LWT - Food Science and Technology 76 (2017) 203e208

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LWT - Food Science and Technology

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

Migration evaluation of silver nanoparticles from antimicrobial edible

coating to sausages
Nicolli Grecco Marchiore a, Isabela Jorge Manso a, Karine Cristine Kaufmann a,
Gislaine Franco Lemes a, Ana Paula de Oliveira Pizolli b, Adriana Aparecida Droval b,

ria Leimann a, *
Lívia Bracht a, Odinei Hess Gonçalves b, Fernanda Vito
a
Departamento Acad^ emico de Alimentos (DALIM), Universidade Tecnolo
gica Federal do Parana
, Campus Campo Moura ~o (UTFPR-CM), via Rosalina Maria
dos Santos, 1233, CEP 87301-899, Caixa Postal: 271, Campo Moura ~o, PR, Brazil
b
s-Graduaça
Programa de Po ~o em Tecnologia de Alimentos (PPGTA), Universidade Tecnolo
, Campus Campo Moura

gica Federal do Parana ~o (UTFPR-CM), via
Rosalina Maria dos Santos, 1233, CEP 87301-899, Caixa Postal: 271, Campo Moura ~o, PR, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Use of silver nanoparticles as an antimicrobial compound directly on foodstuff has been attracted
Received 13 January 2016 attention due to concerns on the total residual silver that remains on the food after preparation. In this
Received in revised form work, silver nanoparticles (AgNPs) were obtained by a green route and applied as edible coating to
23 May 2016
chicken sausages. Antimicrobial activity of the AgNPs were able to inhibit lactic acid bacteria for 30 days,
Accepted 4 June 2016
Available online 6 June 2016
demonstrating that the increase in the shelf life of the sausages was statistically significant (P < 0.05).
The presence of AgNPs also affected the texture of the sausages probably due to the interaction between
silver and phosphorous and sulphur from proteins. After 30 days, lipid oxidation was found to be higher
Keywords:
Antimicrobial activity
in treated sausages than in control samples. Sausages were prepared simulating home cooking and the
Inductive Coupled Plasma with Mass concentration of silver after each step was determined, showing that a simple washing and cooking
Spectroscopy procedure was able to remove most of the silver from sausages. Total silver concentration on the sau-
Lactic acid bacteria sages after that was 5.3 ngAgNPs/gsausage. Also, no appreciable migration of silver nanoparticles from
Chicken sausages sausages surface to its interior was detected.
Texture Profile Analysis © 2016 Elsevier Ltd. All rights reserved.

1. Introduction decreasing sensory characteristics (De Palo, Maggiolino,

Application of edible coatings to meat, poultry, fish, fruits and
vegetables has attracted increasing interest since an array of ad-
ditives can be incorporated to edible coatings formulation
providing new functionalities such as antimicrobial and antifungal
activity (Campos, Gerschenson, & Flores, 2011; Du, Avena-bustillos,
Hua, & Mchugh, 2011). It was demonstrated that food deterioration
caused by pathogens and spoilage bacteria can be significantly
reduced in meat products by antimicrobial-loaded edible coatings
(Alvarez, Ponce, & Moreira, 2013; Brasil, Gomes, Puerta-gomez,
Castell-perez, & Moreira, 2012; Cagri, Ustunol, & Ryser, 2004;
Ferna
ndez-Pan, Carrio

n-Granda, & Mate
, 2014). This is important in
the case of meat sausages because microorganism action in
anaerobic conditions lead to the formation of surface slime thus
* Corresponding author.
E-mail address: fernandaleimann@utfpr.edu.br (F.V. Leimann).
Centoducati, & Tateo, 2013).
Silver ions slowly released from silver nanoparticles (AgNPs)
present broad antimicrobial spectrum against Gram-negative and
Gram-positive bacteria, fungi, protozoa and certain viruses (Kumar
& Münstedt, 2005). However, there is still great concerns on silver
ingestion limits. Silver presents the lowest toxicity among metals to
animal cells, being toxic in concentrations higher than 10 mg L
1
(Leite, 2003; Levin et al., 2009). According to the study developed
by Kittler, Greulich, Diendorf, Ko € ller, and Epple (2010), silver
nanoparticles slowly dissolve into ions in a time scale of several
days. Authors evaluated ions release during 125 days at 5, 25 and
37  C and concluded that the biological action of freshly prepared
and aged nanoparticles are strongly different due to the different
amount of released ions. Hadrup and Lam (2014) reviewed the oral
toxicity of silver ions, silver nanoparticles and colloidal silver. Silver
was detected in body tissues such as the skin epidermis, the
glomeruli and the intestines after exposure to both ionic and
nanoparticulated silver suspensions. In 2010, US EPA (EPA, 2010)

http://dx.doi.org/10.1016/j.lwt.2016.06.013
0023-6438/© 2016 Elsevier Ltd. All rights reserved.
204 N.G. Marchiore et al. / LWT - Food Science and Technology 76 (2017) 203e208
report stressed that the toxicity potential of nanosized silver to
humans depended on the level of exposure and the association
with other nano-Ag containing products (Kim et al., 2015).
AgNPs have been already added to edible coating formulations
to inhibit microbial growth in shitake mushrooms (Jiang, Feng, &
Wang, 2013), minimally processed carrots (Costa, Conte,
Buonocore, Lavorgna, & Nobile, 2012) and asparagus (An, Zhang,
Wang, & Tang, 2008). Unfortunately, little attention was given to
the amount of silver actually ingested or if it is below human
toxicity levels. Interaction between AgNPs and the food matrix is
also worth investigating because silver may migrate to foodstuff
affecting overall properties.
The objective of this work was to investigate silver migration
from an edible coating applied to sausages to the food matrix. Also,
the influence of the silver nanoparticles on bacterial growth,
texture profile and lipid oxidation were determined.
2. Material and methods
2.1. Material
Chicken sausages were acquired from the local market from
Campo Mour~ ao, PR, Brazil (all sausages used were from the same
2 kg package). Soluble starch (Merk, Germany), anhydrous D-
glucose (Isofar, Brazil) and silver nitrate (Proquímios, Brazil) were
used in the silver nanoparticles synthesis. Tryptic soy broth (Bio-
mark, Brazil), Muller-Hinton broth (Biomark, Brazil), MRS agar
(Biomak, Brazil) and saline solution were used in the antimicrobial
analyses. Malonaldehyde bis (dimethyl acetal) (1,1,3,3-
tetramethoxypropane, TMP), 2-thiobarbituric acid, propyl gallate
(Sigma Aldrich, Germany), trichloroacetic acid and EDTA (Vetec,
Brazil) were used in Thiobarbituric acid-reactive substances
(TBARS) assay.
2.2. Silver nanoparticles synthesis
AgNPs were synthesized by the method previously described by
Ghaseminezhad, Hamedi, and Abbas (2012) using D-glucose and
starch as reducing agent and stabilizer, respectively. Initially, silver
nitrate aqueous solution (2 mL, 25 mM) was mixed with starch
solution (50 mL, 1%w/w) and aqueous D-glucose solution (4 mL,
25 mM) was added. Finally, the resultant solution was autoclaved
(Prismatec equipment, Brazil, 121  C and 15 psi) for 15 min forming
the silver colloidal nanoparticles solution.
2.3. AGNPs characterization
Size distribution, polydispersion index (PDI) and z-average size
(Dz) of the synthesized AgNPs were determined by Dynamic Light
Scattering (DLS, Malvern Nanosizer, United Kingdom). Morpho-
logical analysis of AgNPs was performed by Scanning Electron
Microscopy (SEM, Shimadzu SSX 550 Superscan, Japan).
2.4. Sausages coating
AgNPs colloidal solution was diluted to 37.50 mg mL
1 since this
was the minimum inhibitory concentration (MIC) determined by
Pizzoli et al. (2016) against Staphylococcus aureus (ATCC 6538). They
synthesized AgNPs by the same method used in the present work
and determined the MIC of AgNPs against Escherichia coli, Bacillus
cereus, Pseudomonas aeruginosa and Staphylococcus aureus. Since
Staphylococcus aureus was the most resistant microorganism, its
MIC was chosen as reference concentration. All materials used in
the procedure were previously sterilized in an autoclave.
Sausages were immersed during 1 min in the AgNPs solution
(37.50 mg mL
1) and, after that, they were kept over a grid to
remove the excess of solution. For each storage time interval (0, 15
and 30 days), 3 sausages (approximately 135 g aseptically weighed)
were covered with the AgNPs solution and vacuum packaged. For
the control samples the same manipulation was applied except by
the immersion step. Samples were stored at 10 ± 2  C. All treat-
ments were carried out in quadruplicate.
2.5. Microbial analysis
The presence of lactic acid bacteria in the sausages samples was
determined at different time intervals: 0 (just after AgNPs treat-
ment and packaging), 15 and 30 days. Sausages samples were
aseptically weighed (25 g), transferred to sterile plastic pouches
and homogenized with sterile saline solution (225 mL) during
1 min in a stomacher (ITR, MR1204, Brazil). Appropriate dilutions of
the sample homogenates were prepared in sterile peptone water
(0.1%) and inoculated in triplicate in MRS growth media plates
(lactic acid bacteria selective agar). The inoculated culture plates
were incubated in a bacterial culture incubator (Ethik, Brazil) under
aerobic conditions at 37  C for 48 h and finally the plate counts
were determined.
2.6. AGNPs concentration on sausages
Metallic silver concentration was evaluated in sausages during
the storage period (15 and 30 days) and home cooking of the sau-
sages was simulated using the following steps. First, sausages from
one vacuum package were washed with distilled water (1 L). After
that, they were cooked with boiling distilled water (1 L) during
5 min. Finally, the cooked sausages were crushed with distilled
water (1 L) in a domestic blender. The crushed sausages sample was
centrifuged (Mini Spin Plus Eppendorf centrifuge, Germany,
30 min at 14,500 rpm) and the supernatant was collected. The
procedure was adopted in duplicate for all storage time intervals
and the samples (washing water, cooking water and crushed sau-
sages) were submitted to Inductive Coupled Plasma with Mass
Spectroscopy (ICP-MS, Perkin Elmer, NexIon 300 D, Shelton, USA)
analysis. Final results were expressed as metallic silver concentra-
tion (mg0Ag mL
1).
2.7. Texture Profile Analysis
Texture Profile Analysis (TPA) was performed with a TA-XT Ex-
press Enhanced (Stable Micro Systems, United Kingdom) texture
analyzer and the P/36R cylindrical probe was used in the
compression tests. Six pieces from each experimental condition
(2.0 cm diameter, 2.0 cm height) were compressed twice until 50%
of the original sample height. A cross-head speed of 1 mm s
1 was
applied. The following TPA parameters were computed: hardness,
adhesiveness, cohesiveness, springiness, gumminess and
chewiness.
2.8. Assessment of lipid oxidation by TBARS assay
Thiobarbituric acid-reactive substances (TBARS) assay was car-
ried out according to the procedure described by Sallam, Ishioroshi,
and Samejima (2004) with minor adaptations. Sausage samples
(5 g) were mixed with trichloroacetic acid solution (25 mL, 7.5%w/v
TCA, 0.1%w/v propyl gallate and 0.1% EDTA) and homogenized in a
blender for 30 s. After filtration, 5 mL of the filtrate were added to
the TBA solution (5 mL, 0.02 mol L
1) in a test tube. Test tubes were
incubated in a boiling water bath during 40 min then the absor-
bance was measured at 538 nm (UVeVis spectrophotometer, Ocean
Optics, USB650UV, USA). TBA value was expressed as
 N.G. Marchiore et al. / LWT - Food Science and Technology 76 (2017) 203e208
mgmalonaldehyde kg
1
sausage.
2.9. Statistical analysis
The obtained results were evaluated using Student’s t-test
analysis (AgNPs concentration), factorial ANOVA and Tukey’s test
(microbial counts, TPA and TBARS results) at a 5% significance level
(P < 0.05) using the software Statistica 7.0 (Statsoft, USA).
3. Results and discussion
3.1. Silver nanoparticles characterization
The first confirmation that the silver nanoparticles colloidal
dispersion was successfully synthesized was the change in color to
a brown yellowish solution (Sre, Reka, Poovazhagi, Kumar, &
Murugesan, 2015). In Fig. 1, a SEM image of the synthesized
AgNPs is showed and it is possible to observe that AgNPs presented
spherical morphology. Dz and PDI obtained by DLS analyses were
equal to 66 ± 8 nm and 0.37 ± 0.04 respectively. Sankar et al. (2013)
also produced silver nanoparticles with relatively uniform di-
ameters and spherical morphology. Ghaseminezhad et al. (2012)
found a Dz equal to 20 nm for nanoparticles synthesized by the
same method used at the present work. This difference must be
related to AgNPs agglomeration since PDI value obtained is char-
acteristic of large size distributions (Leimann, Cardozo, Sayer, &
Araújo, 2013).
3.2. Lactic acid bacteria counts
The effects of AgNPs treatment and storage time on lactic acid
bacteria (LAB) counts are presented in Fig. 2. AgNPs treated sau-
sages presented significant difference (P < 0.05) when compared to
control sausages just after the treatment (0 day). After 15 days of
storage, AgNPs treated sausages did not present significant bacte-
rial growth. Only after 30 days they reached statistically the same
LAB growth found for control samples at 15 days of storage, indi-
cating that AgNPs treatment successfully controlled the sausages
microbial quality during 15 days. According to Rai, Yadav, and Gade
(2009), bacterial DNA molecules have sulphur and phosphorus as
its major components and AgNPs interact with them affecting
bacteria DNA replication and thus leading to their inactivation.
These results are in accordance with the observations reported
Fig. 1. Scanning Electron Microscopy image of the AgNPs synthesized by modified
polysaccharide method (magnification: 8.000).
205
Fig. 2. Lactic acid bacteria count on sausages treated with AgNPs and control sausages
in function of the storage time intervals (5% significance) (average ± standard devia-
tion, treatments conducted in quadruplicate). Bars marked with different letters pre-
sent significant difference (P < 0.05) by Tukey’s test.
in the literature. An et al. (2008) reported that AgNPs applied to
asparagus prevented significantly psychrotrophic bacteria growth
during 10 days when stored at 2  C and at 10  C, with microbial
counts equal to 4.5 and 6 log CFU g
1 respectively. Jiang et al. (2013)
observed that the AgNPs treatment prevented mesophilic
(4 log CFU g
1), psychrophilic (3 log CFU g
1), pseudomonad
(6.5 log CFU g
1), yeasts and molds (4.3 log CFU g
1) growth on
shiitake mushrooms stored at 4  C for 16 days. Fayaz, Balaji, Girilal,
Kalaichelvan, and Venkatesan (2009) also evaluated AgNPs edible
coatings based on sodium alginate and found that shelf life of
carrots and pears was increased when compared to control with
respect to weight loss and soluble protein content.
3.3. Silver nanoparticles concentration on sausages
AgNPs concentration was determined by ICP-MS for AgNPs
treatment solution and for the samples collected during the sau-
sages preparation steps. Initially it was verified that the concen-
tration of AgNPs on treatment solution applied to the sausages was
equal to 23.47 ± 0.04 mgAgNPs mL
1 and after treating all sausages
the solution remained with 19.27 ± 0.12 mgAgNPs mL
1. One may
conclude that the amount of AgNPs retained at the sausages surface
was equal to 5.3 ngAgNPs g
1 sausage, considering the total weight of
sausages treated with the solution.
Sausages stored for 15 and 30 days were cooked and the AgNPs
concentration was determined. Fig. 3 presents AgNPs concentration
in the water used for sausages washing step (before cooking) and in
the cooking water (Fig. 3(a)) as well as to samples collected from
sausages after cooking, in relation to samples weight (Fig. 3(b)).
The washing step led to the removal of the silver from the
sausages surface for both storage time intervals. It is also worth
noting that the higher amount of AgNPs removed from sausages
was detected in the cooking stage. On the other hand, the cooking
step after 30 days of storage led to a smaller AgNPs concentration,
presenting a significant difference with 15 days of storage
(P < 0.05). This result may be explained by the conversion of
metallic silver (Ag0) into ionic silver (Agþ) which was then released
from nanoparticles (Damm & Münstedt, 2008; Kumar-Krishnan
et al., 2015). According to Sotiriou and Pratsinis (2010), the lower
AgNPs average diameter the higher Agþ ions release from nano-
particles due to the higher total surface area. Agþ ions release rate
206 N.G. Marchiore et al. / LWT - Food Science and Technology 76 (2017) 203e208

probably could be higher at the present work if smaller AgNPs di-
ameters were reached.
When analyzing the AgNPs migration into the sausages, after 15
days (Fig. 3(b)) sausages retained 4.10
3 mg0Ag g
1
sausage and after 30
days this concentration remained unchanged (P < 0.05). Metallic
silver was not absorbed by the sausages with the increase in storage
period, probably remaining only at sausages surface. Although
washing is a common procedure in sausages home cooking, one
may consider including a clear sentence stating the need of
washing the AgNPs treated sausages before cooking in case of
commercial products.
Siqueira et al. (2013) evaluated the acute oral toxicity of AgNPs
in rats to investigate the possibility of their application in edible
coatings composition. Authors observed liver cells degeneration at
1 mg L
1 silver concentration, indicating that the use at this level
must be restricted to the packaging polymeric matrix and not as an
edible coating. At the present work, AgNPs concentration applied to
the sausages was higher than this limit (Fig. 3(b)), however ICP-MS
analysis demonstrated that the actual AgNPs concentration in
sausages after preparation was approximately 4 ng L
1.
3.4. Texture Profile Analysis (TPA)

TPA parameters obtained for control and AgNPs treated sau-

sages are presented at Table 1. Factorial ANOVA demonstrated that
the interaction between storage time and AgNPs treatment was not
significant (P > 0.05). Storage time and the AgNPs treatment did not
significantly affected adhesiveness and springiness (Table 1).
Andre
s, García, Zaritzky, and Califano (2006) evaluated the storage
stability of chicken sausages with low-fat content and found that
adhesiveness and springiness remained statistically constant dur-
ing storage.
Still according to Table 1, it may be observed that storage time
significantly affected (P < 0.05) the following parameters: hardness,
chewiness, gumminess, cohesiveness and resilience. Also, they
were statistically different for 15 days of storage when comparing
to the initial time (0 days) and to 30 days storage for both treat-
ments. Morones et al. (2005) stated that AgNPs interact with pro-
teins that contain phosphorous and sulphur in its structure. It is
possible that the interaction between AgNPs and amino acids was
higher at 15 days due to the higher AgNPs concentration as
detected by IPC-MS (Fig. 3(b)), leading to changes in texture
properties. As Agþ ions were released from AgNPs the intensity of
such interactions were reduced and the texture parameters were
similar to the initial storage condition.

3.5. Sausages lipid oxidation

Table 2 shows lipid oxidation presented as TBARS results. It is

Fig. 3. Metallic silver concentration determined at preparation steps collected sam-
ples: (a) washing and cooking water; (b) residual at sausages after preparation. *Means
significant difference by Students t-test (P < 0.05) (average ± standard deviation,
treatments conducted in quadruplicate).
possible to observe that the treatment with AgNPs presented sig-
nificant difference (P < 0.05) with the control sample only after 30
days. Both samples suffered significant lipid oxidation, however
oxidation on AgNPs treated sausages was fast. After 15 days, this
sample reached a value comparable with the one determined for
control sample at 30 days. The results are in agreement with
findings of Panea, Ripoll, Gonza
lez, Fern

andez-Cuello, and Albertí
(2014) which packaged chicken breasts on packages containing
silver nanoparticles and observed an increase in TBARS values with
storage time. Authors found differences between packaging types
after 10 and 21 days of storage.

Table 1
Texture Profile Analysis (TPA) parameters obtained for control and sausages treated with AgNPs during the storage period (average ± standard deviation, treatments conducted
in quadruplicate).

Parameter 0 days 15 days 30 days

Control AgNPs Control AgNPs Control AgNPs

aA aA aA aA aA
Adhesiveness [N s]
0.26 ± 0.24
0.27 ± 0.19
0.19 ± 0.16
0.40 ± 0.29
0.19 ± 0.16
0.40aA ± 0.29
Springiness [
] 0.92aA ± 0.03 0.92aA ± 0.02 0.92aA ± 0.02 0.923aA ± 0.12 0.92aA ± 0.02 0.923aA ± 0.12
Chewiness [
] 1642.80aA ± 371.10 1559.31aA ± 191.05 1527.32aA ± 235.54 1776.90aA ± 374.92 1527.32aA ± 235.54 1776.90aA ± 374.92
Gumminess [
] 1793.58aA ± 404.51 1740.27aA ± 201.38 1667.26aA ± 257.00 1910.92aA ± 252.71 1667.26aA ± 257.00 1910.92aA ± 252.71
Cohesiveness [
] 0.75aA ± 0.03 0.75aA ± 0.01 0.75aA ± 0.02 0.75aA ± 0.01 0.75aA ± 0.02 0.75aA ± 0.01
Resilience [
] 0.43aA ± 0.03 0.43aA ± 0.01 0.42aA ± 0.02 0.75aA ± 0.01 0.42aA ± 0.02 0.75aA ± 0.01
Hardness [N] 23.54aA ± 6.48 22.81aA ± 2.95 21.72aA ± 3.59 25.27aA ± 3.72 21.72aA ± 3.59 25.27aA ± 3.72
a,b
Means from the same treatment (column) followed by different letters (for each TPA parameter) present significant difference in function of the storage time (P < 0.05);
A,B
Means from the same time period (row) followed by different letters (for each TPA parameter) present significant difference (P < 0.05) by Students t-test in function of the
AgNPs treatment.
 N.G. Marchiore et al. / LWT - Food Science and Technology 76 (2017) 203e208
Table 2
TBARS values for control and sausages treated with AgNPs during the storage period
(average ± standard deviation, treatments conducted in quadruplicate).
Storage time (days) Control (mgMDA kg
1) AgNPs (mgMDA kg
1)
0 0.19aA ± 0.06 0.20aA ± 0.13
15 0.43bA ± 0.02 0.73bA ± 0.15
30 0.65cB ± 0.06 0.96bA ± 0.03
a,b
Means from the same treatment (column) followed by different letters (for each
TPA parameter) present significant difference in function of the storage time
(P < 0.05); A,BMeans from the same time period (row) followed by different letters
(for each TPA parameter) present significant difference (P < 0.05) by Students t-test
in function of the AgNPs treatment.
Although the process of lipid oxidation in meat is thermody-
namically favorable, an activating factor is still necessary to initiate
free radical chain reactions followed by their self-propagation
(German & Kinsella, 1985; Kamil, Jeon, & Shahidi, 2002). Evi-
dence indicates that lipid oxidation in foods may be initiated by a
number of mechanisms (Kubow, 1992) and it is possible that Agþ
released from AgNPs could generate activating factors by oxygen
complexation (Gang, Anderson, Van Grondelle, & Van Santen,
2003; Sant, Weir, & Burrell, 2009) leading to a faster lipid oxida-
tion in a similar mechanism to oxygen iron complexes.
4. Conclusions
Silver nanoparticles (AgNPs) were successfully applied as anti-
microbial agent to control lactic acid bacteria growth on sausages
surface. Lactic acid bacteria counts were reduced as a result of the
AgNPs treatment on sausages. Also, shelf life of the sausages
increased due to the presence of silver nanoparticles. Analyses of
silver concentration during the preparation and cooking step
demonstration that AgNPs concentration may be reduced to
nanogram levels (5.3 ngAgNPs/gsausage) using a simple washing step
before cooking. Texture Profile Analysis parameters (hardness,
chewiness, gumminess, cohesiveness and resilience) were signifi-
cantly affected after 15 days of storage probably due to interactions
between AgNPs and meat proteins. Lipid oxidation was statistically
significant for control and AgNPs treated sausages, however AgNPs
treated sausages suffered a faster oxidation process suggesting that
silver may act as an activation factor in the free radical reactions.
Acknowledgements
Authors thank CAPES (Conve ^nio de Bolsas CP 11/2013 CAPES/
Fundaça~o Arauca
ria), CNPq (Bolsa de iniciaç~
ao em desenvolvimento

gico e inovaça
tecnolo ~o - PIBITI/CNPq 2014/2015) and Fundaça ~o
Arauca
ria (Programa Universal/Pesquisa Ba
sica e Aplicada 24/2012
(Protocolo 37334133700514041013) e Fundaça ~o Arauca
ria) for the
financial support. Also COMCAP Laboratory (Universidade Estadual
de Maringa
dUEM) and LCP (Laborato
rio de Controle de Processos,
Universidade Federal de Santa Catarina e UFSC) for SEM and DLS
analyses, respectively.
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