
artificially contaminated chicken meat stored at 4 ◦ C for 7 days on a tray
lined with the phage pad.
2. Materials & methods
2.1. Bacterial strains and bacteriophages
The bacterial strains used in this study are listed in Supplementary
Table S1. All bacterial strains were incubated in Luria-Bertani (LB) broth
at 37 ◦ C with constant shaking (i.e., 220 rpm) for aeration. If needed,
rifampicin (Rif) was added to the media at a final concentration of 100
μg/mL.
Bacteriophage ФYMFM0293 was previously isolated from raw
chicken meat using a food isolate Salmonella enterica strain YMFM0293
as host bacteria (Lee, Kim, & Kim, 2023). Similarly, phage ФYMFM0433
was isolated from chicken meat using a food isolate S. enterica strain
YMFM0433 and purified by repeated standard plaquing and plaque
pickups (Adams & Comroe, 1956). Phages were propagated by infecting
each host bacterial culture, and phage stocks were prepared by poly­
ethylene glycol (PEG) 6000 precipitation followed by CsCl density
gradient ultracentrifugation, as previously described (Lee et al., 2023).
The prepared phage stocks in SM buffer (50 mM Tris-HCl [pH 7.5], 100
mM NaCl, and 10 mM MgSO4) were stored at 4 ◦ C until further use.
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LWT 197 (2024) 115908
2.2. Morphological and genomic analyses of phages
The phage dilutions were placed on carbon-coated copper grids, and
2% aqueous uranyl acetate (pH 4.0) was applied to negatively stain the
virions. After washing with distilled water, the grid was observed under
a transmission electron microscope (TEM; LIBRA 120®, Carl Zeiss,
Germany) at the National Instrumentation Center for Environmental
Management of Seoul National University. ImageJ (https://imagej.nih.
gov/ij) was used to measure the size of the virions from the captured
TEM images (n = 6).
For genomic analysis, the manually extracted phage genomic DNA
from virions was sequenced using Illumina Miseq. The sequenced reads
were assembled using SPAdes prior to analysis using various bioinfor­
matics tools. Details of phage genome extraction and analysis are
described in the Supplementary methods.
2.3. Determination of antibacterial activity of phages
Bacterial challenge assays were performed as described elsewhere
(Lee et al., 2023) to determine the in vitro antibacterial activity of
phages. Briefly, the culture of the Rif-resistant mutant of Salmonella
YMFM0293 (YMFM0293-RifR) in the early exponential growth phase
was infected with phages at an indicated multiplicity of infection (MOI),
and then bacterial growth was monitored periodically during incubation
at 37 ◦ C by measuring the absorbance of the culture at 600 nm (A600)
using a microplate reader (Tecan, Mannedorf, Switzerland). As a nega­
tive control, an equal volume of SM buffer was added to the culture
instead of phages.
2.4. Determination of ideal air drying time for phage pad preparation
A commercially available absorbent food pad (100 mm × 150 mm;
ECOL Green, South Korea) was purchased from a retail market in Seoul,
South Korea. Each pad consisted of three layers: a central absorbent
nanocellulose layer to absorb food exudates up to ~20 mL, a non-
permeable, poly(lactic acid) (PLA) top layer film to separate food from
the exudate-absorbed nanocellulose layer, and a nonwoven bottom
layer. The phage pad was prepared by impregnating the absorbent food
pad with phage solutions, followed by drying with filtered air as
described in detail below.
To determine the ideal air drying time, the drying rate of the soaked
pad with SM buffer was monitored during air drying. Briefly, an
absorbent food pad was placed on polystyrene polymer (PSP) trays (W
200 mm × L 148 mm × H 28 mm; Living Bank, South Korea), and 15 mL
of SM buffer were spiked uniformly onto the pad using a needle-free
syringe. The wet pad was then dried with filtered air under room con­
ditions (25 ◦ C, 50% relative humidity) in a laminar flow biosafety cab­
inet (BSC). The pad was collected at 2-h intervals from 12 to 16 h and its
weight was measured. The drying rate of the pad was calculated as
follows: [drying rate (%) at time t] = 100 – [(Wt –Wb)/(W0 –Wb)] × 100,
where Wt = (the weight at time t); Wb = (the weight of the unabsorbed
baseline pad); W0 = (the weight at time 0). For complete dehydration, a
pad dried in BSC for 14 h was placed in a desiccator at 25 ◦ C for addi­
tional 48 h. Pad weight was measured at 24 h and 48 h to calculate the
drying rate as described above.
2.5. Determination of water absorbing capacity (WAC) of phage pad
To determine the WAC of the pad, the initial weight of the dried pad
was firstly measured. The pad was then completely immersed in 50 mL
of SM buffer on a PSP tray. The weight of the wet pad with the buffer and
the volume of the remaining SM buffer in the PSP tray were measured
every 30 min until 90 min. The WAC of the pad was calculated as fol­
lows: [WAC (%)] = [(Ww – Wb)/Wb] × 100, where Ww = (weight of the
wet pad); Wb = (the weight of the unabsorbed baseline pad).
I. Lee et al.
2.6. Determination of phage infectivity changes in phage pad
After 15 mL of each phage lysate (~107 PFU/mL) were applied to
absorbent food pads, the phage-absorbed wet pads were dried as
described in section 2.4. At 12, 14, and 16 h of drying, the pad was ho­
mogenized with 150 mL of SM buffer for 2 min using a homogenizer
(BagMixer 400; Interscience, St. Nom, France). The homogenate was
then centrifuged, and the filtered supernatant containing phage particles
eluted from the pad was subjected to the standard plaquing assay to
enumerate phage titers.
To determine the long-term stability of the bactericidal activity of the
phage pad, the change in phage infectivity was monitored during 84
days of storage. To simulate the practical distribution and storage con­
ditions of absorbent food pads in markets, the phage pad prepared with
the phage cocktail (~109 PFU/mL) was packaged in a sterilized poly­
vinyl chloride (PVC) bag and stored in a cardboard box at room tem­
perature. At 0, 7, 14, 28, 56, and 84 days of storage, the number of
infectious phages in the pad was counted as described above.
2.7. Evaluation of anti-Salmonella activity of phage pad
The antimicrobial activity of the phage pad was evaluated as
described elsewhere with some modifications (Silva, Domingues, &
Nerín, 2018). The phage pad was uniformly contaminated with 10 mL of
Salmonella YMFM 0293-RifR (7.5 × 105 CFU/mL PBS). To mimic the
situation of food storage, the contaminated pad was wrapped in plastic
wrap (linear low-density polyethylene; Clean Wrap, South Korea) and
stored in a refrigerator for 48 h. At 0, 24, and 48 h, the pad was ho­
mogenized with 100 mL PBS as described in section 2.6. After centrifu­
gation of the 1 mL homogenate at 16,000×g for 1 min at 4 ◦ C, the
bacterial pellets were washed twice with PBS to remove phages and
resuspended with 1 mL of fresh PBS. The number of Salmonella YMFM
0293-RifR in the suspension was then counted by plating onto fresh
LB-Rif agar plates. An absorbent pad prepared with SM buffer instead of
the phage cocktail was used as a negative control.
2.8. Storage model of Salmonella-contaminated chicken meat
Carved raw chicken meat was purchased from a traditional market in
Seoul, South Korea. Chicken pieces (200 g/experimental group) were
uniformly contaminated with 10 mL of Salmonella YMFM 0293-RifR (5
× 105 CFU/mL) using an atomizer (HDPE, Φ × H = 33 mm × 130 mm,
DAIHAN Scientific) inside the BSC. The contaminated meat was then
placed on the PSP tray lined with the phage pad, and the tray was
wrapped in plastic wrap. During 7 days of storage in the refrigerator at
4 ◦ C, the tray was taken out at the indicated day, and the number of
Salmonella in the lined phage pad was counted as described in section
2.7. An SM buffer-incorporated pad was used as a negative control.
To enumerate the number of Salmonella from the stored chicken
meat, the chicken pieces were homogenized with 200 mL of PBS for 30 s
using Pulsifier II® (Microgen Inc., Camberley, UK). After centrifugation
of the homogenates at 16,000×g for 1 min at 4 ◦ C, the bacterial pellets
were washed twice with PBS and resuspended in 1 mL of PBS. By plating
the suspension onto LB-Rif agar plates, the number of Salmonella YMFM
0293-RifR was counted.
2.9. Statistical analysis
All experiments were performed independently in triplicate unless
mentioned otherwise, and data are expressed as means and standard
deviations (S.D.). GraphPad Prism 8.4.3 (GraphPad Software, USA) was
used for statistical analysis and visualization of results. One-way ANOVA
with Tukey’s multiple comparison test or Student’s t-test was used to
analyze the significance of the difference between groups. A P-value
<0.05 was considered statistically significant.
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LWT 197 (2024) 115908
3. Results and discussions
3.1. Characterization of Salmonella-specific phage ΦYMFM0433
In the present study, we used Salmonella and chicken meat as models
of the target foodborne pathogen and stored food, respectively. Salmo­
nella-infecting bacteriophages were used as antimicrobial agents incor­
porated into the pad. Since the fitness cost for target bacteria to develop
resistance to multiple phages simultaneously is quite high and thus
difficult to achieve, phage cocktails are generally preferred to increase
antibacterial efficacy in phage-mediated biocontrol (C. C. Li, Shi, Sun, &
Zhang, 2022). Therefore, we also investigated the use of a phage cocktail
consisting of different phages recognizing different receptors on the host
Salmonella cell surface. One component phage we used was a virulent
phage ФYMFM0293 that recognizes O-antigens of Salmonella LPS as a
host receptor (Lee et al., 2023).
As the other component of the phage cocktail, a novel phage
ФYMFM0433 was additionally isolated from raw chicken meat.
ФYMFM0433 produced relatively smaller and more turbid plaques than
ФYMFM0293 on the lawn of Salmonella YMFM0293 (Fig. 1A and Sup­
plementary Fig. S1A). Although two phages were similar in their viral
morphology under the TEM as a non-prolate Siphovirus morphotype with
a long flexible tail, the lengths of the virion head and tail differed from
each other (Fig. 1B and Fig. S1B). ФYMFM0433 has a tail of 163.44 ±
10.20 nm, which is ~30 nm longer than ФYMFM0293, and an icosa­
hedral head of 68.19 ± 3.18 nm.
ФYMFM0433 formed single plaques on the lawns of Salmonella
ΔrfaL, ΔrfaG, ΔrfbP, ΔompC, ΔfhuA, ΔlamB rfbP, and ΔflgK mutant
strains (Table S1), indicating that Salmonella LPS, the outer membrane
proteins OmpC, FhuA and LamB, and flagella, which have been reported
as host receptors for some Salmonella phages (M. Kim, Kim, Park, & Ryu,
2014; M. Kim & Ryu, 2011, 2012), were not used as host receptors by
ФYMFM0433. However, the Salmonella ΔbtuB strain could not support
the formation of ФYMFM0433 plaques, whereas the btuB complemen­
tation strain fully restored the plaque formation (Fig. 1C). This result
suggests that ФYMFM0433 uses the vitamin B12 transporter protein
BtuB as its host receptor.
When the host range was tested, single plaques of ФYMFM0433 were
formed by various Salmonella serovars tested, including Salmonella
enterica subsp. enterica serovar Typhimurium, S. Dublin, S. Enteritidis, S.
Heidelberg, S. Paratyphi, and S. Typhi (Table S1). Five food isolates
belong to S. enterica and four other isolates including S. diarizonae were
also sensitive to ФYMFM0433 by forming plaques and growth inhibition
zone, respectively. Compared to ФYMFM0293, which complements the
host range of ФYMFM0433 with Salmonella-specific inhibition, three
Escherichia coli and one Shigella flexneri strain tested were also infected
by ФYMFM0433, indicating the useful polyvalent nature of
ФYMFM0433 with broad host range.
Analysis of the 110,779-bp dsDNA genome and phylogenetic analysis
based on the amino acid sequence of the terminase large subunit sug­
gested that ФYMFM0433 belongs to the family Demerecviridae, sub­
family Markadamsvirinae, genus Epseptimavirus (Fig. 1D and Fig. S2). The
genome of ФYMFM0433 (GenBank accession number PP315292), con­
taining 167 predicted ORFs and 30 tRNA-coding sequences, could be
divided into three functional regions (i.e., pre-early, early, and late re­
gions), similar to other members of the subfamily Markadamsvirinae
(Fig. 1D and Table S2) (Cong et al., 2021; M. Kim & Ryu, 2011). Notably,
genes related to the lysogenic cycle of temperate phages, harmful vir­
ulent/toxic genes, and antibiotic resistance genes were absent from the
genome, supporting the safety of ФYMFM0433 as an alternative anti­
microbial candidate.
3.2. In vitro anti-Salmonella efficacy of phages
To determine the anti-Salmonella efficacy of the phages, bacterial
challenge assays were performed under different MOIs. Treatment with
I. Lee et al. LWT 197 (2024) 115908

Fig. 1. Characteristics of Salmonella phage ФYMFM0433. (A) Plaque morphology of ФYMFM0433 on the host Salmonella YMFM0293 lawn. (B) TEM image of
ФYMFM0433 consisting of an icosahedral head and a flexible long tail. Scale bar, 50 nm. (C) Host receptor determination of ФYMFM0433 by spotting assay. WT, S.
Typhimurium LT2(c); ΔbtuB + pACYC184, btuB deleted LT2(c) with a backbone plasmid pACYC184; ΔbtuB + pMS100, btuB complemented strain using a btuB-
complementing plasmid pMS100. (D) Phylogenetic tree of ФYMFM0433 and ФYMFM0293 based on amino acid sequence comparison of the terminase large subunits
in various phages. Phage taxa are indicated on the right side of the tree. The accession number for each sequence is given in Table S3.

either ФYMFM0293 or ФYMFM0433 could inhibit the in vitro growth of
Salmonella YMFM0293 in an MOI-dependent manner (Fig. 2A and B).
Consistent with the observation that ФYMFM0293 produced larger and
clearer plaques than ФYMFM0433 (Fig. 1A and Fig. S1A), the anti-Sal­
monella activity of ФYMFM0293 is more robust than that of
ФYMFM0433 by rapidly inhibiting the Salmonella growth and main­
taining the inhibition up to ~6 h. ФYMFM0433 showed immediate in­
hibition of Salmonella growth when treated at a high MOI (i.e., MOI of
1), but the growth resumed rapidly at all MOIs tested.
Since ФYMFM0293 and ФYMFM0433 use Salmonella O-antigens and
BtuB, respectively, as host receptors, we expected that a phage cocktail
consisting of these two phages would be more effective in controlling
Salmonella by suppressing the growth resumption, which generally re­
sults from receptor mutation-mediated phage resistance (Hampton,
Watson, & Fineran, 2020; Hasan & Ahn, 2022; H. Kim, Kim, Jee, Heu, &
Ryu, 2022). Indeed, treatment with the phage cocktail at an MOI of 1
suppressed the Salmonella regrowth for at least 12 h (Fig. 2C). The
growth resumption observed at lower MOIs may be due to insufficient
phage titers treated, which were not enough to exert pressure on Sal­
monella to fail to withstand the fitness cost. Based on the host range, host
receptor, and bacterial challenge assay results, we used this phage
cocktail consisting of equal concentrations of ФYMFM0293 and
ФYMFM0433 as an alternative anti-Salmonella agent in the following
experiments.
3.3. Preparation of phage pad with phage cocktail
An absorbent food pad should not interfere with the natural prop­
erties of the food and should not be an economic burden to the customer.
To save the cost and time of the phage pad preparation, we impregnated
a commercial absorbent food pad with the phage cocktail solution and
then dried the pad with filtered air to restore its WAC (Fig. 3A).
To determine the time required for pad drying in the preparation
process, the drying rate of the pad soaked with 15 mL of SM buffer was
calculated during air drying in the BSC. After 14 h of drying, the drying
rate reached ~95%, and did not increase thereafter (Fig. 3B). No sig­
nificant further drying occurred up to the following 48 h of drying in a
desiccator at 25 ◦ C (Fig. 3C), indicating that drying for 14 h under
laminar airflow is sufficient to dry the wet pads. Thus, in the following
experiments, we soaked the absorbent food pad in 15 mL of phage
cocktail solution, and then dried it for 14 h at room temperature in the
BSC to prepare the Salmonella-controlling phage pads.
Since the primary function of the absorbent food pad is to absorb and
retain food exudates, we next evaluated the WAC of the phage pad.

Fig. 2. Bactericidal activity of phages against Salmonella. Salmonella YMFM0293 was challenged with (A) ФYMFM0293, (B) ФYMFM0433, and (C) phage
cocktail at the indicated MOIs, and bacterial growth was monitored periodically up to 12 h. Results are expressed as the mean and S.D. of independent tripli­
cate assays.

4
I. Lee et al. LWT 197 (2024) 115908

Fig. 3. Preparation of phage cocktail-containing absorbent food pad. (A) Schematic of phage pad preparation. (B) Drying rates (%) of the absorbent food pad
impregnated with 15 mL of SM buffer were determined during the 16-h air drying process. (C) To determine the completeness of drying, the pad dried for 14 h was
further dried in a desiccator for 48 h and the change in drying rate (%) was monitored. (D) The water absorbing capacity (WAC, %) of each original commercial food
pad and the prepared phage pad was calculated at 30 min of buffer absorption. Results are expressed as the mean and S.D. of independent triplicate assays, except for
panel (B) where sextuple assays were performed. One-way ANOVA with Tukey’s post-hoc test was used for statistical analysis. Groups with different lowercase letters
are significantly different (P < 0.0001).

When the phage pad was completely soaked in SM buffer for 30 min, a of meat.
total of ~15 mL of SM buffer was absorbed into the pad. This corre­
sponds to 0.1 mL/cm2 pad and a WAC of ~688%, which is approxi­
3.4. Determination of phage stability in phage pad
mately 72% of the WAC of the original commercial absorbent food pad
(Fig. 3D). The amount of buffer absorbed or the WAC of the phage pad
As viral entities composed of a protein scaffold and genetic material,
did not change significantly with increasing soaking time (Fig. S3),
phages are susceptible to denaturation by various factors, including
indicating that the phage pad was able to fully absorb the surrounding
severe changes in pH and temperature beyond critical points and harsh
fluid to its maximum capacity within at least 30 min. Considering that
desiccation stress (Favor, Llanos, Youngblut, & Bardales, 2020; Jońc­
exudates from fresh retail meat cuts generally range from 1 to 3% by
zyk-Matysiak et al., 2019; Kamali, Yavarmanesh, Najafi, & Koocheki,
weight (Offer & Knight, 1988), the WAC of the developed phage pad in
2022a). Since phage denaturation results in a loss of antibacterial effi­
150 cm2 is sufficient to absorb ~15 mL of food exudates from up to 500 g
cacy, we next examined whether the drying process affected the phage

Fig. 4. Stability of infectious phages in phage pad. During the 16 h air drying process of the phage pad impregnated with ~107 PFU/ml of ФYMFM0293 (A) or
ФYMFM0433 (B), the phages remaining in the pad were eluted in SM buffer at 2 h intervals and the number of infectious phages was counted. (C) To simulate the
practical conditions for absorbent food pads in market distribution and storage, the phage pad prepared with ~109 PFU/ml phage cocktail was packaged in a PVC bag
and placed in a cardboard box. During 84 days of long-term storage under indoor conditions, the titer of infectious phages remaining in the pad was determined at the
indicated time points. Mean and S.D. of triplicate assays are shown. One-way ANOVA with Tukey’s post-hoc test was performed, and significantly different groups are
indicated by different lowercase letters (P < 0.0001).

5
I. Lee et al.
infectivity during the phage pad preparation.
When the phage pad of ФYMFM0293 was dried, the phage titer
remaining in the pad was not statistically different from the initial titer,
and it did not change significantly during the 16 h of drying (Fig. 4A). In
contrast, the titer of ФYMFM0433 was reduced by approximately 1.5
PFU/mL within 12 h of pad drying compared to the initial phage titer at
time 0, and this reduced titer was maintained through 16 h of drying
(Fig. 4B). The initial ФYMFM0433 titer at time 0 was also different from
the spiked phage titer (i.e., ~106 vs. ~107 PFU/mL), which may be
attributed to incomplete elution of ФYMFM0433 into the SM buffer due
to entrapment in the absorbent material. Phages classified in the genus
Tequintavirus in the subfamily Markadamsvirinae, such as T5, have been
reported to have L-shaped tail fibers (LTFs) that interact with the
polysaccharide substances (e.g., lipopolysaccharides of host bacteria)
(Garcia-Doval et al., 2015; Heller & Braun, 1982). Given this, the
interaction of the gene product of ORF 148, which was ~53% similar to
T5 LTF at the amino acid sequence level, of Markadamsvirinae phage
ФYMFM0433 with the nanocellulose absorbent material in the pad
might result in the incomplete release of ФYMFM0433 from the pad
(Table S2). ФYMFM0293 did not possess the LTFs (Lee et al., 2023),
which is well consistent with the result that ФYMFM0293 was fully
released as spiked titers from the pad into the elution SM buffer
(Fig. 4A). Although the titer of ФYMFM0433 released into the buffer was
slightly decreased by the drying process, substantial enough numbers of
ФYMFM0293 and ФYMFM0433 remained infectious in the dried phage
pad.
Since absorbent food pads are typically transported and sold in bulk
sizes in PVC bags and stored indoors until used, one of the requirements
for antibacterial absorbent food pads is to maintain their antibacterial
activity during storage in room conditions. Thus, the long-term stability
of the phage pad was evaluated in terms of antibacterial activity during
the 84 days of indoor storage packed in PVC bags. The titer of infectious
phage particles in the phage pad did not change significantly until the
end of the storage and remained stable at its initial value (Fig. 4C). This
result indicates that the developed phage pads packed in PVC bags can
stably maintain their specific antibacterial activity for at least 84 days
under common indoor conditions. The long-term stability of the phages
is superior to other antimicrobial agents that could be relatively easily
inactivated in food pads during storage. For example, essential oils can
be evaporated rapidly from the pads during the storage due to their
volatile nature (X. Li et al., 2021; Sharma, Barkauskaite, Jaiswal, &
Jaiswal, 2021; Zhou et al., 2020). Phage solution is typically lyophilized
with various types of excipients, such as sugars and sugar alcohols,
polymers, etc., to solidify it and thus maintain its long-term potency
under ambient storage and use conditions (Zhang, Zhang, & Ghosh,
2020). In particular, saccharide protectants form a rigid, amorphous
glassy sugar matrix in which phage particles are immobilized to main­
tain their protein structure (Chang et al., 2019; Xu et al., 2023). How­
ever, the phage pads developed in this study achieved the reliable
long-term phage stability without the incorporation of any additives
other than SM buffer. The saccharides of the nanocellulose in the
absorbent materials may contribute to this prolonged phage shelf life by
forming a rigid amorphous matrix that serves to protect the phage
particles from desiccation stress, but further experimental evidence is
needed.
3.5. Evaluation of anti-Salmonella activity of phage pad
To evaluate the potential of the phage pads for Salmonella biocontrol,
Salmonella YMFM0293-RifR cells were spiked directly onto the phage
pad and the number of surviving Salmonella cells was monitored peri­
odically for 48 h of storage at 4 ◦ C. After 24 h, the Salmonella load in the
pad containing ΦYMFM0293 or ΦYMFM0433 at an MOI of 1000 was
reduced by 2.91-log and 1.97-log CFU/pad, respectively, compared to
the control absorbent food pad that containing SM buffer (Fig. 5). In the
subsequent 24 h of storage, Salmonella could not grow significantly in
6
LWT 197 (2024) 115908
the pads, including the control pad, due to refrigeration-mediated Sal­
monella inhibition (Morey & Singh, 2012). The phage cocktail pad was
able to inhibit Salmonella growth to below the detection limit (i.e., 10
CFU/pad) at 24 h and suppress growth resumption by 48 h (Fig. 5). The
phage pad at an MOI of 10,000 also exhibited the efficient control of
inoculated Salmonella on the pad (Fig. S4).
Gouvêa et al. also reported inhibition of Salmonella in absorbent food
pads with a phage cocktail (Gouvêa et al., 2016). Although their pad
remarkably reduced the number of Salmonella by 4.36-log for 12 h of
treatment at 15 ◦ C, in contrast to our result, Salmonella growth resumed
after 24 h. Since we strategically combined two different phages
recognizing Salmonella O-antigens and BtuB as host receptors, respec­
tively, to prepare the phage cocktail, a more efficient control of Salmo­
nella would have been possible compared to the study by Gouvêa et al.
where six phages were combined but each was not fully characterized.
The differences in experimental setting between two studies may also
influence the results. In particular, Gouvêa et al. used Salmonella in
nutrient-rich tryptic soy broth and stored the pad at 15 ◦ C, whereas we
used Salmonella cells resuspended in PBS and stored the pad at a much
lower temperature, 4 ◦ C, which is more unfavorable for Salmonella
growth. The difference in the adsorbent materials used would be another
reason for the difference. All the above factors need to be comprehen­
sively considered in further study to improve and properly evaluate the
antibacterial activity of the phage pad.
3.6. Anti-Salmonella activity of phage pad in Salmonella-contaminated
chicken meat storage model
To determine the practical applicability of the developed phage pad,
the number of Salmonella from the pad placed under artificially Salmo­
nella-contaminated chicken meat was counted during the 7 days of
refrigerator storage (Fig. 6A). When the contaminated chicken meat was
placed on the control pad containing SM buffer, the number of Salmo­
nella transferred from the meat to the pad was reduced by approximately
1-log during the first 24 h of storage. Thereafter, however, the number of
Salmonella increased steadily until the end of the experiment, indicating
that the Salmonella transferred from the chicken meat to the pad were
able to multiply even at 4 ◦ C, using the exudates drained from the
chicken meat as a nutrient source. In contrast, the phage pads, regardless
of their MOI, reduced the number of transferred Salmonella to below the
detection limit throughout the experiment after day 1. This result
demonstrates that the phage pad we developed with the cocktail of two
different phages, ΦYMFM0293 and ΦYMFM0433, could successfully
suppress the growth of Salmonella in the pad that absorbed exudates
Fig. 5. Antibacterial activity of phage pad against directly inoculated
Salmonella at 4◦ C. The phage pad of single phage or phage cocktail was
directly spiked with Salmonella YMFM0293-RifR at an MOI of 1000. During
storage at 4 ◦ C for 48 h, viable Salmonella cells in the pad were enumerated at
24-h intervals. An absorbent food pad prepared with SM buffer was used as a
negative control. Results are presented as the mean and S.D. of three inde­
pendent experiments. At each time point, the statistical difference from the
negative control was analyzed by Student’s t-test. *, P < 0.05; **, P < 0.01;
****, P < 0.0001; n.d., not detected; dashed line, limit of detection.
I. Lee et al.
Fig. 6. Anti-Salmonella activity of phage pad in a model of chicken meat refrigerated storage. Chicken meat artificially contaminated with 5 × 105 CFU/mL of
Salmonella YMFM0293-RifR was placed on a PSP tray lined with the phage pad (MOI of 1000 or 10,000), and the tray was wrapped in plastic wrap. During 7 days of
storage in refrigerator, the number of Salmonella in the phage pad (A) or in the chicken meat (B) was monitored at the indicated time points. An absorbent food pad
prepared with SM buffer served as a negative control. Results are presented as the mean and S.D. of triplicate assays. n.d., not detected.; dashed line, limit
of detection.
from the stored food.
Throughout the refrigerated storage time, the Salmonella population
in the chicken meat loaded on the control pad gradually decreased from
approximately 4-log to 3-log CFU/g (Fig. 6B). Since moisture content is a
critical factor for bacterial survival, the lower viability of Salmonella in
chicken meat (Fig. 6B) compared to exudate (Fig. 6A) may be related to
the lower moisture content of the chicken meat that has gradually dried
during prolonged storage compared to the continuously moist pad (Silva
et al., 2018). A Salmonella reduction pattern was also observed with the
phage pads, but a significantly (P = 0.0184) lower Salmonella load was
achieved with the phage pad at an MOI of 10,000 on day 7 compared to
the control pad (Fig. 6B). This limited reduction in Salmonella load in the
meat itself compared to the exudate may be related to the limited
leaching of the phages from the pad to the meat. Phages are not motile,
and passively diffuse depending on the flow of the surrounding medium
such as liquid and air (X. C. Li, Gonzalez, Esteves, Scharf, & Chen, 2020)
or are transported by their host (Yu et al., 2021). Therefore, retrograde
transfer of phage particles from the absorbent pads to the chicken meat
would normally be difficult to occur unless consumer behavior affects
the upright position of the food packages. On the other hand, if the
contaminated meat does not shed exudate, the function of the pad to
absorb exudate would not be necessary, but at the same time, no path­
ogen reduction can be expected because the exudate that serves as a
medium for pathogen-phage contact is absent.
A non-permeable PLA film on the top of the pad, which prevented
direct contact of the chicken meat with the phage-containing cellulose
absorbent materials, could also influence the result. A previous study of
antimicrobial MC-coated absorbent food pads with a non-permeable
polyethylene top film also showed a similar result: a significant reduc­
tion of Salmonella occurred in ground chicken meat loaded on the MC-
coated pads, but a more pronounced reduction was observed in the
MC-coated pads per se (Ren et al., 2018). Incorporating phages or natural
antimicrobials into the top film layer of the pad would further contribute
to the microbial safety of the packaged food by exerting direct antimi­
crobial activity against the contacted food surfaces (Dai, Huang, Jia,
Fang, & Qin, 2022; Kamali, Yavarmanesh, Najafi, & Koocheki, 2022b).
The combination of phage-based antimicrobial processes, such as
spraying phage solution on fish fillets and scalding poultry carcasses
with thermostable phages, could be an additional strategy to comple­
ment the microbial safety of foods packaged with the phage pads (Lee
et al., 2023; Soni, Nannapaneni, & Hagens, 2010).
The present proof-of-concept study demonstrated the potential of
phage pads in controlling undesirable pathogen growth in food exu­
dates, but some limitations also need to be carefully considered for its
widespread application in the food industry. Since food and food exu­
dates may be contaminated not only with a certain foodborne pathogen
such as Salmonella possessing LPS and BtuB, but also with other common
7
LWT 197 (2024) 115908
contaminants such as lactic acid bacteria, psychrotrophs, and other
Enterobacteriaceae (Ren et al., 2018), phage pads should be prepared
with a phage cocktail consisting of more diverse phages targeting these
bacteria as well to extend their antimicrobial range. The economic
feasibility and scalability of the phage pad is another consideration for
use in large-scale food packaging in the food industry. The phage pad
can be cost-competitive compared to other antimicrobial food pads that
use chemicals, such as encapsulated essential oils (X. Li et al., 2021).
First, the large number of phages can be easily obtained just by propa­
gating using the appropriate host bacteria and their growth medium.
Second, the incorporation of phages into the pad does not require special
facilities and equipment but does require the steps of impregnating and
drying the phage solution. However, these additional steps increase the
production cost compared to the conventional food pad, and the cost
may be passed on to the consumer as a burden. Thus, further studies are
needed on how to produce the phage pads more cost-effectively while
maintaining the efficacy for widespread application in the food industry.
4. Conclusion
In the present study, we developed an antimicrobial absorbent phage
pad by impregnating a phage cocktail onto a commercially available
absorbent food pad and drying it in filtered air. Through a compre­
hensive analysis of the phage genome and host receptor, the phage
cocktail consisting of a novel, BtuB-specific phage ΦYMFM0433 and an
O-antigen-specific phage ΦYMFM0293 was strategically prepared and
incorporated into the pad to effectively control Salmonella. The phage
pad exhibited sufficient WAC (i.e., ~688%, 0.1 mL/cm2 pad) to absorb
meat exudates, and its anti-Salmonella activity persisted for up to 84
days under simulated storage conditions, supporting its practical use.
The phage cocktail incorporated into the pad significantly inhibited the
growth of Salmonella to undetectable levels in the phage pad carried
with contaminated chicken meat during refrigerated storage, and the
Salmonella load in the meat could also be significantly reduced by the
phage pad (MOI = 10,000) on day 7 of storage. With appropriate phage
combinations, phage pads can be proposed as a simple but promising
antimicrobial food packaging system that exerts long-term, specific
antibacterial activity against undesirable microbes in exudates drained
from packaged foods.
Funding
This work was supported by the National Research Foundation of
Korea (NRF) funded by the Ministry of Science and ICT of Korea [grant
number 2019R1C1C1004758].
I. Lee et al.
CRediT authorship contribution statement
Inho Lee: Writing – original draft, Visualization, Validation, Meth­
odology, Investigation, Formal analysis, Data curation, Conceptualiza­
tion. Jieon Lee: Validation, Investigation. Minsik Kim: Writing –
review & editing, Writing – original draft, Visualization, Supervision,
Resources, Funding acquisition, Formal analysis.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2024.115908.
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