CYTA – JOURNAL OF FOOD 2019, VOL. 17, NO. 1, 408–418 https://doi.org/10.1080/19476337.2019.1590461 Bactericidal efficacy of UV activated TiO2 nanoparticles against Gram-positive and Gram-negative bacteria on suspension Carolina Ripolles-Avila , Maria Martinez-Garcia, Anne-Sophie Hascoët and José Juan Rodríguez-Jerez Hygiene and Food Inspection Unit, Department of Food and Animal Sciences, Facultat de Veterinària, Universitat Autònoma de Barcelona, Barcelona, Spain ABSTRACT ARTICLE HISTORY Two different TiO2 nanoparticles, NM101 and NM105, were evaluated against a range of Gram- Received 30 October 2018 positive (Staphylococcus aureus, Bacillus cereus, Lactobacillus casei, Lactobacillus bulgaricus, Accepted 27 February 2019 Lactobacillus acidophilus and Lactobacillus lactis) and Gram-negative (Salmonella enterica var. KEYWORDS Enteridis and Escherichia coli) bacteria. Both NM101 and NM105 TiO2 nanoparticles (UV-exposed TiO2; nanoparticles; or none) had a significant antibacterial activity when the concentration of TiO2 suspension was 100 photocatalytic disinfection; µg mL−1. The activation of the TiO2 NPs led, in all cases, to a shift in the growth curve, revealing UV; food hygiene lower counts as the concentration increased. E. coli was the most significantly affected pathogen by both TiO2 nanoparticles reaching among 2–3 log CFU.mL−1 reduction. In addition, in the case of the PALABRAS CLAVE probiotic bacteria, NM105 TiO2 nanoparticles had similar effects as the bacterial density was TiO2; nanopartículas; desinfección fotocatalítica; reduced by 2–3 log CFU.mL−1. These results may be applied as a potent technology to be included UV; higiene alimentaria in the formulation of new disinfectants. Eficacia bactericida de nanopartículas de TiO2 activadas por luz UV contra bacterias Gram-positivas y Gram-negativas en suspensión RESUMEN Se evaluaron dos nanopartículas diferentes de TiO2, denominadas NM101 y NM105, contra una batería de bacterias Gram-positivas (Staphylococcus aureus, Bacillus cereus, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus acidophilus and Lactobacillus lactis) y Gram-negativas (Salmonella enterica var. Enteridis and Escherichia coli). Ambas nanopartículas de TiO2, NM101 y NM105 (expuestas o no a luz UV) tuvieron una actividad antibacteriana significativa cuando la concentración de suspensión de TiO2 alcanzó los 100 µg mL−1. La activación de las nanopartículas de TiO2 condujo, en todos los casos, a un desplazamiento en la curva de crecimiento microbiano, revelando recuentos más bajos a medida que concentración de TiO2 aumentaba. E. coli fue el patógeno más afectado, alcanzando entre 2-3 log UFC.mL−1 de reducción. Además, en el caso de las bacterias probióticas, las nanopartículas NM105 TiO2 tuvieron efectos similares, ya que la densidad bacteriana se redujo en 2-3 log UFC.mL−1. Estos resultados podrían aplicarse como una potente tecnología a incluir en la formulación de nuevos desinfectantes 1. Introduction this area is nanotechnology. In fact, it has been shown that many nanoparticles (NPs) such as copper oxide, silver and zinc oxide Food safety is an important topic of food industrial devel- possess antimicrobial properties and could be a possible pro- opment (Wang et al., 2016). This is mainly related to food- mising solution (Bondarenko et al., 2013; Duffy, Osmond- borne diseases which have been a widespread health McLeod, Judy, & King, 2018; Sirelkhatim et al., 2015). However, problem in the contemporary world and an important there are some concerns about the residues liberation of these cause of reduced economic productivity (WHO, 2015). nanomaterials to the environment, and how the different organ- Even though disinfection methods commonly employed in isms present in the environment may be affected (Bondarenko the food industry can control microbial pathogens, researchers et al., 2013). With this respect, Kahru and Dubourguier (2010) have shown that there is an increase of bacteria resistant to demonstrated that over seven nanoparticles analyzed (ZnO, Ag, conventional treatments (González-Rivas, Ripolles-Avila, CuO, TiO2, C60-fullerenes, SWCNTs and MWCNTs), TiO2 NPs were Fontecha-Umaña, Ríos-Castillo, & Rodríguez-Jerez, 2018; Zhao, the least environmentally harmful. Furthermore, International Zhao, Wang, & Zhong, 2017). Consequently, there is a necessity Agency for Research on Cancer (IARC) classified TiO2 as to reevaluate traditional methods of disinfection, as well as, to a human carcinogen group 2B although this category is used consider new approaches oriented to improve the reliability and for chemicals for which less than sufficient evidence of carcino- robustness of disinfection whilst eluding the resistance of bac- genicity in experimental animals and also there is limited evi- teria (Li et al., 2008; Özkalelí & Erdem, 2017; Ripolles-Avila, Ríos- dence of carcinogenicity in humans (Jovanović, 2015a). Castillo, & Rodríguez-Jerez, 2018). In this sense, a technology that Furthermore, something to have into consideration is the lack has stimulated a significant interest in finding new possibilities in CONTACT José Juan Rodríguez-Jerez josejuan.rodriguez@uab.cat Hygiene and Food Inspection Unit. Department of Food and Animal Sciences. Facultat de Veterinària, Universitat Autònoma de Barcelona, Travessera dels Turons s/n. Bellaterra, Barcelona 08193, Spain. Supplemental data for this article can be accessed on the publisher’s website. © 2019 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CYTA - JOURNAL OF FOOD 409 of consistent toxicity data of TiO2 NPs due to the diversity of contamination in food, both pathogenic and spoilage microor- materials and test conditions followed by the different research- ganisms, tested on suspension, as a way to reduce the use of ers, which makes a real challenge the fact of conducting risk other disinfectants and contemplate it as an alternative. Two assessment (Jovanović, 2015b). different forms of TiO2 NPs were used in this study, one contain- TiO2 is a semiconductor which upon UV light excitation, it ing purified anatase form and the other containing a mixture of can react with H2O or hydroxide ions adsorbed on the sur- anatase/rutile phase. The ability of all the microorganisms to face to produce hydroxyl radicals (·OH), or reduce O2 to survive photo-inactivation with the UV light was assessed by produce superoxide ions (Banerjee, Muraleedharan, Tyagi, varying the periodicity of irradiation. Furthermore, the turbidity & Raj, 2006). In addition, it has been shown that there is of the two NPs in the different media used in the experiment and a production of reactive oxygen species (ROS), such as the photoactivation of the TiO2 NPs was also evaluated. hydrogen peroxide (H2O2) and singlet oxygen, which are closely related to cell damage, when TiO2 is activated by UV light (Carp, Huisman, & Reller, 2004; Foster, Ditta, 2. Materials and methods Varghese, & Steele, 2011). All of those ROS can cause 2.1. Nanoparticles damage to living cells. Three principal polymorphs of TiO2 exist: anatase, rutile and brookite (Landmann, Rauls, & Two different types of TiO2 nanoparticles named NM101 and Schmidt, 2012). In fact, some studies have revealed that NM105 were employed in this study. The NM101 NPs were anatase form is the most active photocatalyst and, rutile obtained from the nanomaterials repository at the Institute normally less effective (Foster et al., 2011). Nevertheless, for Health and Consumer Protection at the European Miyagi, Kamei, Mitsuhashi, Ishigaki, and Yamazaki (2004) Commission Joint Research (Ispra, Italy) and NM105 NPs have shown that rutile and anatase mixtures have a higher were purchased from Degussa (Frankfurt, Germany). photocatalytic effect than only anatase and also are more Sample NM101 consisted of anatase crystal phase with 7 efficient to generate ROS. ηm size. Besides, sample NM105 was a combination of ana- Matsunaga (1985) reported the effect of photocatalytic inac- tase-rutile phase (80:20 wt/wt) with 21 ηm size. The main tivation of several microorganisms such as E. coli and characteristics of the nanoparticles provided by the indivi- L. acidophilus using nano-TiO2. Since then, research on nano- dual supplier are listed in Table S1. TiO2 photocatalytic effect has been intensively conducted on Suspensions of TiO2 nanoparticles at concentration of 1 mg a wide range of microorganisms to evaluate photocatalytic dis- mL−1 using Nutrient Broth (NB; Oxoid, Hampshire, United infection activities. Furthermore, it would be also worth to eval- Kingdom) and MRS (Oxoid, Hampshire, United Kingdom) were uate the effects of TiO2 embedded in surfaces. As it has been prepared and then vortexed (SLS, Southampton, United proposed by other authors, surfaces made up of light-guiding Kingdom) to mix the preparations in order to have materials, coated with specific semiconductors (i.e. TiO2 NPs) and a homogeneous mixture. Fresh suspensions were prepared stimulated by UV light could provide a new way of disinfection prior to each experiment. (Barthomeuf et al., 2017; Jalvo, Faraldos, Bahamonde, & Rosal, 2017; Yemmireddy & Hung, 2017). In fact, the ability of TiO2 for microbial elimination on photocatalytic self-cleaning/self- 2.2. Bacterial cultures disinfecting surfaces may contribute an additional mechanism Salmonella enterica var. Enteridis (ATCC 13076), Escherichia to the control of disease transmission. Thus, photooxidation coli (ATCC 25922), Staphylococcus aureus (ATCC 29247) and mediated by TiO2 appears to be promising for microbial elimina- Bacillus cereus (ATCC 29247) were purchased as a Culti-Loops tion in areas where the employment of biocides or chemical (Remel Europe, Dartford, United Kingdom). The Culti-Loops cleaning agents is not effective or is restricted by food industry were then streaked onto sterile plate count agar (PCA; regulations (Yemmireddy & Hung, 2015). However, the bacter- Oxoid, Hampshire, United Kingdom) to obtain pure cultures icidal effect of TiO2 NPs seems to be higher when applied in on plates. After streaking, the tips of the Culti-Loops which suspension rather than embedded in surfaces (Kim, Kim, Cho, & contained the microorganisms were separated and depos- Cho, 2003), although comparative effects need to be investi- ited in sterile nutrient broth. Both the inoculated plates and gated. Foster et al. (2011) have shown that a closer contact within broths were incubated at 37 ± 2 ºC during 24 h. After that, the TiO2 and the organism enhances the amount of oxidative the inoculated plates were sealed with Parafilm and kept as damage. On this sense, it has been demonstrated that on water stock cultures at 4 ± 2 ºC for a maximum period of 1 month. suspended TiO2 has a greater effect than TiO2 immobilized on The broth cultures were subcultured into new sterile nutri- surfaces like thin films (Alhaji et al., 2017; Sun, Tay, & Tan, 2003). ent broth every day by transferring 200 µL from the initial This is presumably because of extended contact between the broth culture and incubated at 37 ± 2 ºC for 24 h. microbial cells in suspension and the TiO2 NPs, augmenting the Probiotic strains Lactobacillus casei (ATCC 393), Lactobacillus surface area for ROS production. A number of studies have delbrueckii subsp. bulgaricus (ATCC 11842), Lactobacillus lactis demonstrated the significance of such contact (Caballero, subsp. lactis (ATCC 19435) and Lactobacillus acidophilus (ATCC Whitehead, Allen, & Verran, 2009; Cheng et al., 2009; Horie, 4356) were provided by Nutraceutech, Montreal, Canada. David, Taya, & Tone, 1996). Individual strains were cultivated in MRS liquid medium This study aims to consider the potential of TiO2 for the (Oxoid, Hampshire, United Kingdom) and incubated at 37 ± 2 development of new surface disinfection alternatives to be ºC as static cultures for 48 h. Then, broths were subcultured employed in the food industry. The main purpose was to com- into 20 mL of new sterile MRS liquid medium every 48 h by pare the bactericidal effect of photoactivated and non- transferring 200 µL from the initial broth culture and then were photoactivated TiO2 NPs on a range of bacterial indicators of kept at 37 ± 2 ºC for 48 h more. 410 C. RIPOLLES-AVILA ET AL. 2.3. Inoculum preparation Germany), emitting radiation over 315–400 ηm. The distance within the samples and the lamp was set up to acquire 6.9 Seed cultures of the four pathogens were inoculated into mW cm−2 of UV light incident on the samples. As S. aureus 100 mL nutrient broth and the four probiotic seed cultures was more sensitive to UV light than the rest of microorgan- were inoculated into 20 mL MRS. Cell concentration was isms (see section 3.2), the time of UV light exposure was set adjusted at approximately 107 colony forming units (CFU). up at 6 min, which was the time period that did not com- mL−1 by measuring the absorbance of bacterial suspension promise the growth of the microorganism. Furthermore, UV at 650 ηm using a UV/Vis spectrophotometer (Helios Epsilon, intensities higher than 1 mW cm−2 were always applied, Thermo Scientific, Loughborough, United Kingdom). This cell which has been shown to be sufficient to begin the photo- adjustment was done using the calibration curves obtained catalytic reaction at the established exposure time (Oka by each microorganism. et al., 2008). After the UV exposure, samples were shaken to resuspend the cultures and the optical density was mea- 2.4. Impact of the NPs on the turbidity of the media sured at 650 ηm wavelength on a microplate reader (Spectra MAX M5, Molecular Devices, California, United States). The A turbidity testing of NM101 and NM105 TiO2 was done to microtitre plates were incubated at 37 ± 2 ºC during 24 investigate whether nano-TiO2 would change the turbidity h. After the incubation, samples were shaken again and the of the media through time. A range of 8 different concentra- optical density was measured at 650 ηm wavelength on the tions from 0.78 to 100 µg mL−1 of both NPs were prepared same microplate reader. and measured in nutrient broth and MRS. Figure 1 shows representative results for NM101 TiO2 in NB; which are con- sistent with the results observed for the NM105 TiO2 nano- particle in NB media (supplementary materials). TiO2 showed 2.6. Correlation between optical density (OD) values a different turbidity between concentrations but it did not and CFU.mL−1 change the turbidity of the media over the time. In concor- Serial dilutions of all microorganisms (ranging from 10−1 dance, non-significant differences (P > 0.05) were found until 10−9) were prepared in 200 µL of nutrient broth and between the turbidity achieved by the diverse concentra- MRS broth using sterile microtitre plates and the optical tions of the nanoparticles tested. density was measured at 650 ηm wavelength on a microplate reader. From the dilution 10−3–10−9, 100 µL of aliquots were withdrawn and plated in sterile petri dishes 2.5. Photocatalytic experiments with plate count agar (PCA and MRS, respectively, for patho- The photocatalytic bactericidal tests were performed in ster- gens and probiotic bacteria). The plates were incubated at ile microtitre plates. Twenty microliters of the aqueous sus- 37 ± 2 ºC for 24 h (S. enterica var. Enteridis, E. coli, S. aureus pensions of TiO2 nanoparticles (both NM101 and NM105) and B. cereus) and 48 h (L. casei, L. bulgaricus, L. acidophilus were added to each well in triplicate along with 180 µL of and L. lactis). All microbial counts were reported as CFU.mL−1 bacterial cultures to give final nanoparticle concentration of values. 0.78–100 µg mL−1. In all the tests, four different conditions were examined: (i) presence of TiO2 and UV light, (ii) no TiO2 and presence of UV light, (iii) presence of TiO2 under dark 2.7. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) conditions, (iv) no TiO2 under dark conditions. assay The microtitre plates with bacteria-nanoparticles suspen- sion were located in a noncommercial chamber which was The level of ROS generated by TiO2 NPs was detected by fitted with a UVSPOT 1000 UV lamp (UV Technologies, carboxy-2ʹ,7ʹ-dichloro-dihydro-fluorescein diacetate (DCFH- DA) staining. DCFH-DA is a fluorescent probe that can be deacetylated to form DCFH. DCFH is non-fluorescent unless 0.5 oxidized by ROS to transform DCF. The fluorescence inten- 0.4 sity of DCF can indicate the quantity of ROS produced (Sun et al., 2011). Ten microliters of DCFH-DA probe (Sigma- OD650 0.3 Aldrich, Poole, UK) and 40 µL of sodium NaOH (Sigma- 0.2 Aldrich, Poole, UK) solution were added to the wells of a microtitre plate in triplicate and incubated for 30 min at 0.1 room temperature. After this period, 68 µL of sterile phos- 0 // phate buffered saline (PBS, Sigma-Aldrich, Poole, UK) solu- 0 h 1 h 2 h 3 h 4 h 5 h 6 h 7 h 24 h tion was added to the mixture. In parallel, suspensions of NB 0,78µg/ml 1,56 µg/ml TiO2 NPs (both NM101 and NM105) in nutrient broth and 3,125 µg/ml 6,25 µg/ml 12,5 µg/ml MRS broth were exposed to UV light during 6, 12 and 24 25 µg/ml 50 µg/ml 100 µg/ml min. After the UV exposure, 118 µL of the TiO2 nanoparticle Figure 1. Optical density measurements of different concentrations of NM101 suspension was added to the DCFH-DA+NaOH+PBS mixture TiO2 in nutrient broth over time. Each value corresponds to the mean of three and incubated during 10 min at room temperature. The replicates performed on three separated days (n = 9). The error bars represent investigated range of nanoparticle concentrations was standard deviation. between 0.78 and 100 µg mL−1. After the incubation, fluor- Figura 1. Densidad óptica obtenida a partir de diferentes concentraciones de escence was measured every 1 min until 15 min and then at NM101 TiO2 en caldo nutritivo a lo largo del tiempo. Cada valor corresponde a la media de tres repeticiones realizadas en tres días separados (n = 9). Las 30, 45 and 60 min at 485 ηm (excitation) and 535 ηm barras de error representan la desviación estándar. (emission) wavelengths on a microplate reader. CYTA - JOURNAL OF FOOD 411 2.8. Statistical analysis decrease (P< 0.01 and P < 0.001) in the OD650 at concen- trations of 12.5 µg mL−1 when compared to the control. All experiments were performed three times on three differ- Using the standard curves obtained from the relationship ent days (n = 9). Data from all experiments were analyzed between absorbance and CFU.mL−1, an estimated 2 log using the Minitab statistical package using ANOVA general CFU.mL−1 reduction of the initial concentration of E. coli linear model with subsequent analysis of variance and was obtained when NM101 and NM105 TiO2 was activated Tukey’s test. A P value of <0.05 was considered to be sig- by 6 min UV light exposure. In contrast, when both TiO2 nificant. The global differences were examined followed by NPs were not UV activated (TiO2+ UV-), E. coli was 2.3–3 an analysis of the individual data sets from the two TiO2 log CFU.mL−1 reduced, respectively, for NM101 and NM105. nanoparticles. In the case of S. aureus and B. cereus, a significant decrease in the OD650 value at concentrations of 50 and 100 3. Results µg mL−1 of non-photoactivated (P< 0.001) and photoacti- vated (P< 0.05) NM101 TiO2 was observed when compared 3.1. Effect of UV light over microorganisms with the control. However, when S. aureus and B. cereus A preliminary study was conducted to select the time of were exposed to NM105 TiO2 showed a significant (P< 0.01 exposure of the samples (bacteria + NPs) and to confirm for S. aureus and P< 0.05 for B. cereus) decrease in the that the time of UV exposure would not have impact on the OD650 value just at the highest concentration (i.e. 100 growth of the bacteria. Suspensions of S. enterica var. µg mL−1). Again, estimating the reduction in terms of Enteridis, E. coli, B. cereus and S. aureus in nutrient broth CFU.mL−1 using the standard curves for each microorgan- and suspensions of L. casei, L. bulgaricus, L. acidophilus and ism, in the presence of TiO2 and dark conditions, NM101 at L. lactis in MRS broth were exposed to 3, 6, 12, 24, 36 and 48 100 µg mL−1 reduced 2 log CFU.mL−1 the initial microbial min of UV light with an intensity of 6.9 mW cm−2 incident on load of S. aureus and B. cereus, and NM105 reduced the the samples. The microorganism that showed to be more load of them by 2.69 log CFU.mL−1. With all these results, sensitive to UV light was S. aureus, increasing its sensitivity growth curves of the different pathogens (including Gram- when the time of exposure was raised (Figure 2). Only an positive and Gram-negative) have been observed to be exposure period of 3 and 6 min did not have an impact on significantly affected in a directly proportional way as the growth of S. aureus. Because of that, 6 min of UV applied TiO2 concentration increased. exposure was selected in order to maximize the potential From the data of the impact of photoactivated and non- for photoactivation of nano-TiO2. The microorganism less photoactivated TiO2 NPs on probiotic bacteria, it was evident affected on its growth curve by UV light was S. enterica that there was a dose-dependent decrease in the OD650 var. Enteridis (supplementary materials). In addition, it is values after 48 h of incubation (Figure 5). There was no also important to highlight that UV light did not have any significant difference between photoactivated and non- impact on the turbidity of the culture broths employed in photoactivated TiO2, showing in all cases, the same effects the study (i.e. NB and MRS). As can be observed in Figure 2, on probiotic bacteria (P> 0.05). Furthermore, the OD650 the increase of the exposure time to UV let to no change in values of the probiotic cultures showed no significant the obtained OD650 values on NB and MRS broths. changes during the tests under 0.79 to 25 µg mL−1 NM101 and NM105 TiO2 (P > 0.05). However, using the standard curves to obtain an estimated result of CFU.mL−1, it was 3.2. Photoactivation test with a fluorescent probe apparent that NM105 TiO2 UV activated and non-activated (i.e. dark conditions) induced a very significant 3 log CFU. Results showed that DCF fluorescence intensity slightly mL−1 reduction in the cases of L. casei, L. bulgaricus and increased through the time, increasing in parallel the ROS L. lactis, and 2 log reduction in the case of L. acidophillus. level with the increase of the concentration of TiO2 NPs from Significant, but less pronounced, decreases on the OD650 0.78 to 100 µg mL−1 although non-significantly (P > 0.05) values were also detected in the presence of photoactivated (Figure 3). Moreover, no significant differences (P > 0.05) and non-photoactivated NM101 TiO2 (around 1 log CFU. were found between the ROS level produced after exposure mL−1 reduction in all the cases at the highest concentration). of the UV samples with respect to those subjected to dark NM101 and NM105 TiO2 had a significant difference on conditions, suggesting that there is no need to expose the decreasing the number of probiotic cultures between samples with UV so that the TiO2 NPs could play an anti- them, being NM105 TiO2 the one that had the strongest microbial role. effect (P< 0.01). 3.3. Effect of photocatalytic TiOtio2 NPs on different 4. Discussion microorganisms This study was aimed to evaluate the antibacterial effect of The inhibitory effects of both TiO2 NPs, NM101 and NM105, photoactivated and non-photoactivated TiO2 nanoparticles on pathogenic bacteria is shown in Figure 4. All the patho- on a range of Gram-negative (S. enterica var. Enteridis and gens in nutrient broth decreased their growth in a dose- E. coli) and Gram-positive (S. aureus, B. cerus, L. casei, dependent manner following exposure to NM101 and L. bulgaricus, L. acidophilus and L. lactis) bacteria. NM105 TiO2. The bacterial culture of S. enterica var. Preliminary tests for the correlation between OD650 values Enteridis showed a significant decrease (P< 0.01) in the and CFU.mL−1 (data not shown) were performed in nutrient OD650 value at concentrations of 50 and 100 µg mL−1 of broth and MRS, the growth media used for the cultivation of NM101 and NM105 TiO2, compared to the control suspen- the pathogens and probiotic bacteria, respectively. This was sion. The pathogen E.coli showed a significant growth performed to set up a correlation curve as a basis to 412 C. RIPOLLES-AVILA ET AL. a b 0.5 0.5 0.4 0.4 0.3 OD650 0.3 OD650 0.2 0.2 0.1 0.1 0 // 0 // 0h 1h 2h 3h 4h 5h 6h 7h 24 h 0h 1h 2h 3h 4h 5h 6h 7h 24 h Time of growth Time of growth Nutrient broth UV 3 min Control (without uv) Nutrient broth UV 6 min Control (without uv) c d 0.5 0.5 0.4 0.4 0.3 0.3 OD650 OD650 0.2 0.2 0.1 0.1 0 // 0 // 0h 1h 2h 3h 4h 5h 6h 7h 24 h 0h 1h 2h 3h 4h 5h 6h 7h 24 h Time of growth Time of growth Nutrient broth UV 12 min Control (without uv) Nutrient broth UV 24 min Control (without uv) e f 0.5 0.5 0.4 0.4 0.3 OD650 0.3 OD650 0.2 0.2 0.1 0.1 0 // 0 // 0h 1h 2h 3h 4h 5h 6h 7h 24 h 0h 1h 2h 3h 4h 5h 6h 7h 24 h Time of growth Time of growth Nutrient broth UV 36 min Control (without uv) Nutrient broth UV 48 min Control (without uv) Figure 2. Effect of UV light at different exposure times on the growth curve of S. aureus. The graphs represent: (a) 3 min; (b) 6 min; (c) 12 min; (d) 24 min; (e) 36 min; and (f) 48 min of UV exposure. An every hour measurement was taken until 7 h of incubation and after that, another reading at 24 h of incubation was measured. Each value corresponds to the mean of 3 replicates performed on three separated days (n = 9). The error bars represent standard deviation. Figura 2. Efecto de la luz UV, a distintos tiempos de exposición, sobre la curva de crecimiento de S. aureus. Los gráficos representan: (A) 3 minutos; (B) 6 minutos; (C) 12 minutos; (D) 24 minutos; (E) 36 minutos; y (F) 48 minutos de exposición a luz UV. Se tomó una medición cada hora hasta las 7 horas de incubación y, posteriormente, se realizó otra lectura a las 24 horas de incubación. Cada valor corresponde a la media de 3 repeticiones realizadas en tres días separados (n = 9). Las barras de error representan la desviación estándar. compare the OD values from the experiments with estimates increased, the growth curve of S. aureus was affected. UV of the numbers of bacteria that were represented by these light can cause DNA damage (Hijnen, Beerendonk, & OD values, as other studies did (Kim, 2012; Ripolles-Avila, Medema, 2006). So the use of UV irradiation alone can Ríos-Castillo, Guerrero-Navarro, & Rodríguez-Jerez, 2018; have an impact on the growth of the bacteria but is incap- Yemmireddy & Hung, 2015). Similarly, we described a high able of bacterial disinfection; only strong UV irradiation (UV- correlation was observed between OD650 values and CFU. B and UV-C) can have a disinfecting effect on bacteria (Pham mL−1 for the different bacteria employed for the study. & Lee, 2015; Yu, Lee, Lin, & Huang, 2008). The UV irradiation Investigation of the effect of UV light on the different employed in the study was between the range 315–400 nm, microorganisms used in this study was performed in which has also been used by other authors (Adhikari, Thind, the second instance to identify whether the exposure time Chen, Schraft, & Chen, 2018). The microorganism that we of UV light on the bacteria had an impact on their growth. observed that had greater resistance to UV light was By establishing a UV light exposure time for the activation of S. enterica var. Enteridis, which can be due to the range of the NPs that did not affect the growth of none of the UV light used was considered to be UV-A, and as it is not bacteria, the bactericidal effects were able to be attributed strong it has not had a bactericidal effect against this patho- to the effects of TiO2 photocatalytic activation. Under the gen. Nevertheless, the TiO2 NPs are photoactive and produce tested conditions, S. aureus was shown to be the most reactive oxygen species under natural sunlight (UV-A and sensitive bacteria. When the time of UV light exposure UV-B) (Jovanović, 2015b). CYTA - JOURNAL OF FOOD 413 NM101 2500 Fluorescence intensity 2000 1500 1000 500 0 5 min 15 min 30 min 60 min TiO2 Concentration (µg mL-1) NM105 2500 Fluorescence intensity 2000 1500 1000 500 0 5 min 15 min 30 min 60 min TiO2 Concentration (µg mL-1) Figure 3. DCF fluorescence intensity produced by TiO2 NPs at different concentrations ranging from 0.78 to 100 µg mL−1. The UV exposure time was 6 min, but the results are representative for 12 and 24 min of exposure. The control sample did not contain TiO2. Each value corresponds to the mean of 3 replicates performed on three separated days (n = 9). The error bars represent standard deviation. Figura 3. Intensidad de fluorescencia de DCF producida por nanopartículas de TiO2 a diferentes concentraciones que varían de 0.78 a 100 µg mL−1. El tiempo de exposición a luz UV fue de 6 minutos, pero los resultados son representativos de 12 y 24 minutos de exposición. La muestra de control no contenía TiO2. Cada valor corresponde a la media de 3 repeticiones realizadas en tres días separados (n = 9). Las barras de error representan la desviación estándar. The ability of nano-TiO2 to be photoactivated in the in the assay might be associated to NPs-culture media bacterial cultures was investigated using a DCFH-DA fluor- interactions rather than ROS production. For example, the escent emission assay. This fluorometric probe is primarily enhancement of fluorescence emission could be caused utilized for oxidative stress measurements. Our assay was due to agglomerate formation of the particles in the cell based on the ability of the photoactivated TiO2 NPs to culture medium. Some studies have had similar problems create ROS on the surface of the NPs. Aranda et al. (Aranda et al., 2013; Qi et al., 2012; Sabatini, Pereira, & (2013) and Griffiths et al. (2011) have already demon- Gehlen, 2007). Consequently, this photoactivation test strated the interference of different types of NPs with using DCFH-DA as a fluorometric probe did not provide DCFH-DA fluorescence emission. A modification of the any evidence of TiO2 being photoactivated. Although DCFH-DA test from the original protocol was done in some studies have not used any test to prove photoactiva- order to adapt the assay to the experimental conditions, tion of TiO2 (e.g. Long et al., 2014; Yemmireddy & Hung, for example when the NPs were exposed to UV light the 2015), some others have shown that a degradation test on media used in the assay was culture medium rather than an organic dye, rhodamine B, was very efficient to show if water or PBS. Although increasing the period of UV light the NP was being photoactivated by UV light (e.g. Koizumi exposure increased the fluorescence emission observed in & Taya, 2002; Li et al., 2008; Polo et al., 2011). the assay, non-significant enhancement with increasing TiO2 NPs are a highly effective disinfectant once they are concentrations of either TiO2 NPs was observed. activated by UV light (Kermanizadeh et al., 2013; Kim et al., Therefore, as the fluorescence of the control was not 2003). In water and air systems, TiO2 absorbs photons, result- different to the fluorescence produced by the different ing in the formation of active hydroxyl (·OH) and other ROS concentration of NPs, the increased fluorescence observed (O2−, H2O2, among others) in the presence of O2 and H2 414 C. RIPOLLES-AVILA ET AL. NM 101 on S. enterica var. Enteridis NM 105 on S. enterica var. Enteridis 0.6 0.6 0.5 0.5 0.4 0.4 ** OD650 OD650 0.3 ** 0.3 ** ** 0.2 0.2 ** 0.1 ** 0.1 ** 0 ** 0 0.78 1.56 3.125 6.25 12.5 25 50 100 0.78 1.56 3.125 6.25 12.5 25 50 100 Concentration (µg mL -1) Concentration (µg mL -1) UV Dark Salmonella UV Dark Salmonella NM 101 on E. coli NM 105 on E. coli 0.6 0.6 0.5 0.5 ** *** *** 0.4 *** 0.4 *** *** *** OD650 ** *** OD650 *** 0.3 *** 0.3 *** *** *** 0.2 0.2 0.1 *** 0.1 *** 0 0 0.78 1.56 3.125 6.25 12.5 25 50 100 0.78 1.56 3.125 6.25 12.5 25 50 100 Concentration (µg mL -1) Concentration (µg mL -1) UV Dark E. coli UV Dark E. coli NM 101 on S. aureus NM 105 on S. aureus 0.6 0.6 0.5 0.5 0.4 OD650 0.4 * OD650 0.3 * 0.3 *** 0.2 0.2 *** 0.1 0.1 ** 0 0 0.78 1.56 3.125 6.25 12.5 25 50 100 0.78 1.56 3.125 6.25 12.5 25 50 100 Concentration (µg mL -1) Concentration (µg mL -1) UV Dark S. aureus UV Dark S. aureus NM 101 on B. cereus NM 105 on B. cereus 0.6 0.6 0.5 0.5 0.4 * 0.4 * OD650 OD650 0.3 0.3 0.2 *** 0.2 0.1 * 0.1 *** 0 0 0.78 1.56 3.125 6.25 12.5 25 50 100 0.78 1.56 3.125 6.25 12.5 25 50 100 Concentration (µg mL -1) Concentration (µg mL -1) UV Dark BB. cereus cerus UV Dark BB. cereus cerus Figure 4. OD650 values obtained in the tests of S. enterica var. Enteridis, E. coli, S. aureus and B. cereus with photocatalytic TiO2 NPs in nutrient broth. In the graphs, y-axis shows the OD650 values for each test condition and x-axis shows the different concentrations ranging from 0.78 to 100 µg mL−1 of TiO2 tested (NM101 and NM105). Samples were subjected to UV activation (UV) or non-UV activation (Dark). Each value corresponds to the mean of three replicates (n = 9). The error bars represent standard error of the mean. The graphs provide the P-values obtained by ANOVA analysis. The statistical analysis was performed separately for each experimental condition. Significance indicated by: * = P < 0.05, ** = P < 0.01, and *** = P < 0.001. Figura 4. Valores OD650 obtenidos para S. enterica var. Enteridis, E. coli, S. aureus y B. cereus después de aplicar un tratamiento con nanopartículas de TiO2 en caldo nutritivo. En los gráficos, el eje y muestra los valores de OD650 para cada condición y el eje x muestra las diferentes concentraciones que van desde 0.78 a 100 µg mL−1 de TiO2 (NM101 y NM105). Las muestras se sometieron a activación por UV (UV) o no se activaron (Oscuridad). Cada valor corresponde a la media de tres repeticiones realizadas en tres días separados (n = 9). Las barras de error representan el error estándar de la media. Los gráficos proporcionan los valores de P obtenidos por análisis ANOVA. El análisis estadístico se realizó por separado para cada condición experimental. Significancia indicada por: * = P < 0.05, ** = P < 0.01, y *** = P < 0.001. O. Those ROS produced, particularly H2O2, cannot only trans- under UV light (Dalrymple, Stefanakos, Trotz, & Goswami, verse the cell membranes, but also produce oxidative active 2010; Demidova & Hamblin, 2005; Kubacka, Ferrer, & hydroxyl through a reaction inside bacterial cells that finally Fernández-García, 2012). The damaged cell wall allows TiO2 leads to the disinfection (Fujishima, Rao, & Tryk, 2000; NPs to enter into the cells and releases the cell contents Kikuchi, Sunada, Iyoda, Hashimoto, & Fujishima, 1997). from the cells, resulting in cell death (Long et al., 2014). In There is overwhelming evidence that the mechanism of kill- this sense, we described TiO2 NPs significantly reduced the ing bacteria is due to membrane and cell wall damage populations of all tested Gram-positive and Gram-negative (Foster et al., 2011). Some studies have demonstrated that bacteria in the suspension solutions, being more effective once TiO2 NPs are mixed with the bacterial cultures, TiO2 is NM105 than NM101. Although TiO2 demonstrated antibac- attached to the bacterial cells and damages the cell wall terial activity for each of the evaluated bacterial strains in CYTA - JOURNAL OF FOOD 415 NM 101 on L. casei NM 105 on L. casei 1.2 1.2 1 1 * 0.8 0.8 *** *** OD650 0.6 0.6 *** OD650 0.4 0.4 *** 0.2 0.2 *** 0 0 0.78 1.56 3.125 6.25 12.5 25 50 100 0.78 1.56 3.125 6.25 12.5 25 50 100 Concentration (µg mL-1) Concentration (µg mL-1) UV Dark Lb casei UV Dark Lb casei NM 101 on L. bulgaricus NM 105 on L. bulgaricus 1.2 1.2 1 1 *** 0.8 0.8 *** *** OD650 OD650 0.6 0.6 *** 0.4 *** 0.4 0.2 0.2 *** 0 0 0.78 1.56 3.125 6.25 12.5 25 50 100 0.78 1.56 3.125 6.25 12.5 25 50 100 Concentration (µg mL-1) Concentration (µg mL-1) UV Dark Lb bulgaricus UV Dark Lb bulgaricus NM 101 on L. lactis NM 105 on L. lactis 1.2 1.2 1 1 0.8 0.8 OD650 0.6 OD650 0.6 * 0.4 *** 0.4 *** * *** 0.2 0.2 *** *** 0 0 0.78 1.56 3.125 6.25 12.5 25 50 100 0.78 1.56 3.125 6.25 12.5 25 50 100 -0.2 *** Concentration (µg mL-1) Concentration (µg mL-1) UV Dark Lactobacillus lactis UV Dark Lactobacillus lactis NM 101 on L. acidophillus NM 105 on L. acidophillus 1.2 1.2 1 1 0.8 *** 0.8 OD650 *** OD650 0.6 *** 0.6 0.4 0.4 *** *** 0.2 0.2 *** 0 0 0.78 1.56 3.125 6.25 12.5 25 50 100 0.78 1.56 3.125 6.25 12.5 25 50 100 Concentration (µg mL-1) Concentration (µg mL-1) UV Dark Lb acidophillus UV Dark Lb acidophillus Figure 5. OD values obtained in the tests of L. casei, L. bulgaricus, L. lactis and L. acidophillus with photocatalytic TiO2 NPs in MRS. In the graphs, y-axis shows the OD650 values for each test condition and x-axis shows the different concentrations ranging from 0.78 to 100 µg mL−1 of TiO2 tested (NM101 and NM105). Samples were subjected to UV activation (UV) or non-UV activation (Dark). Each value corresponds to the mean of three replicates (n = 9). The error bars represent standard error of the mean. The graphs provide the P-values obtained by ANOVA analysis. The statistical analysis was performed separately for each experimental condition. Significance indicated by: * = P < 0.05, ** = P< 0.01, and *** = P < 0.001. Figura 5. Valores OD650 obtenidos para L. casei, L. bulgaricus, L. lactis and L. acidophillus después de aplicar un tratamiento con nanopartículas de TiO2 en caldo MRS. En los gráficos, el eje y muestra los valores de OD650 para cada condición y el eje x muestra las diferentes concentraciones que van desde 0.78 a 100 µg mL−1 de TiO2 (NM101 y NM105). Las muestras se sometieron a activación por UV (UV) o no se activaron (Oscuridad). Cada valor corresponde a la media de tres repeticiones realizadas en tres días separados (n = 9). Las barras de error representan el error estándar de la media. Los gráficos proporcionan los valores de P obtenidos por análisis ANOVA. El análisis estadístico se realizó por separado para cada condición experimental. Significancia indicada por: * = P < 0.05, ** = P < 0.01, y *** = P < 0.001. a dose-dependent way, there were no significant differences parahaemolyticus, Actinomyces viscosus, L. acidophilus, (P > 0.05) between the activity of the UV-exposed and non- Streptococcus spp., and coliform bacteria were killed by UV-exposed TiO2. After 24 h of incubation, the biocidal effect photoactivated TiO2 NPs. of TiO2 NPs was evident in the microorganisms tested. These However, according to our results, UV-exposed and non- results are in agreement with studies reported by Kim et al. UV-exposed TiO2 did not show significant differences in their (2003), Rincón and Pulgarin (2003), Rizzo (2009) and Swetha, bactericidal activity. As an inhibition of the bacteria was Santhosh, and Geetha Balakrishna (2010), where, in similar produced with the presence of TiO2 NPs in dark conditions, conditions, S. aureus, E. coli, Salmonella spp.,Clostridium per- this suggests that the NPs were effective and showing fringens spores, Pseudomonas stutzeri, Pseudomonas aerugi- a dose-dependent response even when TiO2 was not nosa, Listeria monocytogenes, Serratia marcescens, Vibrio exposed to UV-A light. The decrease in CFU.mL−1 associated 416 C. RIPOLLES-AVILA ET AL. with the non-photoactivated TiO2 has been shown in other analogous survival ratios when testing different bacteria studies (Lalucat, Bennasar, Bosch, García-Valdés, & Palleroni, (E. coli, S. aureus and P. aeruginosa) with a TiO2 coated implant 2006; Polo et al., 2011; Rincón & Pulgarin, 2004) where they (Tsuang et al., 2008). suggested that the reduction of the bacterial cells can be Regarding the impact of these NPs to human health, it is put down to a fall in pH as a result of the addition of the considered that toxicity and exposure data on TiO2 NPs is photocatalyst. This fall in pH would be associated to both not well defined until now in emerging nanomaterials, conditions (UV activated TiO2 and non-UV activated TiO2) which makes the risk assessment more challenging (Tsang but with the difference that UV-exposed TiO2 would have et al., 2017). However, TiO2 is a food additive approved by both inhibitory effects, the reduction of the pH and the different agencies such as the FDA and can be used in foods photocatalytic activity. Nevertheless, this finding cannot be up to a concentration of 1% (Food and Drug Agency [FDA], justified by the drop in the pH because of UV-exposed and 2015). In this case, the development of integrative technol- non-UV-exposed TiO2 did not show significant differences in ogies to respond to the food industry needs for the cleaning their bactericidal activity. A possible explanation might be and disinfection protocols would include concentrations of that TiO2 nanoparticles are sensitive to photo-oxidation and these NPs lower than this established percentage for food when oxidized they lose their photocatalytic activity (Hu, matrices. Song, Jiang, & Wei, 2015; Lin et al., 2014). The fact that the Considering the results obtained, the potential field of TiO2 NPs had been in use during some time suggests that application of TiO2 NPs can be related to the elimination of light could have damaged the NPs. However, the results pathogenic bacteria that cause food poisoning or bacteria obtained demonstrated that although NPs were slightly acti- that cause food spoilage such as Lactobacilli spp. in meat vated by UV light, they presented an important role as and brewing industries. For example, Matsunaga (1985) antimicrobial for both Gram-positive and Gram-negative demonstrate that Lactobacillus spp. was very sensitive to a Pt- bacteria used in the study, as growth curves were compro- doped TiO2 catalyst and this could be exploited as an alter- mised revealing a bacteriostatic effect, which is something native to eliminate these microorganisms. In this time, Joost interesting to continue investigating. In posterior studies, it et al., (2015) suggested that nanosized TiO2 based novel sur- would be interesting to include microbial viability tests to face coating materials might be very effective for the elimina- observe the real impact of TiO2 NPs as potential disinfec- tion of pathogenic bacteria such as E. coli. In addition, (Long tants. On this sense, the applications of international stan- et al., 2014) suggested that TiO2 NPs may be used as an dards such as UNE-EN 1040 or UNE-EN 1276 could be effective antimicrobial treatment to improve food safety of relevant. Furthermore, it is also interesting to evaluate the meat products. Recently, UV-TiO2 photocatalysis has been effectivity of these nanoparticles once they are embedded studied as an alternative for disinfection of fruits with on surfaces in order to know the impact on biofilms, employ- a delicate texture, demonstrating highly effectivity against ing in vitro models to form mature structures and have E. coli K12 on blueberry fruit surface (Lee et al., 2018). results similar to real conditions (Ripolles-Avila, Hascoët, Guerrero-Navarro, & Rodríguez-Jerez, 2018). Additionally, NM105 TiO2 was more effective than NM101TiO2. Similar findings have been reported by Miyagi 5. Conclusions et al. (2004) who stated that anatase and rutile mixtures All together, our results obtained in this study indicate that have a greater photocatalytic effect than 100% anatase. both UV-exposed and non-UV-exposed TiO2 NPs (both The increased activity of NM105 TiO2 is commonly asso- NM101 and NM105) show, in a dose-dependent manner, an ciated to interactions between the two forms, reducing the antibacterial activity against a range of suspended Gram- recombination between electrons and holes (Foster et al., negative (S. enterica var. Enteridis, E. coli) and Gram-positive 2011). In this regard, Yemmireddy and Hung (2015) reported bacteria (S. aureus, B. cereus, L. casei, L. bulgaricus, L. that among the three TiO2 tested (NM105 and two purified acidophilusandL. lactis) as growth curves are compromised. anatase crystal phase with different size measurements), The data demonstrate that the optimal content of TiO2 NPs in NM105 was found to be the most efficient photocatalyst. a suspension for the disinfection of the bacterial cultures was Furthermore, no significant differences were observed 100 µg mL−1 which was able to reduce an estimated amount between Gram-negative (Salmonella spp. and E. coli) and Gram- of 2–3 log the bacterial population after a 24 h incubation. We positive (S. aureus, B. cereus and the probiotic bacteria) in their suggest that further research into the comparative effects resistance to nano-TiO2 treatment. Some studies have sug- between TiO2 embedded in surfaces and suspended TiO2 gested that Gram-negative bacteria are more sensitive to need investigating. Additionally, the toxicological influence of photocatalytic disinfection than Gram-positive bacteria TiO2 NPs should be evaluated to determine the consequences (Backhaus, Marugán, Van Grieken, & Sordo, 2010; B. Kim et al., of using these NPs in food safety. On the foundation of the 2003; Pal, Pehkonen, Yu, & Ray, 2007). This difference on sensi- results acquired and the literature survey, it can be concluded tivity is normally associated to the differences in the cell surface that the technology has the potential to provide, particularly structure such as cell wall and cell membrane, between Gram- in view of the development of visible light-activated catalysts, negative and Gram-positive bacteria. However, several a forceful weapon to combat both, the transmission of food- researchers have also stated that they found no difference borne diseases and the spoilage of food originated in, for within the bactericidal effect of photoactivated TiO2 on Gram- example, meat and brewing industries by Lactobacillus spp. negative and Gram-positive bacteria. For example, Wong et al. (2006) showed that the sensitivity of some human pathogens (S. aureus, V. parahaemolyticus and L. monocytogenes) to TiO2 was not affected whether the target was Gram-negative or Disclosure statement Gram-positive bacteria. Another investigation reported No potential conflict of interest was reported by the authors. CYTA - JOURNAL OF FOOD 417 ORCID fluorometric and colorimetric dyes. Analytical Chemistry, 83(10), 3778–3785. Carolina Ripolles-Avila http://orcid.org/0000-0003-0032-8893 Hijnen, W. A. M., Beerendonk, E. F., & Medema, G. J. (2006). 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