Preparation of silver nanoparticles/polymethylmethacrylate/cellulose acetate film and its inhibitory effect on Cronobacter sakazakii in infant formula milk

Cronobacter sakazakii is a harmful foodborne patho-gen, and its contaminated food will pose a huge threat to human health. Prevention of C. sakazakii contamination of food is valuable for food safety as well as for human health. In this study, silver nanoparticles (AgNP) were successfully immobilized on the surface of cellulose acetate (CA) and polymethylmethacrylate (PMMA) composite to obtain AgNP/PMMA/CA film. Through the inhibition zone and growth curve experiments, we found that AgNP/PMMA/CA films has excellent antibacterial activity on C. sakazakii . The AgNP/PMMA/CA film can prolong the lag phase of the growth curve of C. sakazakii from 2 to 8 h. The antibacterial films were found to reduce the survival of C. sakazakii in Luria-Bertani and infant formula by combining it with a mild heat treatment (45°C, 50°C, and 55°C). The AgNP/PMMA/CA film combined with 55°C water bath can completely inactivate C. sakazakii in infant formula within 120 min. Finally, the potential mechanism by which AgNP/PMMA/CA films reduce the heat tolerance of C. sakazakii was investigated by quantitative real-time PCR. The results showed that AgNP/PMMA/CA films could reduce the expression of environmental tolerance-related genes in C. sakazakii . The current research shows that AgNP/PMMA/CA film has strong antibacterial activity, and the antibacterial film combined with mild heat treatment can accelerate the inactivation of C. sakazakii and effectively reduce the harm of foodborne pathogens. The AgNP/ PMMA/CA film can be used as a potential packaging material or antibacterial surface coating.


INTRODUCTION
Cronobacter spp., formerly known as Enterobacter sakazakii, is a gram-negative bacillus that parasitizes in the intestinal tract of humans and animals, and is also an important foodborne pathogen (Nazarowec-White and Farber, 1997).Cronobacter sakazakii can cause necrotizing enterocolitis, sepsis, and meningitis in infants with a fatality rate more than 40%, and a small number of survivors would develop very serious neurological complications (Lampel and Chen, 2009).Infants are at risk of contracting C. sakazakii through consumption of infant formula milk powder (IFM) or other utensils contaminated with C. sakazakii (Hunter et al., 2008).Some strains of C. sakazakii present in IFM are biofilm-forming, pathogenic, and are highly resistant to environmental stresses such as high osmotic pressure, low pH, high temperature, oxidation, and desiccation, making it difficult to inactivate C. sakazakii in contaminated food (Ling et al., 2020).Therefore, it is particularly important to effectively prevent and control pathogenic bacteria from the source of pollution.
Nano-inorganic antimicrobial agents are a new type of antimicrobial agents that are more broad-spectrum, durable, and safe than organic antimicrobial agents.Among inorganic antibacterial agents, silver nanoparticles (AgNP) have been widely used due to their low toxicity, high antibacterial activity, and broad antibacterial spectrum (Rai et al., 2009).The researchers also combined AgNP with other polymer materials to form a silver-loaded antibacterial composite material, which can reduce the dissolution of AgNP in the aqueous solution and improve the antibacterial properties of the composite material (Sadeghnejad et al., 2014;Fernando et al., 2018).Jiang et al. (2020) prepared an AgNPloaded nanocellulose fiber and chitosan composite film, and the obtained nanocellulose fiber/AgNP/chitosan film showed an excellent antibacterial activity against Preparation of silver nanoparticles/polymethylmethacrylate/cellulose acetate film and its inhibitory effect on Cronobacter sakazakii in infant formula milk Escherichia coli and Staphylococcus aureus.Wang et al. (2020) combined AgNP with bacterial nanocellulose by reduction and UV irradiation-assisted methods to prepare an environmentally friendly antibacterial membrane, which was effective against E. coli.The film material is also biodegradable, which will reduce the environmental pollution caused by nonbiodegradable synthetic packaging materials.
Bio-based nanocomposites are considered as ideal materials for the preparation of antibacterial food packaging.Cellulose acetate (CA) is an environmentally friendly polymer extracted from cellulose, which can form a film at room temperature, is odorless, nontoxic, biodegradable, insoluble in water, has the small affinity for water vapor, and it is considered to be a promising food packaging material (Gemili et al., 2009;Puls et al., 2011;Gouvêa et al., 2015).Marrez et al. (2019) prepared an AgNP-loaded CA film with strong antibacterial activity against a variety of foodborne pathogens.Beisl et al. (2019) prepared a CA/AgNP filter membrane for water filtration treatment, which reduced the number of E. coli by more than 99.99%.
In addition, polymethylmethacrylate (PMMA) is a common polymer material used to prepare composite antibacterial materials with AgNP, and has been widely used in medical and other antibacterial fields because of its biocompatibility (Alt et al., 2004).Borse et al. (2016) prepared an AgNP-PMMA nanocomposite that showed good bactericidal properties against E. coli and S. aureus.Petrochenko et al. (2017) prepared a PMMA/ Ag composite film, which not only has a good antibacterial effect on E. coli, but also has good biocompatibility.These results indicate that the AgNP-loaded composite films have good antibacterial activity against foodborne pathogens.However, the preparation of antibacterial films using PMMA combined with CA is rarely studied.Hydrogen bonds can form between PMMA and CA, which contribute to specific interactions that promote the miscibility and mechanical properties of the mixture (Bhat and Kumar, 2006).Additionally, the blend of PMMA and CA has good biodegradability, so their use will not cause any environmental pollution (Haider et al., 2019).Therefore, the use of PMMA combined with CA to prepare antibacterial films has a good application prospect.
The objective of this study was to prepare an antibacterial film loaded with AgNP.The physicochemical properties, morphology, and antibacterial properties of the antibacterial films were then evaluated.Finally, the inactivation effect of AgNP/PMMA/CA film combined with mild heat treatment on C. sakazakii in Luria-Bertani (LB) and IFM medium and its potential antibacterial mechanism were explored.

Materials and Reagents
The silver nitrate (AgNO 3 ) and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich Chemical Co.Cellulose acetate was purchased from Sangon Biotech Co. Ltd.Polymethylmethacrylate was purchased from Shanghai Aladdin Biochemical Technology Co. Ltd.Tetrahydrofuran was purchased from Xilong Science Co. Ltd.The infant formula milk powder was purchased from Jiangxi Meilu, Dairy Co. Ltd.The LB medium and agar powder were purchased from Becton Dickinson.The primers were synthesized by TSINGKE Biological Technology.
No animals were used in this study, and ethical approval for the use of animals was thus deemed unnecessary.
Bacterial Strains and Culture Conditions.Cronobacter sakazakii ATCC 29544 was purchased from American Type Culture Collection The strain was stored at −40°C in LB containing 25% (vol/vol) glycerol.The strain was used in the experiment after streaking twice on fresh LB agar containing 1.5% (wt/ vol) agar.Single colonies were selected for activation and subculture for 2 times to allow the bacteria to have sufficient viability before being used in subsequent experiments.
Synthesis of AgNP/PMMA/CA Films.The preparation process is shown in Figure 1, 1 g of PMMA and 2 g of CA were added into 50 mL of tetrahydrofuran, respectively.After dissolution, PMMA solution and CA solution were obtained.Then, 6 mL of mixed solutions of PMMA solution and CA solution with different proportions (5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5) were poured onto a polytetrafluoroethylene plate, and the polytetrafluoroethylene plate was placed in a fume hood to volatilize tetrahydrofuran to obtain the PMMA/CA film.Next, the obtained PMMA/CA film was washed 3 times with deionized water and airdried, and then the film was immersed in 0.01 mol/L NaOH solution at 60°C for 2 h.Subsequently, the film was washed with deionized water for 3 times.The dried film was immersed in a 2 mg/mL of AgNO 3 solution and was water bathed at 60°C for 3 h to obtain the AgNP/PMMA/CA film.Finally, the AgNP/PMMA/ CA films were washed 3 times with deionized water to remove free silver ions.
Characterization of AgNP/PMMA/CA Films.The optical properties of the films were detected by UV-visible absorption (varioskan lux, Thermo Fisher Scientific) to determine whether AgNP were loaded on the films.The surface morphology of the films was observed using a scanning electron microscope (Jasam-6701f, Jeol.Ltd.).The elements in the films were analyzed using scanning electron microscope-mapping.The transmittance of the film at 700 to 3,250 cm −1 was measured by Fourier transform infrared (FT-IR) spectrometer (Nicolet iS50, Madison Thermo Fisher) to determine the interaction between the film components.

Determination of Antibacterial Effect of AgNP/PMMA/CA Films
Determination of the Inhibition Zone.To evaluate the antibacterial effect of AgNP/PMMA/CA films, the films were cut into circular slices with a diameter of 11 mm for the inhibition zone experiment.Briefly, 200 μL of 10 6 cfu/mL of bacterial solution was spread on LB agar plate, and then the AgNP/PMMA/CA films presterilized by UV were placed on LB agar plate coated with the bacterial solution.The control group was tested using PMMA/CA films.Then, the LB agar plate was placed at 37°C for 24 h, and the size of the inhibition zone was measured with a ruler.
Determination of Growth Curve.Evaluation of the inhibitory effect of antibacterial films on the growth and reproduction of C. sakazakii by using 1 cm 2 AgNP/ PMMA/CA films cocultured with bacteria.Briefly, 50 μL of 10 7 cfu/mL bacterial solution (exponential phase) was inoculated into 5 mL of LB or IFM (m/v = 15%) medium.Then, the pre-UV sterilized film was inoculated into 5 mL of LB broth or rehydrated IFM (wt/ vol = 15%), and the test tube was cultured at 37°C, 180 rpm for 30 h.The number of bacteria was counted by plate counting method, and the growth curve was drawn using software according to the counting results.
Effects of AgNP/PMMA/CA Film Combined With Thermal Treatment on C. sakazakii.Due to its high heat resistance, C. sakazakii may be present in the production and processing of milk powder, which may eventually contaminate the product and cause harm to the human health.Therefore, the AgNP/ PMMA/CA film combined with different temperatures (45°C, 50°C, and 55°C) was used to treat the bacterial solution to evaluate the effect of AgNP/PMMA/CA film on the heat resistance of C. sakazakii.Briefly, 50 μL of 10 8 cfu/mL bacterial solution (exponential phase) was inoculated into 5 mL of LB or IFM.Then, 1 cm 2 of AgNP/PMMA/CA film or PMMA/CA film was added to the test tube containing the bacterial solution, and these mixtures were subjected to water bath treatment at different temperatures.Immediately after the water bath, we put the test tube containing the sample solution into the ice water prepared in advance to cool the sample solution.The number of bacteria was then counted using the plate count method.
RNA Isolation and Quantitative Real-Time PCR Assay.Twelve genes (Table 1) related to the environmental stresses tolerance of C. sakazakii were used in quantitative real-time PCR (RT-qPCR) experiments.The bacterial solution cultured overnight was diluted to obtain the initial concentration of C. sakazakii, about 1.0 × 10 9 cfu/mL in 5 mL of LB medium, and the 1 cm 2 of AgNP/PMMA/CA film or PMMA/CA film was added and cultured at 37°C, 180 rpm for 2 h.According to the manufacturer's instructions of AxyPrep Multisource Total RNA Miniprep Kit (Axygen Scientific), the total RNA was extract from the bacteria.Next, total mRNA was reversed transcription to cDNA using Takara PrimeScript RT reagent kit (Takara, Biotech Co. Ltd.).The RT-qPCR reactions were performed using the AriaMx real-time PCR system (Agilent Technologies Inc.), following 10 μL of system containing 5 μL of SYBR Premix ExTaq, 3.2 μL of sterile water, 1 μL of cDNA, and 0.8 μL (10 mM) of forward and reverse primer mixture.The RT-qPCR conditions included pre-degeneration of cDNA at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s and annealing at 56°C for 1 min.Sterile water instead of cDNA template was used as negative control.Three repeated experiments were performed on all samples.
Determination of Silver Migration.Silver migration in AgNP/PMMA/CA films was determined by inductively coupled plasma MS, with some modifications according to the method of Cheng et al. (2021).Briefly, 100 mg of AgNP/PMMA/CA film was immersed in 50 mL of deionized water.On alternate days, for 7 consecutive days, the 100-μL samples were extracted.After that, an equal amount of the water was refilled.Of this sample, 50 μL was added to 300 μL of concentrated nitric acid and 100 μL of perchloric acid, and digested in a water bath at 95°C for 2 h.Calibration curve was drawn for a concentration range of 2.0, 4.0, 6.0, 8.0, and 10.0 μg/kg.

Statistical Analysis
The experiments were independently conducted 3 times.The mean ± standard deviation was calculated from the data obtained from the 3 independent experiments.One-way or 2-way ANOVA were used by SPSS v22.0 (SPSS Inc.) for comparing the results between the different groups.The 2 −ΔΔCt method was used to analyze the RT-qPCR results.Results with P < 0.05 were considered statistically significant.The graph data were processed by GraphPad Prism 8.0.2 software.

UV-Visible Absorption Spectrum of Films
The UV-visible absorption spectrum was used to verify whether AgNP were stable in the film.The results showed that PMMA/CA film was almost no characteristic absorption peak at a 410-nm wavelength, but the AgNP/PMMA/CA film showed it (Figure 2A), which was corresponding to the absorption peak of AgNP (Rolim et al., 2019).The results indicated that the AgNP were stable in the film.

Fourier Transform Infrared Spectrum
Chemical structure and interactions between Ag and CA, and PMMA matrix were evaluated by the FT-IR presented in Figure 2B.Typically, the peak at 1,700 to 1,750 cm −1 was assigned to C=O vibrations from CA and PMMA (Namouchi et al., 2009;Khan et al., 2015).The peak at 2,800 to 3,000 cm −1 and 1,000 to 1,250 cm −1 , respectively, corresponds to C-H and C-O stretching vibration (Khan et al., 2015).However, the normalization of all spectra to the bands located at the 2,800 to 3,000 cm −1 , and obvious intensities decreasing of the peaks at 1,742, 1,380, 1,230, and 1,030 cm −1 were observed by the FT-IR spectra of the AgNP/PMMA/ CA films, which might be attributed to the interactions of the AgNP with the CA matrix (Kendouli et al., 2014).Moreover, the shifts on C=O ester carbonyl group, C-O, and C-H of AgNP/PMMA/CA nanocomposite films were shown, indicating that the AgNP modify the PMMA/CA films structure (Kadhim et al., 2016).The absence of a new peak may be due to the very low content of Ag that was below the detection limit of FT-IR instruments.

Surface Morphology of Films
The scanning electron microscope results are shown in Figures 3A-3C.The surface of PMMA/CA films was found to have a loose porous structure, which is similar to the previous reports (Kamal et al., 2017).We found a lot of AgNP on the surface of AgNP/PMMA/ CA films, and the AgNP particles are chiefly denser in the pores of the film.The loose porous structure on the surface of PMMA/CA films is favorable for the attachment of AgNP.Additionally, the average par-ticle size of AgNP is 61.70 ± 33.7 nm.The scanning electron microscope-mapping results (Figures 3D-3G) show that several elements such as carbon, oxygen, and silver are uniformly distributed on the surface of the film.The mass fractions of carbon, oxygen, and silver, shown in the upper right corner of Figure 3G, are 68.48,30.77, and 0.79%, respectively.The atomic number percentages are 74.70,25.21, and 0.10%, respectively.The above results show that we have successfully reduced AgNP on the surface of PMMA/CA films in situ and prepared AgNP/PMMA/CA films, and the AgNP are uniformly distributed on the surface of the films.

Antibacterial Effect of AgNP/PMMA/CA Films
The antibacterial effect of AgNP/PMMA/CA films against C. sakazakii was evaluated by measuring the inhibition zone size and growth kinetic curve.Figures 4A and 4B were the inhibition zone effects of AgNP/ PMMA/CA films and PMMA/CA films on C. sakazakii, respectively, in which CA: PMMA (vol/vol) = 5:1.The results showed that AgNP/PMMA/CA films had an obvious antibacterial effect on C. sakazakii, whereas PMMA/CA films had no antibacterial effect on C. sakazakii.It showed that PMMA and CA in the film have no antibacterial effect, and the substances that play an antibacterial effect are AgNP.The good antibacterial properties of AgNP/PMMA/CA films are similar to the results in other studies, Son et al. (2006) prepared a AgNP/CA nanofiber by electrospinning technology, which had powerful activity against S. aureus, E. coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae.Beisl et al. (2019) reported the bactericidal application of AgNP/CA film in water treatment and found that the membrane could kill 99.95% of E.  2016) synthesized an Ag-PMMA nanocomposite, which showed good antibacterial performance against gram-negative E. coli, P. aeruginosa, and gram-positive S. aureus, and had a good application prospect in the field of water treatment.
Supplemental Figure S1 (https: / / data .mendeley.com/datasets/ 94b2m8jpjy/ 1; Li, 2022) and Figure 4C showed the results of the inhibition zone of AgNP/ PMMA/CA films with different CA: PMMA ratios.The results in Figure 4C show that when CA: PMMA (vol/ vol) = 5:1, the antibacterial effect of AgNP/PMMA/ CA films is the best.Therefore, CA: PMMA (vol/vol) = 5:1 was chosen to prepare AgNP/PMMA/CA films for subsequent determination of growth kinetics curves.The growth kinetic curves of C. sakazakii under different treatment conditions are shown in Figure 4D.Silver nanoparticles/PMMA/CA films have a significant inhibitory effect on the growth of C. sakazakii in both LB and IFM medium.The AgNP/PMMA/CA film can prolong the lag phase of the growth curve of C. sakazakii from 2 to 8 h.However, PMMA/CA films had no inhibitory effect on the growth of C. sakazakii, which was consistent with the results of the inhibition zone.However, the growth of C. sakazakii in IFM medium is better than that in LB medium.We speculated that the nutrients in IFM are richer than LB, and beneficial for the growth of C. sakazakii, so C. sakazakii can adapt to the stress of AgNP/PMMA/CA films faster.Additionally, some studies have pointed out that IFM contains lactose, protein, and lipids, which help C. sakazakii survive longer under environmental stress (Lin and Beuchat, 2007;Dancer et al., 2009).

Effects of AgNP/PMMA/CA Film Combined with Mild Heat Treatment on C. sakazakii
The survival of C. sakazakii at different temperatures was evaluated as shown in Figure 5 and Table 2.At 45°C, the number of C. sakazakii in the control group and PMMA/CA film treatment group gradually increased, whereas AgNP/PMMA/CA film could significantly inhibit the growth of C. sakazakii in LB and IFM medium (Figures 5A and 5D).At 50°C, the growth of C. sakazakii in the control group and PMMA/CA film treatment group was inhibited, and the number of bacteria tended to be stable, whereas AgNP/PMMA/ CA film could gradually reduce the survival rate of C. sakazakii in LB medium.In contrast, AgNP/PMMA/ CA film did not reduce the number of C. sakazakii in IFM (Figures 5B and 5E), which indicated that C. sakazakii had higher survival ability in IFM.At 55°C, AgNP/PMMA/CA film can inactivate C. sakazakii in a shorter time than the control group and PMMA/ CA film treatment group.In LB and IFM medium, AgNP/PMMA/CA film could inactivate C. sakazakii within 30 min and 120 min, respectively (Figures 5C  and 5F).Consistent with previous results, the survival ability of C. sakazakii in IFM was higher than that in LB.We speculate that the richer nutrient content of IFM can improve the viability of C. sakazakii.This is consistent with the results of the growth curve in Figure 4D, C. sakazakii grows better in IFM medium.Many studies have shown that the composition of the medium affects bacterial growth and that IFM contains lactose, proteins, and lipids that protect bacterial cells from being lysed during drying and heating (Lin and Beuchat, 2007;Dancer et al., 2009).The above results show that AgNP/PMMA/CA film combined with thermal treatment can reduce the survival rate of C. sakazakii, and the survival ability of C. sakazakii in IFM is higher than that in LB.Therefore, in the preparation of IFM, the antibacterial film combined with mild heat treatment can accelerate the inactivation of C. sakazakii and effectively reduce the harm of foodborne pathogens.

Effects of AgNP/PMMA/CA Film on the Expression of Environmental Tolerance-Related Genes in C. sakazakii
Cronobacter spp.contaminates some foods, such as milk powder, due to its high tolerance to environmental stress.Therefore, the reducing environmental stress tolerance of Cronobacter can reduce the threat of foodborne pathogens to human health (Begley and Hill, 2015).Herein, we investigated the effect of AgNP/ PMMA/CA film on the expression of some environmental tolerance-related genes of C. sakazakii, in which the gene groEs and the gene mfla-1165 are closely related to the thermotolerance of C. sakazakii, and the gene groEs can encode a chaperone protein which can facilitate the refolding of misfolded or partially folded proteins (Gottesman et al., 1997).In some reports, the gene groEs can encode a heat shock protein, which can improve the heat tolerance of bacteria (Dong et al., 2018).The gene mfla-1165 is a biomarker related to the heat resistance of C. sakazakii, which is closely related to the heat resistance of C. sakazakii (Riedel and Lehner, 2007).The results of RT-qPCR are shown in Figure 6A and 6B, and the expression of both groES and mfla-1165 was significantly downregulated by AgNP/PMMA/CA film.The genes phoP/phoQ, fur and grxB were confirmed to be related to acid resistance of Enterobacter (Bearson et al., 1997;Ling et al., 2018), and RT-qPCR results (Figure 6C-6J) showed that AgNP/PMMA/CA film could significantly reduce the expression of these genes.Furthermore, Hfq, DnaK, DnaJ, and OmpA have been shown to be involved in the survival of C. sakazakii under environmental stress (Jameelah et al., 2018).According to the RT-qPCR results (Figure 6G-6J), AgNP/PMMA/CA film could significantly reduce the expression of these genes.The sigma subunit of RNA polymerase (RpoS) is a major regulator of C. sakazakii in response to general stress and can regulate metabolism to protect C. sakazakii survival under harsh conditions (Alvarez-Ordóñez et al., 2012).The rpoS gene regulates responses to various environmental stresses, including starvation, hyperosmolarity, high temperature, and low temperature (Hengge-Aronis, 1996).According to the above results, we speculate that AgNP/PMMA/CA film may reduce the survival rate of C. sakazakii in environments such as heat stress by affecting the production, metabolism, and gene expression of C. sakazakii.

Amount of Silver Transferred from AgNP/PMMA/CA Film into the Aqueous Solution
According to Supplemental Figure S2 (https: / / data .mendeley.com/datasets/ 94b2m8jpjy/ 1; Li, 2022), the amount of silver migration from the film into water increased with the passage of time, but the growth rate gradually slowed down, and the migration of silver  within 7 d was lower than the maximum migration of nonapproved substances through functional barriers; as stipulated by the European Commission, the limit is 10 μg/kg (Gallocchio et al., 2016).Therefore, the AgNP/PMMA/CA film is not only used for antibacterial surface coating, but also has application prospects in food packaging and other fields.However, there are still some controversies about the application of AgNP in the food field; because silver is a heavy metal, its accumulation in the human body may cause harm to human health (Tripathi and Goshisht, 2022).In this study, although AgNP were loaded onto the PMMA/ CA film surface and only a small amount of silver migrated into the food matrix, the migration of silver into the food was still a potential hazard, and how to reduce the migration of silver (or prevent migration of silver) is one of the directions for future research.

CONCLUSIONS
We successfully prepared an AgNP-loaded polymer material film (AgNP/PMMA/CA film), and evaluated the inhibitory effect of the film on C. sakazakii.The results showed that AgNP/PMMA/CA film had a good inhibitory effect on C. sakazakii in both LB medium and IFM medium.AgNP/PMMA/CA film combined with heat treatment at 55°C could inactivate C. sakazakii in LB and IFM within 30 and 120 min, respectively.The results showed that the viability of C. sakazakii in IFM was higher than that in LB medium.Further studies showed that AgNP/PMMA/CA film could reduce the thermotolerance of C. sakazakii under heat stress by inhibiting the expression of environmental tolerancerelated genes.Therefore, in the preparation of IFM, the antibacterial film combined with mild heat treatment can accelerate the inactivation of C. sakazakii and effectively reduce the harm of foodborne pathogens.Therefore, we believe that AgNP/PMMA/CA film has broad application prospects in food packaging material or antibacterial surface coating, and AgNP/PMMA/ CA film can reduce the number of bacteria and inhibit their growth.
Figure 3. (A, C) The surface morphology of the AgNP/PMMA/CA film was observed by a scanning electron microscope, and the upper right corner of panel C is the particle size distribution of AgNP.(A) Scale bar = 3 μm; (C) scale bar = 1 μm; (B) the surface morphology of the PMMA/CA film, scale bar = 3 μm; (D-G) scanning electron microscope-mapping analysis of carbon, oxygen, and silver elements on the surface of films, scale bar = 800 nm.AgNP = silver nanoparticles; PMMA = polymethylmethacrylate; CA = cellulose acetate.

Figure 6 .
Figure 6.Fold changes in the transcription of stress tolerance-associated genes in Cronobacter sakazakii in response to AgNP/PMMA/CA film.(A) The mRNA expression levels of C. sakazakii cultured in LB supplemented with AgNP/PMMA/CA film.(B) Heat map of the mRNA expression change.AgNP = silver nanoparticles; PMMA = polymethylmethacrylate; CA = cellulose acetate; values are mean ± SD for n = 3, one-way ANOVA.*P < 0.05, **P < 0.01, ***P < 0.001 compared with the control.
Li et al.: ANTIBACTERIAL FILM: INHIBITORY EFFECT ON FOOD PATHOGENS

Table 1 .
Li et al.: ANTIBACTERIAL FILM: INHIBITORY EFFECT ON FOOD PATHOGENS Genes and primers selected for quantitative real-time PCR (RT-qPCR) 1This gene encodes 16s rRNA, which was used to normalize RT-qPCR data.

Table 2 .
Li et al.: ANTIBACTERIAL FILM: INHIBITORY EFFECT ON FOOD PATHOGENS Combined effects of AgNP/PMMA/CA film and mild heat on Cronobacter sakazakii 1 In a row, an asterisk indicates a significant difference between the PMMA/CA film group or the AgNP/PMMA/CA film group, compared with the control group (P < 0.05).#In a row, a number sign indicates that the PMMA/CA film group is significantly different, compared with the AgNP/PMMA/CA film group (P < 0.05).&In a row, an ampersand indicates that the LB group is significantly different, compared with the IFM group (P < 0.05); values are mean ± SD; n = 3; 1-way ANOVA.