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Migration evaluation of silver nanoparticles from antimicrobial edible coating to sausages
LWT - Food Science and Technology xxx (2016) 1e6
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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 , Campus Campo Moura ~o (UTFPR-CM), via Rosalina Maria gica Federal do Parana Departamento Acad^ emico de Alimentos (DALIM), Universidade Tecnolo ~o, PR, Brazil dos Santos, 1233, CEP 87301-899, Caixa Postal: 271, Campo Moura b ~o em Tecnologia de Alimentos (PPGTA), Universidade Tecnolo , Campus Campo Moura ~o (UTFPR-CM), via s-Graduaça gica Federal do Parana Programa de Po ~o, PR, Brazil Rosalina Maria dos Santos, 1233, CEP 87301-899, Caixa Postal: 271, Campo Moura
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2016 Received in revised form 23 May 2016 Accepted 4 June 2016 Available online xxx
Use of silver nanoparticles as an antimicrobial compound directly on foodstuff has been attracted attention due to concerns on the total residual silver that remains on the food after preparation. In this work, silver nanoparticles (AgNPs) were obtained by a green route and applied as edible coating to chicken sausages. Antimicrobial activity of the AgNPs were able to inhibit lactic acid bacteria for 30 days, 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 in treated sausages than in control samples. Sausages were prepared simulating home cooking and the concentration of silver after each step was determined, showing that a simple washing and cooking procedure was able to remove most of the silver from sausages. Total silver concentration on the sausages after that was 5.3 ngAgNPs/gsausage. Also, no appreciable migration of silver nanoparticles from sausages surface to its interior was detected. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial activity Inductive Coupled Plasma with Mass Spectroscopy Lactic acid bacteria Chicken sausages Texture Profile Analysis
1. Introduction Application of edible coatings to meat, poultry, fish, fruits and vegetables has attracted increasing interest since an array of additives 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; ndez-Pan, Carrio n-Granda, & Mate , 2014). This is important in Ferna the case of meat sausages because microorganism action in anaerobic conditions lead to the formation of surface slime thus
* Corresponding author. E-mail address: [email protected] (F.V. Leimann).
decreasing sensory characteristics (De Palo, Maggiolino, 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 L1 (Leite, 2003; Levin et al., 2009). According to the study developed € ller, and Epple (2010), silver by Kittler, Greulich, Diendorf, Ko 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)
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Please cite this article in press as: Marchiore, N. G., et al., Migration evaluation of silver nanoparticles from antimicrobial edible coating to sausages, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.06.013
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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 Dglucose (Isofar, Brazil) and silver nitrate (Proquímios, Brazil) were used in the silver nanoparticles synthesis. Tryptic soy broth (Biomark, Brazil), Muller-Hinton broth (Biomark, Brazil), MRS agar (Biomak, Brazil) and saline solution were used in the antimicrobial analyses. Malonaldehyde bis (dimethyl acetal) (1,1,3,3tetramethoxypropane, 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.
(37.50 mg mL1) 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 treatments 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 treatment 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 sausages 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 sausages) 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 concentration (mg0Ag mL1). 2.7. Texture Profile Analysis
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). Morphological analysis of AgNPs was performed by Scanning Electron Microscopy (SEM, Shimadzu SSX 550 Superscan, Japan).
Texture Profile Analysis (TPA) was performed with a TA-XT Express 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 s1 was applied. The following TPA parameters were computed: hardness, adhesiveness, cohesiveness, springiness, gumminess and chewiness.
2.4. Sausages coating
2.8. Assessment of lipid oxidation by TBARS assay
AgNPs colloidal solution was diluted to 37.50 mg mL1 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
Thiobarbituric acid-reactive substances (TBARS) assay was carried 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 L1) in a test tube. Test tubes were incubated in a boiling water bath during 40 min then the absorbance was measured at 538 nm (UVeVis spectrophotometer, Ocean Optics, USB650UV, USA). TBA value was expressed as
2.3. AGNPs characterization
Please cite this article in press as: Marchiore, N. G., et al., Migration evaluation of silver nanoparticles from antimicrobial edible coating to sausages, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.06.013
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mgmalonaldehyde kg1 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 diameters 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 characteristic 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 sausages 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 bacterial growth. Only after 30 days they reached statistically the same LAB growth found for control samples at 15 days of storage, indicating 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).
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 deviation, treatments conducted in quadruplicate). Bars marked with different letters present 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 g1 respectively. Jiang et al. (2013) observed that the AgNPs treatment prevented mesophilic (4 log CFU g1), psychrophilic (3 log CFU g1), pseudomonad (6.5 log CFU g1), yeasts and molds (4.3 log CFU g1) 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 sausages preparation steps. Initially it was verified that the concentration of AgNPs on treatment solution applied to the sausages was equal to 23.47 ± 0.04 mgAgNPs mL1 and after treating all sausages the solution remained with 19.27 ± 0.12 mgAgNPs mL1. One may conclude that the amount of AgNPs retained at the sausages surface was equal to 5.3 ngAgNPs g1 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 nanoparticles due to the higher total surface area. Agþ ions release rate
Please cite this article in press as: Marchiore, N. G., et al., Migration evaluation of silver nanoparticles from antimicrobial edible coating to sausages, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.06.013
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probably could be higher at the present work if smaller AgNPs diameters were reached. When analyzing the AgNPs migration into the sausages, after 15 days (Fig. 3(b)) sausages retained 4.103 mg0Ag g1 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 L1 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 L1. 3.4. Texture Profile Analysis (TPA) TPA parameters obtained for control and AgNPs treated sausages 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). s, García, Zaritzky, and Califano (2006) evaluated the storage Andre stability of chicken sausages with low-fat content and found that adhesiveness and springiness remained statistically constant during 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 treatments. Morones et al. (2005) stated that AgNPs interact with proteins 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
Fig. 3. Metallic silver concentration determined at preparation steps collected samples: (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).
Table 2 shows lipid oxidation presented as TBARS results. It is possible to observe that the treatment with AgNPs presented significant 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 lez, Fern findings of Panea, Ripoll, Gonza 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 Control
Adhesiveness [N s] Springiness [] Chewiness [] Gumminess [] Cohesiveness [] Resilience [] Hardness [N]
aA
0.26 ± 0.24 0.92aA ± 0.03 1642.80aA ± 371.10 1793.58aA ± 404.51 0.75aA ± 0.03 0.43aA ± 0.03 23.54aA ± 6.48
15 days AgNPs aA
0.27 ± 0.19 0.92aA ± 0.02 1559.31aA ± 191.05 1740.27aA ± 201.38 0.75aA ± 0.01 0.43aA ± 0.01 22.81aA ± 2.95
Control aA
0.19 ± 0.16 0.92aA ± 0.02 1527.32aA ± 235.54 1667.26aA ± 257.00 0.75aA ± 0.02 0.42aA ± 0.02 21.72aA ± 3.59
30 days AgNPs aA
0.40 ± 0.29 0.923aA ± 0.12 1776.90aA ± 374.92 1910.92aA ± 252.71 0.75aA ± 0.01 0.75aA ± 0.01 25.27aA ± 3.72
Control aA
0.19 ± 0.16 0.92aA ± 0.02 1527.32aA ± 235.54 1667.26aA ± 257.00 0.75aA ± 0.02 0.42aA ± 0.02 21.72aA ± 3.59
AgNPs 0.40aA ± 0.29 0.923aA ± 0.12 1776.90aA ± 374.92 1910.92aA ± 252.71 0.75aA ± 0.01 0.75aA ± 0.01 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); 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. A,B
Please cite this article in press as: Marchiore, N. G., et al., Migration evaluation of silver nanoparticles from antimicrobial edible coating to sausages, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.06.013
N.G. Marchiore et al. / LWT - Food Science and Technology xxx (2016) 1e6 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 kg1)
AgNPs (mgMDA kg1)
0 15 30
0.19aA ± 0.06 0.43bA ± 0.02 0.65cB ± 0.06
0.20aA ± 0.13 0.73bA ± 0.15 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 thermodynamically 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). Evidence 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 oxidation in a similar mechanism to oxygen iron complexes. 4. Conclusions Silver nanoparticles (AgNPs) were successfully applied as antimicrobial 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 significantly 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 ^nio de Bolsas CP 11/2013 CAPES/ Authors thank CAPES (Conve ~o Arauca ria), CNPq (Bolsa de iniciaç~ Fundaça ao em desenvolvimento gico e inovaça ~o - PIBITI/CNPq 2014/2015) and Fundaça ~o tecnolo ria (Programa Universal/Pesquisa Ba sica e Aplicada 24/2012 Arauca ~o Arauca ria) for the (Protocolo 37334133700514041013) e Fundaça financial support. Also COMCAP Laboratory (Universidade Estadual dUEM) and LCP (Laborato rio de Controle de Processos, de Maringa Universidade Federal de Santa Catarina e UFSC) for SEM and DLS analyses, respectively. References Alvarez, M. V., Ponce, A. G., & Moreira, M. D. R. (2013). Antimicrobial efficiency of chitosan coating enriched with bioactive compounds to improve the safety of fresh cut broccoli. LWT e Food Science and Technology, 50(1), 78e87. s, S. C., García, M. E., Zaritzky, N. E., & Califano, A. N. (2006). Storage stability of Andre low-fat chicken sausages. Journal of Food Engineering, 72, 311e319. An, J., Zhang, M., Wang, S., & Tang, J. (2008). Physical, chemical and microbiological changes in stored green asparagus spears as affected by coating of silver nanoparticles-PVP. LWT e Food Science and Technology, 41, 1100e1107. Brasil, I. M., Gomes, C., Puerta-Gomez, A., Castell-Perez, M. E., & Moreira, R. G. (2012). Polysaccharide-based multilayered antimicrobial edible coating enhances quality of fresh-cut papaya. LWT e Food Science and Technology, 47(1),
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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 , Campus Campo Moura ~o (UTFPR-CM), via Rosalina Maria gica Federal do Parana Departamento Acad^ emico de Alimentos (DALIM), Universidade Tecnolo ~o, PR, Brazil dos Santos, 1233, CEP 87301-899, Caixa Postal: 271, Campo Moura b ~o em Tecnologia de Alimentos (PPGTA), Universidade Tecnolo , Campus Campo Moura ~o (UTFPR-CM), via s-Graduaça gica Federal do Parana Programa de Po ~o, PR, Brazil Rosalina Maria dos Santos, 1233, CEP 87301-899, Caixa Postal: 271, Campo Moura
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2016 Received in revised form 23 May 2016 Accepted 4 June 2016 Available online xxx
Use of silver nanoparticles as an antimicrobial compound directly on foodstuff has been attracted attention due to concerns on the total residual silver that remains on the food after preparation. In this work, silver nanoparticles (AgNPs) were obtained by a green route and applied as edible coating to chicken sausages. Antimicrobial activity of the AgNPs were able to inhibit lactic acid bacteria for 30 days, 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 in treated sausages than in control samples. Sausages were prepared simulating home cooking and the concentration of silver after each step was determined, showing that a simple washing and cooking procedure was able to remove most of the silver from sausages. Total silver concentration on the sausages after that was 5.3 ngAgNPs/gsausage. Also, no appreciable migration of silver nanoparticles from sausages surface to its interior was detected. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial activity Inductive Coupled Plasma with Mass Spectroscopy Lactic acid bacteria Chicken sausages Texture Profile Analysis
1. Introduction Application of edible coatings to meat, poultry, fish, fruits and vegetables has attracted increasing interest since an array of additives 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; ndez-Pan, Carrio n-Granda, & Mate , 2014). This is important in Ferna the case of meat sausages because microorganism action in anaerobic conditions lead to the formation of surface slime thus
* Corresponding author. E-mail address: [email protected] (F.V. Leimann).
decreasing sensory characteristics (De Palo, Maggiolino, 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 L1 (Leite, 2003; Levin et al., 2009). According to the study developed € ller, and Epple (2010), silver by Kittler, Greulich, Diendorf, Ko 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.
Please cite this article in press as: Marchiore, N. G., et al., Migration evaluation of silver nanoparticles from antimicrobial edible coating to sausages, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.06.013
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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 Dglucose (Isofar, Brazil) and silver nitrate (Proquímios, Brazil) were used in the silver nanoparticles synthesis. Tryptic soy broth (Biomark, Brazil), Muller-Hinton broth (Biomark, Brazil), MRS agar (Biomak, Brazil) and saline solution were used in the antimicrobial analyses. Malonaldehyde bis (dimethyl acetal) (1,1,3,3tetramethoxypropane, 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.
(37.50 mg mL1) 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 treatments 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 treatment 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 sausages 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 sausages) 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 concentration (mg0Ag mL1). 2.7. Texture Profile Analysis
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). Morphological analysis of AgNPs was performed by Scanning Electron Microscopy (SEM, Shimadzu SSX 550 Superscan, Japan).
Texture Profile Analysis (TPA) was performed with a TA-XT Express 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 s1 was applied. The following TPA parameters were computed: hardness, adhesiveness, cohesiveness, springiness, gumminess and chewiness.
2.4. Sausages coating
2.8. Assessment of lipid oxidation by TBARS assay
AgNPs colloidal solution was diluted to 37.50 mg mL1 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
Thiobarbituric acid-reactive substances (TBARS) assay was carried 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 L1) in a test tube. Test tubes were incubated in a boiling water bath during 40 min then the absorbance was measured at 538 nm (UVeVis spectrophotometer, Ocean Optics, USB650UV, USA). TBA value was expressed as
2.3. AGNPs characterization
Please cite this article in press as: Marchiore, N. G., et al., Migration evaluation of silver nanoparticles from antimicrobial edible coating to sausages, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.06.013
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mgmalonaldehyde kg1 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 diameters 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 characteristic 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 sausages 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 bacterial growth. Only after 30 days they reached statistically the same LAB growth found for control samples at 15 days of storage, indicating 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).
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 deviation, treatments conducted in quadruplicate). Bars marked with different letters present 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 g1 respectively. Jiang et al. (2013) observed that the AgNPs treatment prevented mesophilic (4 log CFU g1), psychrophilic (3 log CFU g1), pseudomonad (6.5 log CFU g1), yeasts and molds (4.3 log CFU g1) 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 sausages preparation steps. Initially it was verified that the concentration of AgNPs on treatment solution applied to the sausages was equal to 23.47 ± 0.04 mgAgNPs mL1 and after treating all sausages the solution remained with 19.27 ± 0.12 mgAgNPs mL1. One may conclude that the amount of AgNPs retained at the sausages surface was equal to 5.3 ngAgNPs g1 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 nanoparticles due to the higher total surface area. Agþ ions release rate
Please cite this article in press as: Marchiore, N. G., et al., Migration evaluation of silver nanoparticles from antimicrobial edible coating to sausages, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.06.013
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probably could be higher at the present work if smaller AgNPs diameters were reached. When analyzing the AgNPs migration into the sausages, after 15 days (Fig. 3(b)) sausages retained 4.103 mg0Ag g1 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 L1 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 L1. 3.4. Texture Profile Analysis (TPA) TPA parameters obtained for control and AgNPs treated sausages 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). s, García, Zaritzky, and Califano (2006) evaluated the storage Andre stability of chicken sausages with low-fat content and found that adhesiveness and springiness remained statistically constant during 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 treatments. Morones et al. (2005) stated that AgNPs interact with proteins 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
Fig. 3. Metallic silver concentration determined at preparation steps collected samples: (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).
Table 2 shows lipid oxidation presented as TBARS results. It is possible to observe that the treatment with AgNPs presented significant 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 lez, Fern findings of Panea, Ripoll, Gonza 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 Control
Adhesiveness [N s] Springiness [] Chewiness [] Gumminess [] Cohesiveness [] Resilience [] Hardness [N]
aA
0.26 ± 0.24 0.92aA ± 0.03 1642.80aA ± 371.10 1793.58aA ± 404.51 0.75aA ± 0.03 0.43aA ± 0.03 23.54aA ± 6.48
15 days AgNPs aA
0.27 ± 0.19 0.92aA ± 0.02 1559.31aA ± 191.05 1740.27aA ± 201.38 0.75aA ± 0.01 0.43aA ± 0.01 22.81aA ± 2.95
Control aA
0.19 ± 0.16 0.92aA ± 0.02 1527.32aA ± 235.54 1667.26aA ± 257.00 0.75aA ± 0.02 0.42aA ± 0.02 21.72aA ± 3.59
30 days AgNPs aA
0.40 ± 0.29 0.923aA ± 0.12 1776.90aA ± 374.92 1910.92aA ± 252.71 0.75aA ± 0.01 0.75aA ± 0.01 25.27aA ± 3.72
Control aA
0.19 ± 0.16 0.92aA ± 0.02 1527.32aA ± 235.54 1667.26aA ± 257.00 0.75aA ± 0.02 0.42aA ± 0.02 21.72aA ± 3.59
AgNPs 0.40aA ± 0.29 0.923aA ± 0.12 1776.90aA ± 374.92 1910.92aA ± 252.71 0.75aA ± 0.01 0.75aA ± 0.01 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); 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. A,B
Please cite this article in press as: Marchiore, N. G., et al., Migration evaluation of silver nanoparticles from antimicrobial edible coating to sausages, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.06.013
N.G. Marchiore et al. / LWT - Food Science and Technology xxx (2016) 1e6 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 kg1)
AgNPs (mgMDA kg1)
0 15 30
0.19aA ± 0.06 0.43bA ± 0.02 0.65cB ± 0.06
0.20aA ± 0.13 0.73bA ± 0.15 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 thermodynamically 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). Evidence 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 oxidation in a similar mechanism to oxygen iron complexes. 4. Conclusions Silver nanoparticles (AgNPs) were successfully applied as antimicrobial 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 significantly 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 ^nio de Bolsas CP 11/2013 CAPES/ Authors thank CAPES (Conve ~o Arauca ria), CNPq (Bolsa de iniciaç~ Fundaça ao em desenvolvimento gico e inovaça ~o - PIBITI/CNPq 2014/2015) and Fundaça ~o tecnolo ria (Programa Universal/Pesquisa Ba sica e Aplicada 24/2012 Arauca ~o Arauca ria) for the (Protocolo 37334133700514041013) e Fundaça financial support. Also COMCAP Laboratory (Universidade Estadual dUEM) and LCP (Laborato rio de Controle de Processos, de Maringa Universidade Federal de Santa Catarina e UFSC) for SEM and DLS analyses, respectively. References Alvarez, M. V., Ponce, A. G., & Moreira, M. D. R. (2013). Antimicrobial efficiency of chitosan coating enriched with bioactive compounds to improve the safety of fresh cut broccoli. LWT e Food Science and Technology, 50(1), 78e87. s, S. C., García, M. E., Zaritzky, N. E., & Califano, A. N. (2006). Storage stability of Andre low-fat chicken sausages. Journal of Food Engineering, 72, 311e319. An, J., Zhang, M., Wang, S., & Tang, J. (2008). Physical, chemical and microbiological changes in stored green asparagus spears as affected by coating of silver nanoparticles-PVP. LWT e Food Science and Technology, 41, 1100e1107. Brasil, I. M., Gomes, C., Puerta-Gomez, A., Castell-Perez, M. E., & Moreira, R. G. (2012). Polysaccharide-based multilayered antimicrobial edible coating enhances quality of fresh-cut papaya. LWT e Food Science and Technology, 47(1),
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Please cite this article in press as: Marchiore, N. G., et al., Migration evaluation of silver nanoparticles from antimicrobial edible coating to sausages, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.06.013