Journal of Food Engineering 161 (2015) 8–15
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Priming with nano-aerosolized water and sequential dip-washing with hydrogen peroxide: An efficient sanitization method to inactivate Salmonella Typhimurium LT2 on spinach Ming Zhang a, Jun Kyun Oh b, Szu-Ying Huang a, Yan-Ru Lin a, Yi Liu a, M. Sam Mannan a, Luis Cisneros-Zevallos c,⇑, Mustafa Akbulut a,b,⇑ a
Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX 77843-3122, United States Department of Materials Science and Engineering, Texas A&M University, College Station, TX 77843-3003, United States c Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843-2133, United States b
a r t i c l e
i n f o
Article history: Received 11 December 2014 Received in revised form 13 March 2015 Accepted 18 March 2015 Available online 27 March 2015 Keywords: Nano-aerosol Hydrogen peroxide Liquid sanitizer Sanitization Fresh produce
a b s t r a c t This work investigates the efficacy of a combined sanitization approach involving pretreatment of nanoaerosolized water followed by H2O2 dip-washing against Salmonella Typhimurium LT2 on spinach. The addition of a pretreatment step utilizing nano-aerosolized water for 5 min significantly increased the sanitization efficacy: The number of surviving bacteria was 5.2 ± 0.8 CFU/g, 4.7 ± 0.2 CFU/g, and 1.1 ± 1.1 CFU/g for H2O2 dip-washing only, H2O2 spraying only, and for the combined treatment, respectively. The enhanced efficacy of the combined treatment was attributed to the ability of nano-aerosolized water to reach and fill crevices that tended to form micro airpockets with the liquid (bulk) sanitizer. Overall, these findings may suggest that a short priming step involving nano-aerosolized water may be incorporated in traditional liquid sanitization approaches to enhance their effectiveness. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Fresh produce is a significant element of a healthy diet (Van Boxstael et al., 2013). In recent decades, there has been an increased consumption and larger scale production of fresh produce, leading to an increase in the number of outbreaks caused by foodborne pathogens associated with them (Olaimat and Holley, 2012). A recent study reported that foodborne pathogens gave rise to 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths per annum between 2000 and 2006 in the United States (Scallan et al., 2011). Contamination of fresh fruits and vegetables by human pathogens can occur during growth, harvest, transport and further processing and handling (Alegre et al., 2010; Issa-Zacharia et al., 2011; Marti et al., 2013; Sánchez et al., 2012). Sanitization of fresh produce plays an important role to reduce the occurrence of foodborne illness. Currently, there are several intervention methods available to inactivate microorganism on the whole and fresh cut ⇑ Corresponding authors at: Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843-2133, United States (L. Cisneros-Zevallos) and Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX 77843-3122, United States (M. Akbulut). E-mail addresses: lcisnero@tamu.edu (L. Cisneros-Zevallos), makbulut@tamu. edu (M. Akbulut). http://dx.doi.org/10.1016/j.jfoodeng.2015.03.026 0260-8774/Ó 2015 Elsevier Ltd. All rights reserved.
produce (Bermúdez-Aguirre and Barbosa-Cánovas, 2013; Goodburn and Wallace, 2013; Zhang et al., 2011, 2013). Those based on liquid/aqueous sanitizers such as electrolyzed water (Issa-Zacharia et al., 2011; Park et al., 2002), chlorine dioxide (López-Gálvez et al., 2010; Trinetta et al., 2012), chlorinated water (Bermúdez-Aguirre and Barbosa-Cánovas, 2013; Waters and Hung, 2014), hydrogen peroxide (Hassan et al., 2013; Huang et al., 2012), peroxyacetic acid (Neo et al., 2013; Vandekinderen et al., 2009) and organic acids (Nguyen and Yuk, 2013; Park et al., 2011) are the most common ones in the industry due to their ease of application and low cost. These sanitizers are often applied by dipping or spraying. However, past studies have shown that their efficacy can largely be influenced by the surface property of the produce (Fransisca and Feng, 2012; Ukuku and Fett, 2006). For some surface types and topographies, the aqueous sanitizers may not able to deliver the required amount of lethal chemical components to the microorganism at the protected sites (such as crevices) on the surface or sub-surface of fresh produces (Burnett and Beuchat, 2001). As such, considering the increasing number of outbreaks, there is an increasing need to develop effective sanitization methods to inactivate pathogens on produce surfaces. In an effort to improve aqueous chemical intervention methods and increase penetration of aqueous sanitizers on surfaces, aerosolized antimicrobials were introduced (Kim et al., 2014; Lee et al.,
M. Zhang et al. / Journal of Food Engineering 161 (2015) 8–15
2007; Park et al., 2012; Tayel and El-Tras, 2010). Aerosolization has been commercially used for room disinfection in hospitals, for medical treatment of respiratory disease, for dispersal and agricultural application of pesticides, and for delivering consumer products such deodorants and painting. There is a limited amount of work related to the aerosolization of sanitizers for food systems in the literature (Ganesh et al., 2010; Huang et al., 2012; Oh et al., 2005; Zhang et al., 2007). In the present study, we investigated a novel technology combining the spraying of nano-aerosolized water first and followed by dipping in hydrogen peroxide solution. The hypothesis behind this approach is that nano-aerosolized water can fill the protected sites (i.e. crevices) on the surface or sub-surface of fresh produces, thereby enhancing the delivery of the lethal aqueous chemicals to the target microorganisms at the protected sites by bridging and diffusion. As a model microorganism, Salmonella enterica subsp. enterica serovar Typhimurium LT2 (S. Typhimurium LT2) was used because Salmonella spp. are the most commonly reported bacteria causing food-borne illnesses according to the data from the US-CDC (Sivapalasingam et al., 2004). As a model produce, spinach was selected due to its economic importance, large production and consumption (Lucier and Jerardo, 2007), the presence of a rough surface which may provide protected sites for microbial protection (Burnett and Beuchat, 2001; DeFlaun and Mayer, 1983) and to its association to recent foodborne illness outbreaks (Arnade et al., 2009; Calvin, 2007; Wendel et al., 2009). 2. Materials and methods 2.1. Preparation of produce surface Spinach was purchased from a local grocery store (College Station, TX, USA). From the batch of spinach, spinach leaves were carefully selected to ensure uniformity in maturity, size, weight, color, and physical appearance. The selected leaves were gently washed with running water to eliminate and remove all the dirt, sand, and grime. Then, the leaves were dried using tissue paper. 2.2. Characterization of surface topography In order to determine spinach topography in 3-D, optical slicing with confocal microscopy (Leica TCS SP5, Leica Microsystems Inc., IL, US) was used. In these measurements, the separation between observation planes was set at 0.547 lm. The obtained images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). 2.3. Growth and preparation of microorganisms Rifampicin-resistant Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (S. Typhimurium LT2, ATCC 700720; American Type Culture Collection, Manassas, VA, USA) was obtained from the Department of Animal Science Food Microbiology Laboratory culture collection at Texas A&M University (College Station, TX, USA). Preparation of S. Typhimurium LT2 can be found in a previous paper (Zhang et al., 2014). Briefly, working cultures of S. Typhimurium LT2 were obtained by transferring from tryptic soy agar slant (TSA; Becton, Dickinson and Co., Sparks, MD, USA) to 9 mL tryptic soy broth (TSB; Becton, Dickinson and Co.). After incubation at 37 °C for 24 h, the culture was transferred to a fresh TSB and incubated for another 24 h. And then bacterial cell were collected by centrifugation at 2191g and washed with peptone water for three times. After the final cycle, the pellet was suspended in 9.0 mL 0.1% (w/v) of peptone water and used immediately. The inoculum
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concentration was 8.0 ± 0.4 1010 CFU/mL, confirmed via plating on TSA with rifampicin (80 lg/mL). 2.4. Inoculation of spinach leaves with bacterial organisms First, 2 mL of bacterial inoculation with a concentration of 8.0 ± 0.4 1010 CFU/mL was added into sterile polystyrene Petri dish (100 mm 15 mm, Thermo-Fisher Scientific, Inc.). Second, to ensure that only one side of spinach was inoculated with microorganisms, spinach leaf was placed on top of the bacterial suspension in the Petri dish. After 5 min bacterial contact, the leaf was carefully removed and air-dried at room temperature in a fume hood for 15 min. Finally, the unexposed edges of the leaf were cut away with scalpel to obtain the leaf piece that is uniformly and completely exposed to the bacterial suspension. The leaf pieces were weighed, which was used to calculate bacterial concentration per spinach weight. In this study, the mass of leaf pieces ranged from 0.5 g to 0.8 g. 2.5. Generation and characterization of nano-aerosols Fig. 1 shows the schematic illustration of the electrospray aerosol generator, which consisted of a high voltage generation source and a liquid flow system (Lian et al., 2010). The high voltage generation source, which involved a function generator (DS-345, Stanford Research System, Sunnyvale, CA, USA) and a voltage amplifier (Trek Model 610E, Trek Inc., Lockport, New York, USA) was electrically connected to the spray nozzle (capillary). The liquid flow system consisted of an infusion syringe pump (KDS 220, KD Scientific, Holliston, MA, USA), with four 2.5-mL injection syringes. The liquid reaching the nozzle tip formed a Taylor cone, which emitted a liquid jet through its apex (Mejia et al., 2009). Due to Coulomb repulsion, small and highly charged droplets were formed and resist aggregation (Deng et al., 2006). In electro-spraying, it is possible to control the aerosol size by adjusting liquid flow rate and applied voltage: Generally, aerosol size decreases with increasing voltage and with decreasing liquid flow rate (Lian et al., 2010). In this study, voltage was set to be 9 kV and flow rate was 1.2 mL/hr to obtain nano-sized aerosols. The size of resultant aerosols was measured using a laser diffraction particle analyzer (SprayTec, Malvern Inc., Worcestershire, UK). 2.6. Sanitization methods The spinach leaves contaminated by bacteria was sanitized with three different approaches. The first approach involved dipping in 3% hydrogen peroxide H2O2 (Bulk H2O2) for 2 min. The second approach relied on electrospraying 3% hydrogen peroxide H2O2 (Aerosol H2O2) on the contaminated leaf for 5 min. The third approach, which is the main focus of this paper, involved two steps: electrospraying leaf with nano-aerosolized water for 5 min (priming stage) and sequential dipping in 3% H2O2 immediately for 2 min (Combined treatment). As control experiments, electrospraying aerosolized Milli-Q water on the contaminated leaf for 5 min (Aerosol water) or no treatment (Control) were used. 2.7. Enumeration of inoculum organisms Plate count method was used to measure the number of surviving S. Typhimurium LT2 cells on the treated spinach leaves in order to compare the efficiency of sanitization approaches. First, the treated spinach samples were placed in 9 mL test tubes containing 0.1 % (w/v) of peptone water. Then, the tubes were shaken on a Mini Shaker (VWR International, LLC) at 900 rpm for 10 min. Next, serial dilutions of the suspension were made and plated on TSA supplemented with 80 lg/mL rifampicin. Survivors were enumerated
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Plastic Tube
Signal Generator
Capillary Tubes Multiple Syringe Pump Aerosol Spinach
High Voltage Amplifier
Malvern Laser Characterization
Fig. 1. Experimental setup for generating nano-aerosol and priming (sanitizing) spinach leaves contaminated by bacteria.
following 24 h aerobic incubation at 37 °C. The minimum concentration that could be observed was 9 CFU/sample, corresponding to 1.1–1.3 log CFU/g.
Redmond, WA, USA) and mean separation tests performed using a two-tail distribution t-test (p < 0.05). 3. Results and discussion
2.8. Determining ability of sanitization approaches to reach crevices of spinach To study the wetting behavior and the mechanism how the two-step sanitization method interacts with the crevices of spinach surfaces, we developed a technique relying on confocal microscopy, fluorescent solution, and spinach replica surfaces. Here, replica instead of real surface was used because spinach was not transparent enough for confocal microscopy studies. Soft lithography technique was used to create a transparent, exact surface replica of spinach surfaces. In this technique, the first step was to pour poly(vinyl alcohol) (PVA, Mw = 11,000– 31,000 g mol 1, ca. 10 wt.%) solution onto the surface of a spinach. After water completed evaporated at room temperature, the PVA film, containing the inverse spinach topography, was gently peeled off. Next, PDMS pre-polymer (Sylgard 184, Dow Corning, Midland, MI, USA) and a curing agent at a weight ratio of 10:1 were mixed and immediately poured onto the PVA film. PDMS film was degassed and cured in a vacuum oven at 65 °C for 5 h. Finally, PDMS replica was obtained by peeling off the cured PDMS from the PVA film. The surface topography and hydrophobicity of the PDMS replica was characterized by SEM and confocal microscopy, and contact angle tensiometry, respectively. After the replicas were obtained, we treated spinach replicas with fluorescein disodium salt hydrate in water solution (10 3 wt.%) by three different approaches mimicking the sanitization approaches described above: (a) rinsing in bulk fluorescein solution for 15 min and then rinsed in DI water for 10 s; (b) spraying with nano-aerosol fluorescein solution for 5 min; and (c) spraying with water nano-aerosol for 5 min and then rinsed in bulk fluorescein solution for 5 min follow by rinsing in DI water for 10 s. After the treatments, a confocal microscope (Leica TCS SP5) was used to determine the distribution of fluorophores on the replica surface. The excitation laser wavelength was 488 nm, and the observed wavelength ranged from 500 nm to 550 nm. Here, the expectation is that when there is liquid–solid contact, some fraction of fluorescein in solution will deposit (physisorb) on produce surfaces. On the other hand, when micro airpockets form, there is no liquid–solid contact at these locations, and hence, no fluorescein deposition. 2.9. Statistical analysis All experiments were replicated at least three times. The results were presented as means ± standard deviation. Analyses of variance (ANOVA) were conducted using Microsoft ExcelÒ (Microsoft Corp.,
3.1. Characterization of aerosols and surface topography of spinach To better understand how bacteria and aerosols interact and can position themselves on spinach surfaces, we measured the size of aerosols and characteristic length scales of crevices occurring on the spinach surfaces. Fig. 2 displays confocal microscopy images of spinach surface. The analysis of these images revealed that the spacing between asperities (i.e. the dimensions of crevices) was 32.1 ± 4.8 lm in length, 3.8 ± 0.9 lm in width, and 4.4 ± 2.5 lm in depth. Typically, S. Typhimurium LT2 are 1–2 lm in length and 0.5–0.7 lm in diameter (Zhang et al., 2013). Accordingly, the crevices of spinach surfaces are large enough to accommodate bacteria in them, and thus may protect bacteria against outside influences and hinder the sanitization of fresh produce surfaces. Fig. 3 shows the particle size distributions of water and hydrogen peroxide aerosols. The curves, both of which were Gaussian (r2 = 0.95 for H2O and r2 = 0.90 for H2O2), were centered on 204 nm and 188 nm with full widths at half maximum (FWHM) of 81 nm and 110 nm for water and hydrogen peroxide, respectively. The results confirm that both types of aerosols were smaller than bacteria and much smaller than the crevices of the spinach surfaces. Therefore, such aerosols could possibly reach the bottom of the crevices. 3.2. Bactericidal effect of sanitization method Fig. 4 shows the number of surviving bacteria upon different type of surface treatments. The log number of bacteria on spinach after drying without any further treatment was 6.2 ± 0.7 log CFU/g. Upon the treatment of aerosol water, the number did not change significantly, (5.8 ± 0.1 log CFU/g) (p < 0.05). Upon the treatment of hydrogen peroxide (3 wt.%) nano-aerosol, the log number was 4.7 ± 0.2 log CFU/g, which means a 1.5 ± 0.7 log reduction in the number of surviving bacteria. For the case of bulk hydrogen peroxide treatment, the number of surviving bacteria on spinach was 5.2 ± 0.8 log CFU/g. The comparison of sanitization efficacy using bulk and aerosolized hydrogen peroxide treatments indicates that there is no statistical difference between these two methods. On the other hand, the number of surviving bacteria on spinach was only 1.1 ± 1.1 log CFU/g after the combined treatment involving priming surfaces with nano-aerosol followed by H2O2 dipping. The number of bacteria was slightly smaller than the lowest number that can be observed by the method (1.2 log CFU/g). For the combined treatment, the log reduction was 5.1 ± 1.3 in comparison to the control and 4.1 ± 1.4 in comparison to the H2O2 bulk
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a
b
50 µm
Fig. 2. Confocal microscopy images of spinach surface: (a) Top view and (b) ‘‘Extended depth of field’’ view. Here, by optically sectioning the spinach surface, the in-focus information at the surface is first acquired over a range of images, which is then processed to generate a single in-focus image using ImageJ with ‘‘Extended Depth of Field’’ plug-in (Forster et al., 2004). The scale bar is 50 lm.
Fig. 3. The particle size distribution of water and hydrogen peroxide aerosols measured by light scattering.
treatment only. Overall, the combination treatment significantly increased the sanitization efficacy compared to H2O2 wash only and aerosol H2O2 only. Huang et al. (2012) studied sanitization efficacy of H2O2 microaerosols against two strains of Escherichia coli O157:H7 on spinach. They observed that a post-treatment of aerosolized H2O2 following a pre-treatment of bulk H2O2 (i.e. dipping) did not increase the sanitization efficiency compared to the sanitization involving treatment with bulk H2O2 solutions only. After washing with bulk H2O2 solutions, Log reductions were 1.5 ± 0.2 and 1.6 ± 0.1 for the two strains; and the post-treatment with aerosolized H2O2 did not increase the reductions. Log reductions of the two strains after directly treated with aerosolized were 1.2 ± 0.3 and 1.1 ± 0.2 for 2.5% H2O2 while 1.1 ± 0.4 and 1.2 ± 0.4 for 5% H2O2. Considering our result and that of Huang et al., we may deduce that a pre-treatment of aerosol followed by a post-treatment of bulk H2O2 is likely to provide a different mechanism of sanitizer transport and delivery from a post-treatment of aerosol following a pre-treatment of bulk H2O2. Oh et al. (2005) studied the sanitization efficiency of microaerosols on lettuce treated for pro-longed periods of time. The aerosol system was peroxyacetic acid with a concentration of 40 ppm and a size of 5.42- to 11.42-lm. Log reductions of Salmonella Typhimurium were 0.3, 3.3 and 4.5 after 10, 30 and 60 min of treatment while log reductions of E. coli O157:H7 were 0.8, 2.2 and 3.4, respectively. Their short time treatment results are comparable with our findings on nano-aerosol treatment only. However, we do not have data to compare long term effects as we did not perform experiments with very long treatment times (e.g. 30 min and 60 min) because such long treatment times are not desirable for the produce industry.
3.3. Mechanism of enhanced efficiency for combined sanitization approach
H2O2
H2O2 Combined treatment
Fig. 4. Number of survivors on spinach after: no treatment (Control), spraying with nano-aerosol water (Aerosol water), dipping in 3% H2O2 (H2O2 Bulk), spraying with nano-aerosols of 3% H2O2 (Aerosol H2O2), and spraying with nano-aerosol water followed by sequential dipping in 3% H2O2 (Combined treatment). Treatments with same letters are not significantly different (p < 0.05).
When liquid sanitizers come into contact with a produce surfaces, micro airpockets can form between the crevices on the surface and sanitizer. Such micro airpockets are believed to be responsible for inefficient delivery of sanitizers to microorganisms at protected sites on the surface or sub-surface of fresh produces. Because the size of nano-aerosols is much smaller than the dimensions of crevices occurring on produce surface, nano-aerosols can readily reach to depths of the crevices. This is the main hypothesis behind using nano-aerosols (or micro-aerosols) for sanitizing produce surfaces. To better test this hypothesis and better understand
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Fig. 5. Polydimethylsiloxane (PDMS) surface topography replica of spinach leaf using a soft lithography technique: (a) SEM image, (b) Confocal microscopy image, and (c) ‘‘Extended depth of field’’ view of confocal microscopy image. The scale bar is 20 lm in (a) and 50 lm in (b) and (c).
Fig. 6. Confocal images of PDMS replica of spinach treated by: (a) bulk fluorescein solution, (b) aerosolized fluorescein solution, and (c) aerosolized water and bulk fluorescein solution.
the mechanism of aerosol transport to the proximity of produce surface, we relied on an experimental approach involving spinach replica surfaces, a fluorescent solution, and confocal microscopy (described in Materials and Methods). As can be seen from SEM micrographs, fine features of spinach surface such as stomata and crevices were successfully replicated using the abovementioned soft lithography technique (Fig. 5a). The amplitude and space parameters of surface roughness on the replica was characterized via confocal microscopy (Fig. 5b and c). The quantification of these images indicated that the average crevice width, length, and depth were 32.7 ± 5.5 lm, 4.2 ± 1.2 lm, and 4.8 ± 1.1 lm, respectively. The comparison of surface textures of the real spinach and replica surfaces revealed that the characteristics dimensions of these textures were not statistically different according to Student’s t-test (p < 0.05). To further compare the real spinach and replica surfaces, the wetting characteristics of water on these surfaces were measured. It was found that the static contact angle of water was 81 ± 7° for the real spinach and 107 ± 5° for the replica. While the slight difference in surface hydrophobicity can result in variations in the volume of trapped air (micro airpocket), it is unlikely to cause a total change from the appearance to disappearance of micro airpockets. This is especially reasonable considering previous works indicating that metastable Cassie-state could be observed at angles as low as 74° when micro-texture of overhang structure is present (Cao et al., 2007, 2008). Additionally, wetting failure can also occur on hydrophilic surfaces having contact angles as low as 40° and big crevices (e.g. depth of 3 lm) when the wetting velocity is high enough to create a kinetically-trapped gas–liquid–solid composite interface (Zhao et al., 2014). Hence, the replica surfaces are appropriate to obtain
a qualitative understanding of how nano-aerosols can reach and fill crevices. Fig. 6 shows the confocal microscopy images of these transparent replicas after treating with (a) bulk fluorescein solution, (b) aerosolized fluorescein solution (see supplementary information for size distribution), or (c) aerosolized water followed by bulk fluorescein solution where all treatment approaches were followed by rinsing in DI water for 10 s to remove unattached fluorescein solution. Upon treating the replica surface with bulk fluorescein solution and rinsing, the fluorescein signal (green1 areas) could only be observed on the top areas of the surface while there was no fluorescein molecule reaching to the bottom of the crevices (Fig. 6a). In other words, this finding can be interpreted as follows: if we used bulk H2O2 instead of bulk fluorescein solution and there were bacteria at the bottom of the crevices, bulk H2O2 would not be able to reach there and sanitize the surface. For the surface treated by aerosolized fluorescein solution, fluorescein signal was observed both on the top areas and crevices (valleys) (Fig. 6b). This result indicates that accessibility of aerosols into the crevices is much higher than bulk solution. However, it is important to note that the fluorescence signal was stronger on the top areas and some areas of valleys had no signal (Fig. 6b), suggesting that the total volume of aerosols reaching into crevices is small compared to the total volume of crevices. Some larger bright spots were also observed presumably due to the aggregation of aerosol fluorescein solution before or after reaching replica surface.
1 For interpretation of color in Fig. 6, the reader is referred to the web version of this article.
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For the surface treated by the combined treatment (i.e. a pretreatment (priming) with aerosolized water and treatment with bulk fluorescein solution), fluorescein signal could clearly be seen throughout the surface including on the stomas and crevices (Fig. 6c). These results clearly support our finding in that the combined treatment method has much higher probability to reach and sanitize the bacteria in the crevices of produce surfaces. It is also important to note that the fluorescence signal in the crevices (valleys) was even stronger than that on top areas. This is most likely because fluorescein dye on top areas is easier to remove during rinsing in water. These behaviors can be explained by Wenzel and Cassie–Baxter states. Liquid in intimate contact with a rough surface can be either in Wenzel state (Wenzel, 1936), in which liquid is in intimate contact with the solid asperities and the surface is completely wet, or in Cassie-Baxter state (Cassie and Baxter, 1944), in which liquid rest on the tops of the asperities and the surface is incompletely wet with some gas trapped. The Cassie–Baxter state exists when the following two criteria are met: Contact line forces overcome body forces of unsupported droplet weight and the microstructures are tall enough to prevent the liquid that bridges microstructures from touching the base of the microstructures (Extrand, 2004). If an aqueous liquid is placed on a hydrophobic rough surface (i.e. asperities with narrow and deep crevices), the Cassie– Baxter state is more likely to occur and the air is trapped between liquid and asperities. However, if the droplet size is smaller than the crevice size, the liquid could reach the base of the crevices without any hindrance. Even if the crevices are not fully filled with liquid, the small amount of liquid within the crevices can serve as a bridge connecting bulk liquid with the liquid in the crevices. Thus, in the presence of aerosolized drops, a transition from Cassie– Baxter state to Wenzel state can take place. The confocal microscopy results can be further-interpreted as follows: When bulk liquid sanitizer is applied on a contaminated spinach surface having narrow and deep crevices, the liquid cannot completely wet the surface (i.e. Cassie–Baxter state) and the trapped gas could protect bacteria from sanitization (Fig. 7a). However, if the surface is pre-treated (primed) with nano-aerosolized water, the crevices are filled with water fully or partially. Then, by bridging and connecting with the liquid in the crevices, the applied liquid sanitizer can fully wet the produce surface (i.e. Wenzel state). This means that bacteria will no longer be protected from sanitization (Fig. 7b).
(a)
The only question remaining is why there was no significant improvement in the sanitization efficacy when H2O2 aerosols were used in comparison to the bulk H2O2 treatment. It is known that is H2O2 is not a very stable compound and can decompose to water and oxygen. The rate of decomposition is dependent on the temperature and on the concentration of the peroxide, as well as the pH and the presence of impurities and stabilizers (Lin and Gurol, 1998; Rice and Reiff, 1927; Takagi and Ishigure, 1985). While the decomposition of dilute solutions is slow at room temperature and atmospheric pressure, the decomposition rate of aerosolized H2O2 can be much larger due to its high chemical potential (Campbell et al., 2002) and high surface area to volume ratio (Albanese et al., 2012). Additionally, aerosol H2O2 was positively charged during the generation of aerosol, which could also increase its decomposition rate since H2O2 decomposition can be catalyzed by some positive ion (De Laat and Gallard, 1999; Haber and Weiss, 1934). Thus, a significant part of aerosol H2O2 may decompose before it reaches the produce surface. 3.4. Application prospect Nanoaerosol technologies have recently attracted a lot of attention due to the preliminary findings indicating sanitization efficiency (Klems and Johnston, 2013; MacMillan et al., 2012). This work contributes towards an advancement in efficiency of such technologies through the use of combined treatment involving pre-spraying with nano-aerosols and post-dipping in sanitizer solution. While the majority of the research at this front is done at a laboratory scale as this work, some prior works have already extended some nano-aerosol approaches up to larges scales (Huang et al., 2012; Oh et al., 2005). We anticipate that for this work, the design of capillary tube arrays to increase the total volumetric flow rates and areal coverage will be the key step for the scale-up of this work to the industrial or practical applications. An important consideration for any sanitization approach is the treatment time. Due to the large processing volumes, fresh produce industry, in particular for spinach, typically spend 2 min on the sanitization step. For the described technology to gain acceptance from fresh produce industry, the treatment time should be decreased while maintaining similar bacterial inactivation potential. In this study, we have only tested the proof-of-concept with a fixed position and dimension of capillary tube, flow rate, and applied voltage. We envision that by extensively optimizing the Air Pocket
Bacteria
Produce Surface Sanitizer
Cassie-Baxter State
(b)
Nanoaerosols
Wenzel State Fig. 7. Differences in the wetting behavior of spinach surfaces, which is (a) directly exposed to bulk H2O2 solution and (b) first exposed to water nano-aerosols, and then to bulk H2O2 solution. The former case leads to the formation of micro airpockets while the latter case does not. This difference is presumably responsible for the enhanced efficacy of the combined treatment approach described above against bacteria.
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parameter space, we can increase the mass flux of nanoaerosols, thereby decreasing the treatment time. Another issue to consider for all nanoaerosol technologies is if these alter the surface chemistry of produce in any way and if so, as a consequence of this, how the quality and shelf life of produce changes. The addressing of these questions require long term studies carefully characterizing the nanoaerosol treated produce with multiple techniques, which we plan to study in a future work. 4. Conclusion In the present study, we report a combined method involving a pre-treatment of nano-aerosolized water and sequential treatment of H2O2 dip-washing to sanitize produce surfaces for the first time. The efficacy of the combined method was better than that of H2O2 dipping wash only and spraying H2O2 nano-aerosol only: Treatment with 3% H2O2 dipping for 2 min only decreased the number of S. Typhimurium LT2 from 6.2 ± 0.7 to 5.2 ± 0.8 log CFU/g on spinach surface; while spray with 3% H2O2 nano-aerosol for 5 min only decreased the number to 4.7 ± 0.2 log CFU/g. On the other hand, the combined method (initial electrospraying with nano-aerosols of water for 5 min first and sequential dipping in 3% H2O2 for 2 min) decreased the number of S. Typhimurium LT2 to 1.1 ± 1.1 log CFU/g. In addition, our confocal microscopy studies showed that when a liquid drop is applied on a surface having a spinach surface texture, micro airpockets can form. Therefore, the enhanced efficacy of the developed approach was attributed to the fact that nano-aerosols can reach bacteria in protected sites (crevices) on spinach surfaces while bulk liquids cannot always reach such sites. This is important because during an aqueous liquid-based sanitization treatment, as those applied commercially, micro airpockets may form on rough surfaces protecting bacteria from sanitization. Priming surfaces with nano-aerosolized water followed by a traditional liquid-dipping treatment can fill the crevices and gaps on the surfaces and enable lethal aqueous chemical compounds to reach protected bacteria, thereby increasing sanitization efficacy. Acknowledgements The authors are grateful for financial support from USDA National Institute of Food and Agriculture for funding through Agriculture and Food Research Initiative Competitive Grand No. 2011-67017-30028. The authors want to thank Dr. Matthew Taylor and Keila Perez in Department of Nutrition and Food Science, Texas A&M University, for their assistance with the experiments. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jfoodeng.2015.03. 026. Reference Albanese, A., Tang, P.S., Chan, W.C., 2012. The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annu. Rev. Biomed. Eng. 14, 1–16. Alegre, I., Abadias, M., Anguera, M., Oliveira, M., Viñas, I., 2010. Factors affecting growth of foodborne pathogens on minimally processed apples. Food Microbiol. 27 (1), 70–76. Arnade, C., Calvin, L., Kuchler, F., 2009. Consumer response to a food safety shock: the 2006 food-borne illness outbreak of E. coli O157: H7 linked to spinach. Appl. Econ. Perspect. Policy 31 (4), 734–750. Bermúdez-Aguirre, D., Barbosa-Cánovas, G.V., 2013. Disinfection of selected vegetables under nonthermal treatments: chlorine, acid citric, ultraviolet light and ozone. Food Control 29 (1), 82–90.
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