Survival and reduction in foodborne bacteria using methyl cellulose film doped with europium oxide nanoparticles

Abstract The study validated the efficacy of methyl cellulose films doped with different concentration of Eu2O3 nanoparticles to inactivate foodborne pathogens. Eu2O3 nanoparticles were added to the methyl cellulose solution with different weight percentages (0.0, 0.5, 0.75, 1.0, 1.25, and 1.5 wt%). X‐ray diffraction patterns for the prepared films were studied. A significant lower count of E. coli, S. typhimurium, and S. aureus (p ≤ .05) inoculated in MC films doped with Eu2O3 nanoparticles compared with pure MC film could be achieved. The findings acquired verify the impact of prepared MC films doped with Eu2O3 nanoparticles on the test strains.

with other decontamination strategies to achieve satisfactory safety rates.
Developing of naturally occurring polymer with the film formation capacity and antimicrobial properties to improve health, safety, shelf life, and biomedical application, gains a considerable regard nowadays (Fernandez-Saiz, Lagaron, Hernandez-Muñoz, & Ocio, 2008;Irkin & Esmer, 2015;Malhotra, Keshwani, & Kharkwal, 2015). Despite the common use of cellulose and its derivatives in food packaging, researchers look to improve its antimicrobial properties in the future. Cellulose is one in all the foremost various and biodegradable compound that insoluble in water and most organic solvent (Coffey, Bell, & Henderson, 1995;El-Kader & Ragab, 2013).
Substitution of hydroxyl groups within the backbone of cellulose by some functional groups makes it water soluble such as methyl groups in methyl cellulose (MC). Methyl cellulose has an excellent film formation capacity, water solubility, and efficient oxygen and lipid permeability (Chevillard & Axelos, 1997;Nasatto et al., 2015). In literature, there are various studies about preparation of functional methyl cellulose nanocomposite films and their physicochemical (Tunç & Duman, 2010), antibacterial (Tunç & Duman, 2011), mechanical, and gas barrier (Tunc, Duman, & Polat, 2016) properties.
A great interest for biomedical applications has been emphasized to develop biopolymers by adding nanoparticles to its matrix. Doping biopolymers with nanoparticles is the concern of the research and industrial world, as they exhibit physical, chemical and antimicrobial enhancement (Carbone, Donia, Sabbatella, & Antiochia, 2016;Li, He, Li, & Zhang, 2015;Muthulakshmi, Rajini, Rajalu, Siengchin, & Kathiresan, 2017). The use of rare earth elements nanoparticles as a dopant for different biopolymers can be considered as a way to develop bilateral.
In our study, we will use Eu 2 O 3 nanoparticles as a dopant in the MC matrix with different concentrations to form a thin films, which were then defined by x-ray diffraction process. The prepared MC films were examined to evaluate their consequences on the used foodborne test strains.

| Preparation of MC films doped with Eu 2 O 3 nanoparticles
MC of 2% aqueous solution at 20°C was provided by LOBA Chemie, India, with viscosity of 350-550 cP and pH values of 5.5-8.0. Eu 2 O 3 nanoparticles was supplied by Sigma-Aldrich, its density was 7.42 g/ ml at 20°C, and the particle size was less than 150 nm. All glasswares were thoroughly cleaned in aqua region and rinsed copiously with double distilled water.
To prepare thin film of MC, incorporated with Eu 2 O 3 nanoparticles, two grams of MC were dissolved in 100 ml double distilled water at 50°C using a magnetic stirrer overnight. Eu 2 O 3 nanoparticles were added to the MC solution with different weight percentages (0.00, 0.50, 0.75, 1.00, 1.25, and 1.50 wt%) and were stirred for 12 hr at 50°C. The solution was cast in stainless-steel plates with diameter 12 cm and then dried in open air at room temperature for 3 days until solvent was nearly evaporated. The obtained films were of suitable thickness ≈ 100μm.

| X-ray diffraction
The amorphous/crystalline nature of methyl cellulose/Eu 2 O 3 nanocomposite films was checked by using DIANO X-ray defractometer equipped with Cu-K α radiation (λ = 1.54056 A o , operation voltage = 30 kV).

| Preparation of test strains
E. coli, S. typhimurium, and S. aureus were obtained from the Microbiology laboratory of Molecular Diagnostic and Personalised Therapeutics unit (MDXPTU), Hail University. Strains were originally isolated from chicken meat samples, then were identified biochemically, serologically, phenotypically, and genotypically. Strains were saved in the MDXPTU Biobank at −80°C. Each strain was cultivated separately in Tryptic soy broth (Difco) at 37°C for 24 hr. The cells were harvested by centrifugation 5,000 g/10 min and were washed twice then were resuspended to a final cell density of 7 log cfu/ml (OD 600 0.2) using sterile saline (0.85% NaCl).

| Bacterial inoculation
MC films doped with different concentration of Eu 2 O 3 nanoparticles were tested antimicrobially against MC control film. All the tested MC films were cut aseptically to form an area of 1 cm 2 , each area was inoculated with 10 µl of test strains (8 log cfu/ml concentrations) (E. coli, S. typhimurium and S. aureus). The inoculated MC films were kept in bio-safety cabinet to dry for 2 hr.

| Survival and reduction in test strains
The survival and reduction in the different test strains inoculated on the MC films doped with different concentration of Eu 2 O 3 nanoparticles were determined against the pure MC film. About 10 ml of sterile phosphate-buffered saline was added to the Inoculated MC films (area of 1 cm 2 ) in sterile tubes. Vigorous shaking was carried out to the tubes using vortex for 3 min. A 10-fold serial dilution was prepared. Dilutions were plated in duplicate onto Tryptic Soya agar and Mueller-Hinton agar (Difco) that was subsequently incubated at 37°C for 24 hr.

| Pulse field gel electrophoresis (PFGE)
PFGE was conducted to identify the clonal relatedness of the test strains after inoculation on MC films. Already optimized protocol following Standard Operating Procedure (SOP) for PulseNet PFGE using a CHEF-Mapper (Bio-Rad Laboratories) was used.

| Statistical analysis
The mean values with standard error of the means (SEM) were calculated. One way analysis of variance (ANOVA) at 95% level of confidence and least Significant difference (LSD) post hoc were done to determine significant differences (p < .05 was considered as significant). The XRD pattern of the MC homopolymer shows a semicrystalline structure revels three peaks (Figure 1b), a sharp one at 2ϴ = 8° corresponds to the trimethylglucose-type crystalline order (Kato, Yokoyama, & Takahashi, 1978). A broad peak with maximum at 2ϴ = 21° indicates the intermolecular structure of MC (Rangelova et al., 2011), and a weak peak appeared at 2ϴ = 13.3° which indicates a more hydrated structure (Liebeck, Hidalgo, Roth, Popescu, & Böker, 2017).
The decrease in the degree of crystallinity of MC films doped with 1.25, and 1.50 wt% Eu 2 O 3 NPs was due to the saturation effect.
From the data on the degree of crystallinity, it was noticed that the values of the degree of crystallinity for the composite samples are higher than that of the MC homopolymer.

| Antimicrobial activity
The obtained results in this study revealed that MC films doped with different concentration of Eu 2 O 3 nanoparticles have been shown to possess potential antibacterial activity against the used foodborne test strains that previously isolated from chicken meat products.
A significant lower count of E. coli, S. typhimurium, and S. aureus The obtained reductions were ranged from 3.3 to 4.54 log cfu/ cm 2 for E. coli, 3.42 to 4.42 log cfu/cm 2 for S. typhimurium and 3.52 to 4.19 log cfu/cm 2 for S. aureus (Figure 3). These results were statistically significant (p < .05). Moreover, E. coli test strains had the highest log reduction compared with the other test strains (Figure 4). (Tunç & Duman, 2011)  It was assumed that gram-negative bacteria are more reactive to environmental modifications than gram-positive cells (Shigehisa, Ohmori, Saito, Taji, & Hayashi, 1991).
The initial adherence of the test strains to MC films was studied.
The adherence ability of the test strains to MC films was reduced significantly (p < .05) in MC films doped with different concentration of Eu 2 O 3 nanoparticles compared with pure MC film. Moreover, MC film doped with 1.5 wt% Eu 2 O 3 nanoparticles was the most effective concentration that could reduce the adherence of test strains and these findings confirmed what has been achieved in this study ( Figure 5). Another confirmatory method to emphasize our obtained data in this study was done through testing the week attachment of the test strains against the prepared MC films. The number of recovered bacterial cells after 30 min of attachment to MC films followed by three times rinsing in phosphate-buffered saline were recorded ( Figure 6). S. aureus strains exhibited weak attachment to MC films F I G U R E 1 X-ray diffraction patterns of (a) pure Eu 2 O 3 nanoparticles, (b) MC film doped with different concentration of Eu 2 O 3 nanoparticles compared with other test strains and this was confirmed by the large number of S. aureus strains compared to other strains recovered from the homogenate (Figure 6).
The obtained results showed that S. aureus strains were less adhere to MC films surface compared with other test strains and these findings could be attributed to the nonmotile feature of S. aureus (nonflagellate cocci) compare with the other flagellate test strains. Flagella are clearly implicated in the attachment of bacteria (Notermans & Kampelmacher, 1974). Bacterial flagella endow the organism with motility and the ability to respond to a chemotactic stimulus (Lillard, 1985). S. aureus cells showed more hydrophobic features compared with E. coli that were found to be moderately hydrophilic (Burks et al., 2003;Mitik-Dineva et al., 2009). S. aureus cells have a hydrophobic nature which due to the extreme negative charge and the existence of hydrophobic teichoic and lipoteichoic acid in their cell wall (Canepari, Boaretti, Lleo, & Satta, 1990;Gross, Cramton, Götz, & Peschel, 2001).
Our findings reported the potential antimicrobial activity of MC films doped with Eu 2 O 3 nanoparticles. The concentration of the Eu 2 O 3 nanoparticles required to perform activity against foodborne microorganisms is important to use it effectively in food package. A reduction with 5 log in viability is important to attain for better foodborne pathogen reduction (Food & Administration, 2004;Olaimat & Holley, 2012). However, MC films doped with 1.5 wt% Eu 2 O 3 nanoparticles showed a greater effect to reduce foodborne pathogen from food product's surface.
The clonal relatedness of the test strains was studied after inoculation on MC/Eu 2 O 3 nanocomposite films using PFGE. The achieved findings showed that the clonal relatedness of test strains have not been influenced after inoculation on MC/Eu 2 O 3 nanocomposite films (Figures 7 and 8).

CO N FLI C T O F I NTE R E S T
All authors declare that there is no conflict of interest.