Development of Titanium Dioxide (TiO2) Nanocoatings on Food Contact Surfaces and Method to Evaluate Their Durability and Photocatalytic Bactericidal Property Veerachandra K. Yemmireddy, Glenn D. Farrell, and Yen-Con Hung

Titanium dioxide (TiO2 ) is a well-known photocatalyst for its excellent bactericidal property under UVA light. The purpose of this study was to develop physically stable TiO2 coatings on food contact surfaces using different binding agents and develop methods to evaluate their durability and microbicidal property. Several types of organic and inorganic binders such as polyvinyl alcohol, polyethylene glycol, polyurethane, polycrylic, sodium and potassium silicates, shellac resin, and other commercial binders were used at 1:1 to 1:16 nanoparticle to binder weight ratios to develop a formulation for TiO2 coating on stainless steel surfaces. Among the tested binders, polyurethane, polycrylic, and shellac resin were found to be physically more stable when used in TiO2 coating at 1:4 to 1:16 weight ratio. The physical stability of TiO2 coatings was determined using adhesion strength and scratch hardness tests by following standard ASTM procedures. Further, wear resistance of the coatings was evaluated based on a simulated cleaning procedure used in food processing environments. TiO2 coating with polyurethane at a 1:8 nanoparticle to binder weight ratio showed the highest scratch hardness (1.08 GPa) followed by coating with polycrylic (0.68 GPa) and shellac (0.14 GPa) binders. Three different techniques, namely direct spreading, glass cover-slip, and indented coupon were compared to determine the photocatalytic bactericidal property of TiO2 coatings against Escherichia coli 0157:H7 at 2 mW/cm2 UVA light intensity. Under the tested conditions, the indented coupon technique was found to be the most appropriate method to determine the bactericidal property of TiO2 coatings and showed a reduction of 3.5 log CFU/cm2 in 2 h.

Abstract:

Keywords: antimicrobial test, binding agent, E. coli, food contact surface, physical stability, TiO2 coating

A simple approach to create physically stable and bactericidal TiO2 nanocoatings was developed on food contact surfaces of stainless steel using different binding agents. The developed TiO2 nanocoatings might help to minimize microbial cross-contamination and ensure safe food processing environment.

Practical Application:

attention for several industrial applications (Rai and others 2010). In this context, nanotechnology-based advanced oxidation processes involving photocatalytic titanium dioxide (TiO2 ) nanoparticles (NPs) have shown great promise as an effective non-targeted disinfectants for killing a wide range of microorganisms. TiO2 has been recognized as the most promising photocatalyst due to its appropriate electronic band structure, photostability, chemical inertness, low cost, ready availability, and capable of repeated use without substantial loss of catalytic activity (Ibhadon and Fitzpatrick 2013). TiO2 is one of the most commonly used heterogeneous photocatalyst utilized as a self-cleaning and selfdisinfecting material for coating materials in many applications. It has a more helpful role in environmental purification due to its nontoxicity, photo-induced super hydrophobicity and antifogging effect (Haghi and others 2012). Over the last decade, there is an increased interest in the application of TiO2 photocatalytic disinfection technique for the purpose of food safety and quality enhancement (Manreet and Hayata 2006; Chawengkijwanich and Hayata 2008; Chorianopoulos and others 2011). TiO2 has been approved by the American Food and Drug Administration MS 20150241 Submitted 2/9/2015, Accepted 6/1/2015. Authors Yemmireddy, (FDA) for use in human food, drugs, cosmetics, and food contact Farrell, and Hung are with Dept. of Food Science and Technology, Univ. of Georgia, materials (Chorianopoulos and others 2011). TiO2 photocatalysts 1109 Experiment Street, Griffin, GA, 30223–1797, U.S.A. Direct inquiries to generate strong oxidizing power when illuminated with UV-A author Hung (E-mail: [email protected]). light of wavelength less than 385 nm. The bactericidal properties

Microbial cross-contamination is a major issue in the food processing environment leading to several foodborne outbreaks and illnesses in the recent times. Cross-contamination through both food contact and non-food contact surfaces such as knifes, cutting boards, working surfaces, equipment and the processing environment is well reported. Also, increased popularity of fresh and minimally processed foods poses additional risk of cross-contamination from food contact and nonfood contact surfaces (Llorens and others 2012). Conventional sanitation and disinfection procedures that are widely followed in the industry are not sufficient to address the emerging risks of microbial cross-contamination involving resistant pathogens. In addition, many of the established sanitizers and disinfectants lack residual effect for extended period of protection and generate toxic disinfection by-products (Meylheuc and others 2006). Modification of surfaces with antimicrobial agents to prevent the growth of harmful microorganisms has received much

R  C 2015 Institute of Food Technologists

doi: 10.1111/1750-3841.12962 Further reproduction without permission is prohibited

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Introduction

TiO2 antimicrobial nanocoatings . . . of TiO2 were attributed to the high redox potential of the reactive oxygen species (ROS) such as hydroxyl radical (. OH), superoxide ions (O2 - ), and hydrogen peroxide (H2 O2 ) formed by the photoexcitation (Foster and others 2011). Several techniques have been proposed to immobilize TiO2 NPs on hard surfaces for the purpose of photocatalytic disinfection (Visai and others 2011). Sol-gel synthesis of NPs and subsequent dip, spin, or spray coating is the most widely reported procedure in the literature. Other coating methods such as electrochemical deposition, electrophoretic coating, chemical vapor deposition, sputtering, and plasma spraying were complicated and costly for practical application (Kasanen and others 2011). Alternatively, a simple direct coating of the NPs using wet chemical approaches was also reported. However, poor adhesion on the substrate and the lack of physical stability of the developed coatings is the major issue when using direct coating methods (Mills and Lee 2002; Keshmiri and others 2004; Han and others 2012). Thus, binding agents are usually necessary for direct coating in order to form strong adhesion between the NP and the substrate. Either organic polymer or inorganic binding materials have been used for photocatalyst immobilization (Du and others 2008; Lim and others 2009; Henderson 2011). However, no extensive stability studies have been conducted in the past to evaluate the durability of nanocoatings when they are intended to use in food processing environment (Bastarrachea and others 2015). In addition, several methods have been proposed to determine the bactericidal activity of photocatalytic nanocoatings with each having their own advantages and limitations. For use in food safety applications, the antimicrobial coatings expected to have characteristics such as long lasting efficacy, ease of fabrication, durability, and no toxicity. With appropriate binding agents, physically stable and durable TiO2 nanocoatings with strong bactericidal property can be developed on food contact surfaces. Hence, the main goal of this study was to identify appropriate binders to create stable TiO2 nanocoating on stainless steel surfaces and identify methods to evaluate physical stability and bactericidal property of TiO2 nanocoatings. Specific objectives include:

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i. To identify the most promising binders to create TiO2 coatings on stainless steel. ii. To develop a simple method for coating TiO2 NPs on stainless steel. iii. To evaluate the physical stability and the durability of the TiO2 coatings. iv. To identify a suitable testing method to determine the bactericidal property of TiO2 coatings.

Materials and Methods Selection of TiO2 NPs and binders for coating  TiO2 (Aeroxide P25, Sigma-Aldrich, St. Louis, Mo., U.S.A.) NPs with an approximate particle size of 21 nm and specific surface area of 50 m2 /g as per suppliers specifications were used for developing nanocoatings in this study. Ten different types of polymeric, silicate, and resin-based organic and inorganic binding agents: (i) polyvinyl alcohol (PVA; P0469, TCI America, Portland, Oreg., U.S.A.), (ii) Polyethylene glycol (PEG) (P0903, TCI America), (iii) Ludox AS-40 (LAS-40) (Sigma-Aldrich, Co.), (iv) 2 types of   potassium silicates (PS1 and PS6; KASIL 1 and KASIL 6, PQ Corp., Valley Forge, Pa., U.S.A.), (v) 2 types of sodium silicates   (SSO and SSN; O and N , PQ Corp.), (vi) Ceramabind 830 R

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(C830; Aremco Products, Inc., Valley Cottage, N.Y., U.S.A.), (vii) Ceramabind 643-1 (C643-1) (Aremco Products), (viii) Water based oil-modified polyurethane (B; 23025, Miniwax , Miniwax Comp., Upper saddle river, N.J., U.S.A.), (ix) Water-based  polycrylic (C; 23333, Miniwax , Miniwax Comp.), (x) Shellac (A; 00304, Zinsser Co., Inc. Somerset, N.J., U.S.A.) were tested to evaluate their feasibility to incorporate in coating solution to achieve physically stable TiO2 coatings. R

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Substrate selection and preparation Stainless steel (Type 304, finish #2B, 25 mm2 ) coupons were used as model food contact surface for coating. Prior to coating, each coupon was slightly roughened to increase the coating adhesion and achieve a high level of bond strength by using an electric sander fitted with a P100 fine grit sand paper for 1 min on each side of the coupon. Roughening is a common practice in developing surface coatings for better attachment of the coating onto the metallic substrates. Later, the surface roughened coupons were degreased 1st by washing in acetone followed by ethanol and finally rinsed with deionized water. The cleaned stainless steel coupons were dried in a hot air oven at 60 °C for 30 min before used for coating. Screening of binders for developing stable TiO2 coating Several preliminary experiments were conducted in order to select binding agents that are most suitable to develop physically stable TiO2 coatings on stainless steel. In the 1st stage of experiments, TiO2 coatings were prepared by using 2 types of organic binders PVA and PEG. Several suspensions of TiO2 NPs mixed with PVA or PEG binders at 1:1 to 1:5 NP to binder weight ratios were prepared by using ethanol as solvent. Stainless steel coupons were dip coated with the resultant suspensions using an Instron (Model #5544, Instron Corp., Canton, Mass., U.S.A.) operated with a dipping speed of 10 mm/s, residence time of 10 s and a withdrawal speed of 0.5 mm/s. In this manner single or multiple coatings of TiO2 were applied on each coupon based on the viscosity of coating suspension and uniformity of the coated film. The coated coupons were dried in a hot air oven at 60 °C for 1 h. The dried coupons were visually inspected for coating uniformity and washed under running water for about 5 to 10 min in order to quickly assess the adherence behavior and physical stability of the coatings. Even though heat treatment after coating helped to increase the adherence of the coating to stainless steel surface, heat treatment also resulted in formation of clumps on the TiO2 nanocoating. The results of these experiments showed that the TiO2 nanocoatings with PVA and PEG as binders are nonuniform in nature and unable to withstand washing under running water (data not shown). In the 2nd stage of coating experiments, inorganic binders such as Ludox AS-40 (LAS-40), potassium (PS1 and PS6) and sodium (SSO and SSN) silicates from a commercial source as well as 2 other commercial binders of unknown composition (C830 and C643-1) were used for TiO2 nanocoating. Twenty different paste formulations (5 different binders at 4 different NP to binder ratios) were prepared for coating by mixing TiO2 NPs with each binder at 1:1 to 1:4 NP to binder weight ratios. About 1 g (±0.15) of the TiO2 paste was weighed and painted on each coupon using a Crayola paint brush so as to form a layer of uniform TiO2 coating. TiO2 coatings with potassium and sodium silicates (PS1, PS6, SSO, and SSN) and C830 were air-dried at room temperature for about 1 h; while the TiO2 coatings with LAS-40 and C643-1 were air-dried at room temperature for 2 h and then

TiO2 antimicrobial nanocoatings . . .

Compositionc Sample

codea

Type of

TA4 TA8 TB8 TB16 TC8 TC16

binderb

A A B B C C

(weight basis)

TiO2 NPs

Binder

1 1 1 1 1 1

4 8 8 16 8 16

a

TiO2 nanocoatings with binders A, B, and C. b Binders A, B, and C are shellac, polyurethane, c

and polycyclic, respectively.

Composition of the coating suspension.

cured at 93 °C for 1 h in a hot-air oven as per the manufacturer guidelines. Increasing the concentration of binder (that is, less NPs to binder ratio), increased the viscosity of TiO2 pastes and physical stability of the resultant coatings. TiO2 coatings with potassium and sodium silicates at 1:4 weight ratio was found to be physically more stable upon scratching with pencil points of 2H hardness. However, these coatings were not stable upon washing in running water followed by sonication in water bath for 15 min. Based on these results it was found that the TiO2 coatings with tested inorganic binders helped to achieve uniform and physically stable coatings (data not shown). However, TiO2 coatings with these binders are not suitable for application in moist conditions encountered in food processing environment. In the 3rd stage of coating experiments, polymer-based sealers polyurethane (B) and polycrylic (C), as well as a natural resin, shellac (A), were tested for their feasibility to incorporate in TiO2 coatings. The composition of different TiO2 coatings with binders A, B, and C are presented in Table 1. Based on the nature of each binder, the viscosity of coating solution increased with decreasing NP to binder ratio to a point where it is not feasible for coating. Hence, the reported compositions in Table 1 were selected to achieve feasible viscous suspensions for coating. Suspensions for coating were prepared by mixing TiO2 NPs with binders A, B, and C at 1:4 to 1:16 (TiO2 : binder) weight ratios in a mortar for about 15 min. Stainless steel coupons were then dip coated with the resultant suspensions as described earlier. The coated coupons were air-dried over night at room temperature. These coated coupons were found to be uniform and physically stable upon scratching with pencil points of 2H hardness. Also, washing under running water as well as sonication in water bath for about 15 min did not remove the coating from the substrate significantly. Hence, TiO2 nanocoatings with the binders A, B, and C were selected for further studies to evaluate their physical stability.

Surface characteristics of TiO2 nanocoatings In order to maintain consistency in use of samples for evaluating both physical stability and bactericidal properties of the nanocoatings, an indented stainless steel coupon having dimensions 46 × 12.5 × 1.25 mm3 and surface area of 540 mm2 were used for the TiO2 coatings with the binders A, B, and C, respectively. A sample of 0.25 g of the coating solution at 1:4 to 1:16 NP to binder weight ratio were poured into the well of stainless steel indentation to form a uniform layer of TiO2 coating. The coated coupons were dried in air overnight at room temperature. This approach of coating is referred as solution deposition technique. The thickness of the coatings was measured by using a thickness  gauge (Elcometer 345) at 8 different locations on each coupon. Film morphology and microscopic structure of the coating surface R

was characterized by a variable pressure scanning electron microscope (VPSEM, Zeiss 1450 EP) with accelerating 25 kV. The SEM images were further analyzed using an image processing software (Paint. NET) to estimate the area ratio of coated surface covered by the NPs compared with the binder.

Measurement of physical stability of TiO2 nanocoatings The hardness of the coatings was evaluated with the help of a scratch test based on the ASTM G171-03 method (ASTM 2009). Briefly, Instron fitted with a hemispherical diamond tip indenter of 76.5 µm having a conical apex angle of 120° (J&M Diamond Tool, Inc., Rumford, R.I., U.S.A.) and an antivibration table was used for this test. Three linear scratches of at least 5 mm length at 2 mm apart from each other were made on each coating with an applied load of 1 to 3 N. The width of each scratch was measured at 3 different locations equidistance from each other using a digital microscope pro (Celestron LLC, Model # 44308). Scratch hardness number (HSp ) was calculated as per the standard using following equation: H S p = kP/w 2 where HSp is the scratch hardness number (GPa), k is the geometrical constant (24.98), P is the applied normal force (grams-force), W is the scratch width (µm). In addition, adhesive strength of the coatings was evaluated using a scotch tape test by following ASTM D3359-02 test method B (ASTM 2002). Six parallel cuts of about 20 mm length at 2 mm apart were made through the coating in one steady motion using a straight edged metal guide and a sharp razor blade as described in ASTM D3359-02. Similarly, another 6 cuts were made through the coating at a 90° angle to the previous cuts to make a lattice pattern of small squares of about 0.5 × 0.5 mm2 dimensions on the coating. Later, about 25 × 50 mm2 pressure-sensitive adhesive tape (Permacel 99, Permacel, New Brinswick, N.J., U.S.A.) was applied over the lattice pattern and smoothed into the place by using a pencil eraser over the area of the incisions to ensure good contact with the coating. Adhesive tape was then removed by pulling it off rapidly with a constant force at close to a 180˚ angle. The possible crumbling at the edges of the cuts is a measure of the coating adhesion strength and is ranked from 5B to 0B according to the descriptions and illustrations provided in the ASTM standard (Table 2). A cleaning procedure commonly used in food processing environment was simulated by means of an in-house developed reciprocating test (Figure 1). In this test set-up, a texture anTM alyzer (TA. XT Plus , Texture Technologies Corp, Scarsdale, N.Y., U.S.A.) was fitted with a moving head consisting of scrubby TM side of sponge (3M Scotch-Brite , Heavy-Duty scrub sponge, St. Paul, Minn., U.S.A.) as 4 individual brushes to simulate cleaning procedure (Figure 1B). Briefly, the weight of each coated coupon was measured using a calibrated balance before subjecting to cleaning procedure. Later, a 2 mL of diluted detergent solution (Dawn UltraTM , Procter & Gamble, Cincinnati, Ohio, U.S.A.) was poured onto each TiO2 coated coupon. The coupons were then subjected to cleaning procedure using the test set-up described earlier at a moving head speed of 20 mm/s for up to 500 cycles (that is, 1000 to-and-fro motions) with an applied load of 1 N on each coupon. The cleaned coupons then were air-dried overnight at room temperature and the weight of each coupon was measured again. The difference in the weights of TiO2 coated Vol. 80, Nr. 8, 2015 r Journal of Food Science N1905

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Table 1–Composition of different TiO2 nanocoatings.

TiO2 antimicrobial nanocoatings . . . Table 2–ASTM D3359-02 classification of adhesion test results.

mately 107 CFU/mL bacterial cells. Cell concentration was adjusted by measuring the absorbance of bacterial suspension at 600 nm using a UV/Vis spectrophotometer and confirmed by plating 100 µL portions of the appropriate serial dilution on tryptic soy agar (Difco Laboratories, Franklin Lakes, NJ, U.S.A.) plates incubated at 37 °C for 24 h. Prior to antibacterial activity tests, TiO2 coated coupons were pre-sterilized under UVC light for 1 h in a biosafety cabinet. The sterile coupons were placed in a 90 mm diameter petri-dish containing moist filter paper to maintain humidity during the treatment. Bacterial culture was inoculated on each TiO2 coupon as follows: (i) direct spreading method: A drop of 100 µL inoculum was spread on the surface of TiO2 coating using a sterile loop based on the direct spreading technique, (ii) glass cover-slip method: A drop of 100 µL inoculum was spread on the surface of TiO2 coating as before and then a glass cover-slip of same size as stainless steel coupon was placed on top of the bacterial culture, (iii) indented coupon method: A 300 µL inoculum was pipetted into the well of indented TiO2 coated coupon to cover entire indented coated surface. The samples were then illuminated from above with a UV-A light system (American DJ, Model UV Panel HPTM , LLUV P40, Los Angeles, Calif., U.S.A.) at 2 mW/cm2 intensity. The intensity of light reaching the surface of the coating was mea sured using a UV radiometer (UVP , Upland, Calif., U.S.A.). Plain stainless and only binder coated stainless steel coupon under UV-A light were used as 2 independent controls. After 2 h UV treatment, TiO2 coated coupons were immersed in 10 or 30 mL (for indented coupon technique) PBS solution containing 0.1% tween 80 and vortexed for 30 s to resuspend the bacteria. A viability count was performed by appropriate dilution and plating on E. coli O157:H7 selective Sorbitol MacConkey agar (SMAC) and incubation at 37 °C for 24 h. All the experiments were conducted in triplicate. R

coupons before and after cleaning was measured to determine the wear resistance of the TiO2 coatings.

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Measurement of bactericidal property of TiO2 nanocoatings In order to select most appropriate test method to determine photocatalytic bactericidal activity TiO2 nanocoatings, 3 different techniques: (i) direct spreading, (ii) glass cover-slip, and (iii) indented coupon were investigated. TiO2 coating with binder A on stainless steel coupons (25 mm2 ) were used in direct spreading and glass cover-slip techniques; while indented stainless steel coupons (540 mm2 ) as described earlier were used for the indented coupon method. Further, E. coli has been widely studied bacteria in several of the photocatalytic disinfection experiments involving TiO2 NPs. However, the susceptibility of pathogenic strains of E. coli to photocatalytic disinfection is not well reported. As a reason, a 5 strain cocktail of E. coli O157: H7 isolated from different sources: E009 (beef), EO932 (cattle), O157-1 (beef), O157-4 (human), and O157-5 (human) was used as a test pathogen in this study. Each bacterial stain was cultured separately in 10 mL of tryptic soy broth (Difco, Becton Dickinson, Sparks, Md., U.S.A.) and kept on a shaking incubator at 230 rpm and 37 °C for 16 h. Following the incubation, bacterial cells were harvested by sedimentation at 4000 x g for 12 min and resuspended in a sterile phosphate-buffered saline (PBS, pH 7.2). An equal volume (2 mL) of each strain suspension was combined to obtain a 10 mL of a 5 strain cocktail containing approxiN1906 Journal of Food Science r Vol. 80, Nr. 8, 2015

Statistical analysis Data were analyzed by the analysis of variance (ANOVA) procedure using Statistical Analysis System (SAS/STAT 9.3, 2011). T-tests were used for pairwise comparisons. Least significant difference of means tests was done for multiple comparisons, and all tests were performed with a level of significance 0.05.

Results and Discussion Effect of binders on TiO2 coatings Figure 2 shows the images of different TiO2 coatings with binders A, B, and C prepared by suspension deposition technique. As seen in the figure, all the nanocoatings were uniform and strongly adhered to the stainless steel substrate. We found that increasing the binder concentration in the coating resulted in smoother surfaces with fewer visible aggregates as seen in the image of sample TB16 when compared with sample TB8. Also, no cracks were formed on the TiO2 coating with binders A, B, and C. However, TiO2 coating with binder A at 1:4 NP to binder weight ratio (that is, TA4) showed formation of cracks along the edges of the coating. As the concentration of binder A further increased (TA8), no visible cracks were noticed on the coating. Thickness of TiO2 coatings with binders A, B, and C at different NP to binder weight ratios were shown in Table 3. Thickness of nanocoatings ranged from 50 to 107 µm. In general, increasing the concentration of binder in the coating decreased the thickness of nanocoatings. At the same NP to binder composition (for example at 1:8 NP to binder weight ratio), thickness of TiO2 coating

TiO2 antimicrobial nanocoatings . . . with binder C (97 µm) was found to be significantly higher than the thickness of TiO2 coatings with binders A (74 µm) and B (51 µm). The difference in the thickness of these nanocoatings may be attributed to the differences in the viscosity of coating solutions formulated using different binding agents and the relative proportion of NPs to binder concentration in each type of nanocoating. Li and others (2009) reported that the thickness of TiO2 membranes developed with PVA binder on stainless steel decreased with decreasing the molar concentration of TiO2 in the casting solution. They found that the thickness and micropores of the coatings can be controlled by simply adjusting the concentration of casting solutions instead of applying multiple coats. Similar results were reported by Cerna and others (2011) for TiO2 coated layers with varying levels of PEG. Since photocatalytic antimicrobial activity of TiO2 is a surface-dependent phenomenon due to generation of ROS, the thickness of the coating becomes insignificant. However, thickness of the coating may have a significant effect on the physical stability and other structural properties of the TiO2 coatings.

Figure 2–Images of TiO2 nanocoatings with shellac (A), polyurethane (B), and polycrylic (C) binders at different NP to binder concentrations. Where, TA4 (TiO2 coating with binder A at 1:4 NP to binder weight ratio), TA8, TB8, TC8 (TiO2 coating with binder A, B, and C at 1:8 NP to binder weight ratios), TB16, TC16 (TiO2 coating with binder B and C at 1:16 NP to binder weight ratios).

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Surface characteristics of TiO2 coatings Figure 3 shows scanning electron micrographs of surface of different TiO2 coatings. SEM images give us a detailed look at appearance of the deposited coatings at a micro level. At the same level of NP to binder concentration, TiO2 coatings with binders A, B, and C have shown completely different structural characteristics as seen in Figure 3. TiO2 coating with binder A (TA8) is more compact in nature with aggregated clumps on the surface (Figure 3B). While, the surface of TiO2 coating with binder B (TB8) is compact with several microscopic pores throughout the coating (Figure 3C). Whereas, TiO2 coating with binder C (TC8) resulted in a compact structure without aggregated clumps with larger but fewer number of pores on the surface (Figure 3E). Inset of the respective SEM images of TA8, TB8, and TC8 shows the structural arrangement of the TiO2 coatings at nanoscale. Upon analyzing these SEM micrographs at nanoscale to estimate the surface coverage of NP compared with binder showed a 40%,

21%, and 39% coverage of binder and 60%, 79%, and 61% coverage of TiO2 NPs for coatings TA8, TB8, and TC8, respectively (Figure 3). However, the actual number of TiO2 NPs that are exposed on the surface of the coating were just a fraction of total

Figure 1–In-house fabricated wear resistance tester (A) Cleaning heads fitted to a texture analyzer to perform reciprocating motion. (B) Enlarged image of cleaning heads and platform to fit coated coupons in place. Vol. 80, Nr. 8, 2015 r Journal of Food Science N1907

TiO2 antimicrobial nanocoatings . . . Table 3–Physical stability results of TiO2 nanocoatings with different binders. Coating typee TA4 TA8 TB8 TB16 TC8 TC16

Thickness (µm) 107 74 51 50 97 56

± ± ± ± ± ±

17.34a 11.67c 8.09c 7.04c 2.35ab 8.98c

Hardnessf (GPa) 0.15 0.14 1.08 0.88 0.68 0.55

± ± ± ± ± ±

0.02d 0.11d 0.25a 0.11ab 0.08bc 0.06c

Adhesion ratingg 3B 3B 4B 4B 4B 2B

Wear resistanceh (mg) 5.53 3.47 14.0 5.18 1.67 1.53

± ± ± ± ± ±

0.86b 1.94b 2.03a 2.87b 0.83b 0.29b

Mean values with the same superscript in the same column are not significantly different (P > 0.05). e TiO2 coatings with binders A, B, and C at 1:4 to 1:16 NP to binder weight ratio. f Scratch hardness number at 2 N based on ASTM G171-03 method. g Adhesion rating (5B: Superior; 0B: Inferior) based on ASTM D3359-02 method-B. h Weight loss in milligrams after subjecting to simulated washing procedure.

percent coverage of TiO2 NPs. For example in Figure 4, if we analyze the magnified image of TiO2 coating with binder C (TC8) at nanoscale; about 39% of the coating surface was covered with the binder (dark black region), 58% of the surface was covered by the unexposed TiO2 NPs which are partly shielded by the binder particles (blurred grey region), and rest of the 3% of the surface was covered by the exposed TiO2 NPs (bright white spots). Further increasing the concentrations of binders B and C in the coating (that is, TB16 and TC16) resulted in a more compact surface structure with fewer number of pores as shown in Figure 3(D) and (F). This shows that the type and the amount of binder used in the coating has a significant effect on the morphological and structural properties of the TiO2 coating. This phenomenon is more obvious when readymade TiO2 NPs were mixed with different binding agents for coating. Also, it is expected to generate some irregularities and nonuniformity in the surface of coating while using the solution deposition technique in the indented stainless steel coupon. However, the results of this study help to prove the concept of developing durable antimicrobial nanocoatings on food contact surfaces using appropriate binding agents.

Physical stability of TiO2 coatings Different test procedures were adopted in order to estimate the physical stability of the TiO2 coatings for use in food processing environment. Adhesion strength of the TiO2 coatings was determined by following ASTM D3359-02 standard and the results were reported in Table 3. TiO2 coatings with binder B (TB8 or TB16) showed the highest adhesion rating of 4B. Here, rating 4B indicates that less than 5% of the coating has been removed from the surface as represented in Table 2. On the other hand, TiO2 coating with binder C at 1:16 NP to binder weight ratio (TC16) showed the lowest adhesion strength of 2B which means more than 65% of the coating has been delaminated from the surface. Further, increasing the concentration of NPs in the coating formulation to 1:8 NP to binder weight ratio (TC8) significantly enhanced the adhesion strength of the coating up to 4B. A similar trend was observed for TiO2 coating with binder A (TA8 or TA4). This indicates that depending upon the type of binder, there exists an optimum concentration of NPs and binder in the coating in order to achieve highest adhesion to the substrate. For the tested binding agents in this study, a coating suspension at a

N: Nanoscale Food Science Figure 3–Scanning electron micrographs of the surface of TiO2 coatings with binders A, B, and C at different NP to binder concentrations (Inset: magnified image of a part of coated surface at lower scale). Where(A) is the TiO2 coating with binder A at 1:4 NP to binder weight ratio (TA4), (B), (C), and (E) are TiO2 coating with binder A (TA8), B (TB8), and C (TC8) at 1:8 NP to binder weight ratios, respectively. Figure 3(D) and (F) are TiO2 coating with binder B (TB16), and C (TC16) at 1:16 NP to binder weight ratios, respectively.

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TiO2 antimicrobial nanocoatings . . . except for TB8. In addition, subjective analysis of the coatings after wear testing revealed that all the TiO2 nanocoatings looked physically very stable with slight scratch marks on the surface. However, after the wear test was followed by drying, formation of cracks and peeling-off of the coatings from the substrate was noticed for TB8. This might be attributed to the weak intermolecular bonds between the NPs and the binder at 1:8 NP to binder weight ratio for TB8.

Effect of test method on bactericidal activity results of TiO2 coatings Shellac (binder A) is a food-grade natural resin most commonly used in the food industry for several applications. Also, based on the structural characteristics of the TiO2 coatings with binder A, no significant difference in the total coverage of TiO2 NPs was observed with decreasing NP concentration in the coating (Figure 2). In addition, these coatings exhibited good physical stability on stainless steel surface. For this reason, TiO2 coating with binder A was selected as a representative nanocoating to identify a suitable testing method to determine the bactericidal property. The most widely reported direct spreading and glass cover-slip techniques were compared with an indented coupon technique developed in this study. The results of antimicrobial activity of TiO2 coatings are shown in Table 4. Under tested conditions, control samples with plain stainless steel coupon and only binder coated stainless steel coupon under UV-A light showed a reduction in between 1.5 and 2.5 log CFU/cm2 . No significant difference (P > 0.05) in the reduction among control samples was observed for direct spreading, and indented coupon techniques.

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concentration of 1:8 NP to binder weight ratio was found to be the optimum for exhibiting the highest adhesion strength. The hardness of the coating was determined as per ASTM G171-03 standard using a scratch resistance test. Preliminary experiments were conducted to determine the maximum normal force that can be applied on the surface of TiO2 coating and a normal force of 2 N was found to be the optimum to determine and compare the scratch resistance of different TiO2 coatings developed in this study. Control samples with only binder coating failed to withstand the scratch resistance test. Scratch hardness of TiO2 coatings ranged from 0.14 GPa for sample TA8 to 1.08 GPa for sample TB8 (Table 3). TiO2 coatings with binder B showed highest scratch hardness followed by coatings with binder C and A, respectively. Scratch resistance of the TiO2 coatings developed in this study using different binders were found to be comparatively much higher than the chemical vapor deposited TiO2 coatings on stainless steel substrate which was 6.5 GPa at 40 mN (Sobczyk-Guzenda and others 2013) and sol-gel dip coated TiO2 coatings on polycarbonate sheets which was 0.5 ± 0.04 GPa at 25 µN (Yaghoubi and others 2010). Wear resistance of the TiO2 nanocoatings after simulated cleaning procedure was reported in Table 3. The weight loss (mg) after 1000 cycles of simulated cleaning procedure was expressed as wear resistance. The weight loss of the TiO2 nanocoatings after wear testing ranged from 1.53 (for TC16) to 14 (for TB8) mg. TiO2 coatings with binder C had the highest wear resistance (less weight loss) followed by TiO2 coatings with binders A and B, respectively. In general, increasing the binder concentration in the coating increased the wear resistance. However, the difference is not statistically significant (P < 0.05)

Figure 4–SEM image of TiO2 coating with binder C at 1:8 NP to binder weight ratio (TC8) showing regions of binder, surface exposed TiO2 NPs, and unexposed TiO2 NPs that are partly covered by the binder. Vol. 80, Nr. 8, 2015 r Journal of Food Science N1909

TiO2 antimicrobial nanocoatings . . . Table 4–Bactericidal activity of TiO2 nanocoatings using different test methods. Log reduction (CFU/cm2 ) at 2 mW/cm2 for 2 h Treatment SS controld Binder controle TA16f TA8g

Direct spreading 2.42 ± 0.04b 2.45 ± 0.39b 3.31 ± 0.48a 3.15 ± 0.47a

Glass Cover-slip 1.47 ± 0.27b 2.15 ± 0.32a 2.96 ± 0.64a 2.76 ± 0.60a

Indented coupon 2.19 ± 0.10c 2.31 ± 0.15c 3.04 ± 0.07b 3.57 ± 0.48a

Mean values with the same superscript in the same column are not significantly different (P > 0.05). d Plain stainless steel coupon under UVA. e Only binder A coated coupon under UVA. f TiO2 coating with binder A at 1:16 NP to binder weight ratio. g TiO2 coating with binder A at 1:8 NP to binder weight ratio.

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This shows that the binder coating itself had no significant antimicrobial property and the observed reduction was only be attributed to the effect of UV-A light. However, a significant difference (P ࣘ 0.05) in the reduction between the 2 different control samples was observed in case of glass cover-slip technique. Also, it should be noted that there was no significant difference (P > 0.05) in reduction between binder control and the TiO2 coatings at different NP concentrations for the glass cover-slip technique. For the other 2 methods, TiO2 coatings showed significantly higher microbial reductions when compared with the control samples. This shows that the glass cover-slip technique may not be suitable for the determination of bactericidal property of TiO2 nanocoatings, especially when TiO2 coatings were created using a binder. This can be explained based on 2 possible reasons: (i) A cover slip on the inoculated coupon helps to achieve uniform coverage of bacterial cells on the surface of the nanocoating. However, it also inhibits the presence of catalyst such as atmospheric oxygen which otherwise plays an important role in the heterogeneous photocatalysis involving TiO2 to generate ROS, (ii) The amount of surface occupied NPs were limited when nanocoatings were prepared by mixing with a binding agent as explained in the surface characteristics of nanocoatings in this study (Figure 3). In such a case, a cover-slip on the inoculated nanocoating promotes only localized reactions on the coated surface and reduces the efficacy of photocatalytic bactericidal property of the nanocoating. In addition, leakage of inoculated bacterial culture from the sides of the coupon is difficult to avoid by using a cover-slip technique. Similar, concerns has been expressed by Mills and others (2012) and they suggested using an alternative approach such as a simple well system into which a standard volume of the bacterial suspension is applied to the sample which lies at the bottom of the well. Increasing the concentration of NPs in the TiO2 coating from 1:16 to 1:8 NP to binder weight ratio did not significantly increase in the reduction observed using the direct spreading technique. As per the SEM image analysis results of this study, it is expected to achieve higher log reduction for coatings with more NPs due to more surface coverage by TiO2 NPs. However, this did not happened using the direct spreading technique. This may be due to non-uniform coverage of the inoculum on the entire surface of TiO2 coating when using direct spreading technique. Whereas, the indented coupon technique showed a significant increase (P < 0.05) in the microbial reduction by increasing the concentration of NPs in the coating (TA16 compared with TA8). Even though there was no significant difference in the reduction within the same sample among the 3 tested techniques, the indented coupon technique helped to achieve more consistent results by minimizing variations in the determination of TiO2 antimicrobial property. A similar technique has been used by Cushnie and others (2010). Mills and others (2012) reported that the advantage of this type of N1910 Journal of Food Science r Vol. 80, Nr. 8, 2015

approach is that it allows the bacterial suspension to be accurately deployed to a known area of surface under investigation. As per our observation the major benefits of using the indented coupon technique are: (i) to achieve uniform coverage of inoculated bacterial cells on the entire surface of the coating, (ii) to minimize the sample to sample variation and hence decreases the standard deviation, (iii) to achieve more available surface area, and (iv) to allow the presence of oxygen for efficient photocatalytic disinfection to takes place. Under tested conditions, the results of current study suggest that the indented coupon technique is a more appropriate method to determine bactericidal efficacy of photocatalytic TiO2 coatings.

Conclusions This study investigated the effect of various food processing environmental factors on the physical stability of TiO2 coatings with different binders. This study has identified 3 promising binding agents to develop physically stable TiO2 coatings on food contact surfaces. Image analysis of the coated surfaces revealed that increasing the binder concentration in the coating decreased the exposed TiO2 NPs on the surface which may reduce the bactericidal property of TiO2 coatings. TiO2 coatings with polyurethane as a binder showed the highest scratch resistance followed by coating with polycyclic and shellac, respectively. TiO2 coatings with polyurethane and polycrylic at 1:8 NP to binder weight ratio showed the highest adhesion to the substrate. Overall, TiO2 coating with polycrylic showed the highest physical stability followed by nanocoating with polyurethane and shellac. An indented coupon technique was found to be the most appropriate to test the bactericidal property of TiO2 coatings. Follow-up studies need to be conducted to determine optimum conditions to exhibit the highest bactericidal property by the developed TiO2 coatings under repeated use conditions.

Acknowledgments Funding for this study was provided by Agriculture and Food Research Initiative grant no 2011-68003-30012 from the USDA Natl. Inst. of Food and Agriculture, Food Safety: Food Processing Technologies to Destroy Food-borne Pathogens Program(A4131).

Author contributions Authors V. K. Yemmireddy and Yen-Con Hung designed experiments and wrote the manuscript. V. K. Yemmireddy performed all the experiments and conducted data analysis. Author Glenn D. Farrell provided technical assistance in designing and fabricating instruments for physical stability assessment.

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