Food Microbiology 49 (2015) 74e81

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Inactivation and potential reactivation of pathogenic Escherichia coli O157:H7 in bovine milk exposed to three monochromatic ultraviolet UVC lights Fugui Yin a, b, Yan Zhu b, Tatiana Koutchma b, *, Joshua Gong b a b

Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, 410125, China Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, N1G 5C9, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 October 2014 Received in revised form 6 January 2015 Accepted 31 January 2015 Available online 7 February 2015

The ultraviolet (UVC) light irradiation has been recently studied as a novel non-thermal treatment method for milk. However, the potential reactivation of microorganisms following exposure to UVC light in milk medium was not studied yet. Therefore, the present study was conducted to determine the inactivation and reactivation of pathogenic Escherichia coli O157:H7 following exposure to UV light at three monochromatic wavelengths (222, 254 and 282 nm) in bovine milk. The results showed that inactivation of E. coli O157:H7 following exposure to the UV light at 254 nm was higher (P < 0.05) than that following exposure at 222 and 282 nm at the same UV fluence of 5, 10 and 20 mJ/cm2. The reactivation of E. coli O157:H7 was increased as the incubation time and temperature increased regardless of the UV light sources under dark incubation phases. The evaluated reactivation ratios of E. coli O157:H7 following exposure to the UV light at 254 nm in milk were lower (P < 0.05) than that following exposure at 222 nm after 1 to 6, 2 to 5 and 5e6 h incubation at 4, 20 and 37  C, respectively. Furthermore, at most incubation time points, the reactivation ratio of E. coli O157:H7 following exposure to these three UV light sources were lower (P < 0.05) than that of non-UV treated cells regardless of the incubation temperature. The lowest reactivation ratios of E. coli O157:H7 were observed after milk exposure to the UV light at 254 nm at 4  C incubation when compared to that following exposure to the UV light at 222 and 282 nm. © 2015 Published by Elsevier Ltd.

Keywords: UVC light Inactivation Reactivation Escherichia coli O157:H7 Milk

1. Introduction Thermal pasteurization process has long been employed to provide safety of milk and other dairy products (Grant et al., 1996). However, heat treatment may cause substantial changes in flavor and nutritional composition, particularly induce enzyme inactivation, lipid oxidation and protein denaturation in food products including milk (Choudhary and Bandla, 2012). In this regard, there is a growing interest to develop non-thermal alternative strategies for inactivation of the pathogenic bacteria in the milk (Krishnamurthy et al., 2007; Bandla et al., 2012; Christen et al., 2013a). Ultraviolet (UV) irradiation is considered a promising nonthermal method, as it is lethal to most types of microorganisms

* Corresponding author. Tel.: þ1 226 217 8123; fax: þ1 226 217 8181. E-mail address: [email protected] (T. Koutchma). http://dx.doi.org/10.1016/j.fm.2015.01.014 0740-0020/© 2015 Published by Elsevier Ltd.

(Bintsis et al., 2000; Koutchma, 2009). In addition, UV is energyefficient and cost-effective in comparison with other disinfection methods (Guerrero-Beltr and Barbosa-C, 2004). The effectiveness of UV light in the biological inactivation primarily due to the fact that UV light irradiation could induce physical shifting of electrons and breaking bonds in deoxyribonucleic acid (DNA) as well as DNA pyrimidine bases mis-pairing in most microorganisms with greater effect at the wavelengths between 250 and 260 nm (Lopez-Malo and Palou, 2005; Oteiza et al., 2005). UV light can also induce damage of proteins in cell membrane (Suzuki, 1985; Eischeid and Linden, 2011; Schenk et al., 2011) and affect the metabolic n and Mackey, processes critical to survival of microorganisms (Paga 2000). The US Food and Drug Administration (FDA) regulations have approved the use of UV-light for fresh juices and it has been successfully commercialized in food industry (US FDA, 2000). Additionally, increasing research has also been conducted to evaluate the potential of UV light as a non-thermal alternative to thermal pasteurization for dairy products (Matak et al., 2005;

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Reinemann et al., 2006; Krishnamurthy et al., 2007; Bandla et al., 2012; Christen et al., 2013a). Particularly, recent research demonstrated that the UV light at the wavelength of 253.7 nm is capable of reducing vegetative bacteria in human milk with no change of main nutritional components and immune response stimulating-related active ingredients in comparison to pasteurization method (Christen et al., 2013a, b), therefore shed a light on the promising prospect of UV light irradiation on pasteurization of dairy products. However, since bacteria and other microorganisms have been inevitably exposed to the UV irradiation from the sunlight in their long evolutionary history, the UV defensive systems has evolved and therefore enabled the UV-inactivated microorganisms to reverse the UV-induced damages and regain activity (Kalisvaart, 2004; Quek and Hu, 2008). Generally, there are two ways for bacteria reversing the UV light-induced damage on DNA, namely photo-reactivation (Hu and Quek, 2008; Guo et al., 2009) and nucleotide excision reactivation (dark reactivation) (Thoma, 1999; Salcedo et al., 2007). Photo-reactivation is light dependent, which requires specific wavelengths of light ranging from 300 to 500 nm to complete the reactivation (Salcedo et al., 2007), while dark reactivation is a multistep and light-independent process, which includes two different repair mechanisms, including base excision repair and nucleotide excision repair (Sinha and H€ ader, 2002; Rastogi et al., 2010). The reactivation of microorganisms flowing exposure to the UV light at 254 nm in water as well as 222, 254 and 282 nm in apple juice has been reported already (Oguma et al., 2002; Jungfer et al., 2007; Yin et al., 2015); however, similar research is still limited available in dairy products. Due to the differences of nutritional components and pH values between apple juice products and milk (Bernabucci et al., 2013; Christen et al., 2013a; Yin et al., 2015), we speculate that the reactivation of microorganisms following exposure to similar UV light sources in these mediums would be different although further evidences are needed. Bovine milk is a biological fluid containing a lot of nutritional and bioactive components, namely proteins, carbohydrate, lipids, fatty acids, minerals as well as some bioactive antibodies, peptides and immunoglobulins (Singh et al., 2002). It is not only nutritious and healthy for human and other mammals but also a favorable medium for microbial growth. In addition, these components may affect the optical characteristics of fluid, such as scattering and turbidity particularly, and can further affect the penetration of UV light as well as UV absorption coefficient (Guerrero-Beltr and Barbosa-C, 2004; Falguera et al., 2011). Besides, the specific characteristics of bovine milk, namely nourishing with different nutrients as well as stable and close to neutral pH value (Bernabucci et al., 2013), may affect the reactivation of microorganisms following exposure to UV light irradiation. In addition, during commercial production bovine milk can be detained for several days for further processing, distribution and selling prior to consumption by the customers, particularly, at refrigerated (4  C) or ambient conditions (20  C) (Koutchma and Barnes, 2013). Besides, to optimize the economic profits, longer shelf life of milk products is also expected, for example extended shelf-life (ESL) milk. There is a chance that during production and storage period, microorganisms in UVC treated milk may undergo reactivation and proliferation in dark phase. Therefore, it is necessary to explore the inactivation and reactivation characteristics of microorganisms following exposure to UV light in dairy products. Recently, two novel monochromatic UVC light sources with the wavelength of 222 and 282 nm have been developed for bacterial disinfection (Pennell et al., 2008; Wang et al., 2010). Particularly, the UV radiation at 222 nm is the most effective for the inactivation of Bacillus subtilis spores (Matafonova et al., 2008; Rahmani et al., 2010) and that at 282 nm also exhibited promising bactericidal effect on

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tested Escherichia coli strains (Matafonova et al., 2012, 2013). However, the inactivation and reactivation potential of foodborne pathogens during refrigerated and ambient storage following exposure to these novel UV light sources in milk was not reported. Escherichia coli (E. coli) O157:H7, the Shiga toxin-producing bacterial strains, are often associated with devastating or lifethreatening systemic manifestations (Oteiza et al., 2005). Several milk-borne disease outbreaks are attributed to bovine milk contaminated with E. coli O157:H7 (Control and Prevention, 2007; Denny et al., 2008; Guh et al., 2010). Notably, UV irradiation induced expression of Shiga toxin gene as well as its transfer from E. coli O157:H7 to Non-Pathogenic E. coli was also observed (Pacheco and Sperandio, 2012; Yue et al., 2012). Thus, it is important to explore the effectiveness of UV irradiation induced bacteriostatic inactivation and reactivation along with associated potential risks before the application of UV irradiation as a non-thermal pasteurization method for milk. Therefore, the present study sought to investigate the inactivation efficacy of monochromatic UV lights at the wavelengths of 222, 254 and 282 nm against a pathogenic E. coli O157:H7 strain as well as to examine the potential reactivation of this specific strain in bovine milk during dark incubation phases at refrigerated and ambient temperatures following exposure to these three UV light sources. 2. Materials and methods 2.1. Test microorganisms The test microorganism was E. coli O157:H7 strain EDL 933, which was obtained from the microbial collection of Dr. Magdalena Kostrzynska (Guelph Food Research Centre, Agriculture and AgriFood Canada, Guelph, ON, Canada). This particular strain produces both Shiga toxin 1 and Shiga toxin 2 (Shaikh and Tarr, 2003) and is associated with hemorrhagic colitis and other severe disease conditions (Lee et al., 2007). Particularly, the inactivation and reactivation characteristics of this strain have been studied after exposure to UV light in some fruit juices (Oteiza et al., 2010; Yin et al., 2015), and it is also commonly used as an indicator in disinfection studies (Marouani-Gadri et al., 2009; Habimana et al., 2010). 2.2. Test medium The test medium was the pasteurized whole bovine milk (containing 3.25% milk fat) (Beatrice Companies Inc. Toronto, ON, Canada) with vitamin D concentration to supply 45% of the recommended human daily value, and purchased locally and stored at 4  C for 24 h before performing the tests. 2.3. Characterization of the UV light absorption in bovine milk The absorption spectrum of bovine milk tested in the light wavelength range from 200 to 300 nm was measured with a Cary 300 Bio UV/Vis Spectrophotometer (Varian Inc., Walnut Creek, CA, USA) using quartz demountable cuvettes (NSG Precision Cells, Inc., Farmingdale, NY, USA) with a path length of 0.005 cm. The absorption coefficient for UV light wavelength for path length of 1 cm was determined according to equation (1).

Absorbancei

ð1 cmÞ

¼

Absorbancei ð0:005 cmÞ 0:005 ðversus path lengthÞ

(1)

where Absorbancei(1 cm) represents the absorption coefficient of bovine milk tested in response to different UV light sources for path

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length of 1 cm; Absorbancei(0.005 cm) represents the absorbance of bovine milk tested with different UV light sources measured at the path length of 0.005 cm; i represents the UV wavelength of either 222, 254 or 282 nm, respectively. 2.4. Monochromatic UV light generating unit and UV fluence rate determination Three monochromatic UV lamps were fixed on the top of the bench top unit and used as UV light sources for the wavelengths of 222, 254 and 282 nm as previously described (Zhu et al., 2014; Yin et al., 2015). The lamp emitting light at 222 nm was filled with KrCl* mixture, while that emitting at 282 nm was filled with XeBr* (Healthy Environment Innovations LLC. Dover, NH, USA). A low pressure mercury lamp (15W, GE/Hitachi, Wilmington, NC, USA) was employed to generate UV light at the wavelength of 254 nm. The lengths for the lamps emitting at 222, 254 and 282 nm are 20, 29 and 20 cm, respectively. Milk samples (20 mL) were transferred into a sterile Petri dish (f ¼ 5.5 cm), stirred and placed directly below each UV lamp for irradiation. The UV lamps overlaid the middle axis of the Petri dish, and the distances from sample surface to the lamps emitting at 222, 254 and 282 nm were 5, 7.5 and 5 cm, respectively. To stabilize the temperature and prevent overheating, the lamps emitting at 222 and 282 nm were aircooled during operation. UV fluence was calculated by taking into account the geometry of the UV light experimental setup as well as the output power of the lamps. Briefly, each UV lamp was considered as a composition of continuous point light sources. The UV fluence rate has been calculated using the equation developed by Yin et al. (2015) for any small volume in the liquid sample that receives the UV light exposure from the point on the lamp at a specific distance from the center of the lamp. The estimated average UV fluence rate was then calculated from serial fluence rates of small volumes with equal increments along the proposed axis of x, y and z of the samples using the equation described by Yin et al. (2015). 2.5. Inactivation of E. coli O157:H7 following exposure to UV light Inactivation of E. coli O157:H7 following exposure to each monochromatic UV lights in the tested bovine milk was determined according to the protocol described previously (Yin et al., 2015) with minor modification. Briefly, to ensure sufficient cell density, the frozen pure E. coli O157:H7 stock was incubated under optimal conditions in trypticase soy broth (TSB, Difco, Sparks, NV, USA) at 37  C. A 20 h culture in stationary phase was used to represent the growth phase most typically observed in the environment. Prior to the experiment, a suspension of E. coli O157:H7 was centrifuged (ThermoFisher Scientific Inc. Asheville, NC, USA) at 400  g for 10 min, and the supernatant was aseptically drawn off. The pellet was re-suspended in 2 mL 0.01 M phosphate buffered saline (PBS, pH ¼ 7.2), vortexed (Fisher Genie 2, VWR Canada, Mississauga, Canada) and centrifuged again at 400  g for another 10 min, and then aseptically drawn off the supernatant. The above procedures were repeated twice. The pellet was re-suspended in 187 mL bovine milk to obtain the original E. coli O157:H7 population of 2  107 CFU/mL. The sample was kept at 20  C for 30 min to acclimatize the bacteria to the new environment. Thereafter, 20 mL of the suspension (milk þ E. coli O157:H7) was transferred to a sterile Petri dish (f ¼ 5.5 cm) and placed directly below the lamps for UV treatment. Before the exposure to UV light was conducted, the UV lamp emitting at 254 nm was allowed to stabilize by turning on and pre-heated for at least 30 min. The sample temperature was kept at 20  C during UV light exposure. To overcome the limited penetration of UV light in the tested bovine milk and insure samples absorbed UV light irradiation uniformly, the samples

were continuously mixed with a magnetic stirrer (IKA® Works, Inc., Waterbury, Connecticut, USA) at 220 rpm during UV light irradiation. The UV fluence and corresponding UV irradiation times for each UV lamp are summarized in Table 1. Each UV light treatment condition was conducted in triplicate and the whole trial was also repeated twice. After UV irradiation was completed, the samples were collected and the surviving populations of E. coli O157:H7 were enumerated by a spread plating method. Each sample was spread onto two Tryptic soy agar (TSA, Difco, Sparks, NV, USA) plates and incubated aerobically at 37  C for 18 h and the CFU counting was determined using plates with appropriate dilutions. The bacterial population was expressed as Log CFU/mL milk. Two samples were collected prior to UV treatment and served as controls. The UV inactivation efficiency of E. coli O157:H7 was expressed as the Log reduction of the bacterial population after exposure to UV lights. 2.6. Reactivation of E. coli O157:H7 under dark incubation phase Reactivation of E. coli O157:H7 following exposure to each UV light under dark incubation phase was conducted as previously described (Yin et al., 2015). To get approximate 2 Log reduction of E. coli O157:H7 after exposure to UV light at three wavelengths, the irradiation time for the UV wavelength at 222, 254 and 282 nm were calculated as 17.1, 5.0 and 9.6 min, respectively, according to previous description (Zhu et al., 2014). After UV treatments were completed, samples were immediately collected to seven culture tubes with 1.5 mL volume sample per tube. Each tube was covered with aluminum foil and incubated at 4, 20 and 37  C for the assigned incubation time (0e6 h). The non-UV treatment sample with E. coli O157:H7 population of 2  105 CFU/mL (positive control) was obtained by further dilution of the original milk sample with E. coli O157:H7 population of 2  107 CFU/mL. At each incubation time point, the surviving population of E. coli O157:H7 was also enumerated by the spread plating method. Due to the fact that the application of the same UV fluence from the same UV source does not exactly lead to 2 Log reductions in bacteria counts, the following equation (2) was applied to calculate the bacterial reactivation ratio at each incubation time point as previously described (Quek and Hu, 2008).

% reactivation ¼

Nt  N0  100% Ninitial  N0

(2)

where Nt is the population of E. coli O157:H7 at incubation time t after exposure to UV light, N0 is the population of E. coli O157:H7 immediately after exposure to UV light (t ¼ 0), and Ninitial is the initial population of E. coli O157:H7 before exposure to UV light. 2.7. Statistical analysis All experiments were performed at least in duplicate. The data on inactivation of E. coli O157:H7 following exposure to

Table 1 Calculated exposure time (min) to achieve similar fluence at three UV light wavelengths in bovine milk. UV wavelength (nm)

UV fluence (mJ/cm2) 5

10

20

222 254 282

6.0 4.0 2.9

11.9 7.9 5.7

23.8 15.9 10.5

Note: The volume of tested bovine milk for UV irradiation treatment was 20 mL. The distance between UV lamps with the wavelengths of 222, 254 and 282 nm and the sample surfaces were 5, 7.5 and 5 cm, respectively.

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different UV light irradiation with different UV fluence was analyzed in a split-plot design for repeated measures using the general linear model (GLM) procedure of SAS 9.13 (SAS Institute, Inc., Cary, NC, USA). The statistical model included the effect of UV light source as the main plot, and the effects of UV fluence and UV light source  UV fluence interaction as the subplot. The comparisons among UV light source with same UV fluence were made when a significant F test (P < 0.05) for the UV light source  UV fluence interaction was observed. The data on the reactivation of E. coli O157:H7 at same incubation time point but with different UV light source irradiation were also analyzed as a split-plot design for repeated measures. The statistical model included the effect of UV light source as the main plot, and the effects of incubation time and the UV light source  incubation time interaction as the subplot. The comparisons among treatments with same incubation time were made when a significant F test (P < 0.05) for the UV light source  incubation time interaction was observed. The data on variation of the reactivation of E. coli O157:H7 under same UV light source irradiation but different incubation time during dark incubation phase were analyzed with one-way ANOVA analysis model of SAS 9.13 (SAS Institute, Inc.). The multiple comparisons were tested by the TukeyeKramer method to determine significance of differences among treatment means. A P < 0.05 was required for statistical significance. 3. Results 3.1. UV light absorbance of bovine milk and UV exposure time at three monochromatic wavelengths The absorption coefficients (a) of the tested bovine milk for the UV wavelengths at 222, 254, and 282 nm were a222 ¼ 805.2/cm, a254 ¼ 476.2/cm and a282 ¼ 467.6/cm, respectively (Fig. 1). The absorption coefficient was decreasing with wavelength increase, indicating the higher penetration depth of UV photons at the longer UV wavelengths. The average fluence rates at the sample surface for UV wavelengths at 222, 254, and 282 nm were 0.014, 0.021 and 0.029 mW/cm2, respectively, as calculated for the thickness of 0.25 cm of the milk sample according to previous description (Zhu et al., 2014). In order to achieve similar UV fluence among three UV lamps at 5, 10 and 20 mJ/cm2, the exposure time was calculated for each wavelength using corresponding average fluence rate and is summarized in Table 1. It can be seen that with the increase of the UV wavelength, less exposure time was required to achieve similar value of the UV fluence.

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3.2. Inactivation of E. coli O157:H7 following exposure to three monochromatic UV wavelengths Generally, inactivation of E. coli O157:H7 in the tested bovine milk was affected (P < 0.05) by both UV wavelength and UV fluence. Consequently, comparisons of the means among UV wavelengths with same UV fluence were made. The reduction of E. coli O157:H7 following exposure to UV light at 254 nm (1.81, 2.38 and 2.95 Log) was higher (P < 0.05) than that at 222 nm (0.72, 1.56 and 2.40 Log) and 282 nm (1.34, 1.70 and 2.09 Log) at the UV fluence of 5, 10 and 20 mJ/cm2. The bacterial reduction after exposure milk to UV light at 282 nm (1.34 Log) was higher (P < 0.05) than that at 222 nm (0.72 Log) at the UV fluence of 5 mJ/cm2, while that at 222 nm (2.40 Log) was higher (P < 0.05) than at 282 nm (2.09 Log) at the UV fluence of 20 mJ/cm2 (Fig. 2). Thus, the UV inactivation of E. coli O157:H7 in milk was the most effective at 254 nm. 3.3. Reactivation of E. coli O157:H7 in dark incubation phase 3.3.1. Effect of UV wavelength Reactivation of E. coli O157:H7 in dark incubation phase following exposure to UV light at 222, 254 and 282 nm were affected (P < 0.05) by both the UV light wavelength and UV light wavelength  incubation time interaction. Generally, the population of E. coli O157:H7 increased as the incubation time extended after exposure to each UV light source regardless of the incubation temperature. At 4  C, the reactivation ratio of E. coli O157:H7 following exposure to UV light at either 222, 254 or 282 nm was lower (P < 0.05) than the control during 1e6 h incubation; particularly, that following the exposure at 254 nm was also lower (P < 0.05) than that at 222 nm during the same incubation time (Fig. 3). At 20 and 37  C, the reactivation ratio of E. coli O157:H7 following exposure to the UV light at 254 and 282 nm was lower (P < 0.05) than that in the control sample in 1e6 h incubation (Figs. 4 and 5). In particular, that following exposure at 254 nm was lower (P < 0.05) than at 222 nm during 2e5 h incubation at 20  C. 3.3.2. Effect of temperature The reactivation ratio of E. coli O157:H7 during dark incubation phase following exposure to the UV light at the wavelengths of 222, 254 and 282 nm was also affected (P < 0.05) by both incubation temperature and incubation temperature  incubation time interaction. Consequently, comparisons of the means among incubation temperatures at same incubation time point were made (The data was the same as indicated in Figs. 3e5 but with different statistical model). The reactivation ratio of E. coli O157:H7

Fig. 1. UV absorbance of bovine milk at different UV light wavelengths.

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Fig. 2. Effect of UV light wavelength on the inactivation of Escherichia coli O157:H7 in tested bovine milk ( ), UV light at 222 nm; ( ), UV light at 254 nm; ( ), UV light at 282 nm a, b mean values under the same UV fluence, with unlike letters were significantly different (P < 0.05, n ¼ 12). The population of E. coli O157:H7 in the tested bovine milk before UV light treatment was 2  107 CFU/mL.

Fig. 3. Reactivation of Escherichia coli O157:H7 following exposure to UV light irradiation in tested bovine milk in dark phase incubated at 4  C. ( ), UV light irradiation at 222 nm; ( ), UV light irradiation at 254 nm; ( ), UV light irradiation at 282 nm; ( ), Control (no UV irradiation applied). a, b, c, d mean values within the same incubation time with unlike letters were significantly different (P < 0.05, n ¼ 8). The population of E. coli O157:H7 in the tested bovine milk before UV light irradiation was 2  107 CFU/mL and the same as that in Figs. 4 and 5.

Fig. 4. Reactivation of Escherichia coli O157:H7 following exposure to UV light irradiation in tested bovine milk in dark phase incubated at 20  C ( ), UV light irradiation at 222 nm; ( ), UV light irradiation at 254 nm; ( ), UV light irradiation at 282 nm; ( ), Control (no UV irradiation applied). a, b, c mean values within the same incubation time with unlike letters were significantly different (P < 0.05, n ¼ 8).

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Fig. 5. Reactivation of Escherichia coli O157:H7 following exposure to UV light irradiation in tested bovine milk in dark phase incubated at 37  C ( ), UV light irradiation at 222 nm; ( ), UV light irradiation at 254 nm; ( ), UV light irradiation at 282 nm; ( ), Control (no UV irradiation applied). a, b, c mean values within the same incubation time with unlike letters were significantly different (P < 0.05, n ¼ 8).

following exposure to the UV light at 222, 254 and 282 nm at 37  C was higher (P < 0.05) than that incubated at both 4 and 20  C after 1e6 h incubation, that following the exposure at 222 nm at 20  C was higher (P < 0.05) than that at 4  C after 3e6 h incubation, and that following the exposure at 254 and 282 nm at 20  C was higher (P < 0.05) than that at 4  C after 1e6 h incubation, respectively. 4. Discussion Similar to human milk, bovine milk is challenging to treat with UV light due to its much higher absorption coefficients of 805.2, 476.2 and 467.6/cm at the wavelengths of 222, 254 and 282 nm when compared to that of apple juice with 30.25, 23.70 and 16.15/ cm at the same wavelengths (Christen et al., 2013a; Yin et al., 2015) or drinking water with 0.02 and 0.06/cm at the 254 nm, respectively. In this regard, to achieve sufficient UV doses for microbial inactivation in milk, longer UV exposure time is needed (Christen et al., 2013b) in addition to efficient delivery of UV photons to the whole volume of opaque liquids. Previous research indicated that the inactivation of E. coli O157:H7 is UV dosage-dependent at the wavelength of 254 nm (Oteiza et al., 2005; Zimmer-Thomas et al., 2007), and similar observations were also observed for the inactivation of E. coli O157:H7 (strain EDL 933) when exposed to the UV lights at the wavelengths of 222 and 282 nm in apple juice (Yin et al., 2015) and bovine milk in the present study. Typically, the higher the UV dosage is, the lower the number of the pathogen survives. In addition, the wavelength of UV sources also affected the inactivation efficiency of UVC light on E. coli O157:H7. Notably, at the same UV dosage, the UV light at 254 nm appeared to be more efficient than that at 222 and 282 nm to inactivate E. coli O157:H7 in bovine milk; while the UV light at 222 nm was more efficient than that at 254 and 282 nm to inactivate the same pathogenic bacterial strain in apple juice (Yin et al., 2015). Such phenomenon is partially due to the differences in the mechanisms that the UV light at these wavelengths induced bacterial inactivation. The UV light at 254 nm mainly caused bacterial DNA damage (Lopez-Malo and Palou, 2005), while that at 222 and 282 nm mainly destroy the protein molecules of microorganisms (Schmid, 2001). In this regard, we speculate that the components such as protein and free amino acids, fat in bovine milk may contribute to extremely high absorption coefficient and competitively absorbed more photons from the UV lights at 222 and 282 nm during UV light processing. As a result, only number of limited

photons from UV sources is available to destroy the bacterial proteins crucial to death, thus decreased the inactivation efficiency of E. coli O157:H7; however, further evidences and research are needed. In the present study, it was found that reactivation of E. coli O157:H7 following exposure to different UV lights was occurred and steadily increased as the post-treated incubation time extended regardless of the UV light sources and incubation temperature. The highest reactivation ratios of E. coli O157:H7 were observed in the samples treated with the UV light at 222 nm at either 4, 20 and 37  C when compared to that treated at 254 and 282 nm, however, which were still significantly lower than that of non-UV light treated samples (the control), indicating that UV light induced cell damages cannot be fully recovered in 6 h. Interestingly, contrary to the current observation, the negative reactivation of E. coli O157:H7 in apple juice with pH value of 3.5 was previously observed following exposure to the same UV light sources in dark incubation phase (Yin et al., 2015). In addition, such observation was also reported for E. coli O157:H7 following exposure to the UV light at 254 nm in buffer (saline) even incubated for 48 h under dark phase (Sommer et al., 2000). The reasonable explanation could be that bovine milk is nutrient-rich with a stable close to neutral pH value ranges from 6.5 ~ 6.7 (Bernabucci et al., 2013), which provides optimal conditions for cell repair and proliferation comparing to that of apple juice and saline. Furthermore, the reactivation efficiency of E. coli O157:H7 following exposure to the UV light at 254 nm was lower (or at least numerically lower) than that following exposure at 222 and 282 nm in the present study, implying that the repair efficiency of damaged DNA is lower that of damaged proteins in bovine milk medium although the potential mechanism is still not understood (Costa et al., 2007; Chondrogianni et al., 2014). Increase in incubation temperature was previously reported to negatively affect the reactivation of several E. coli strains (STCC 471, STCC 4201, and STCC 27325) under either photo or dark incubation phase following exposure to the UV light at 254 nm. Similar results were also observed for E. coli O157:H7 in apple juice following exposure at 222, 254 and 282 nm (Yin et al., 2015). The higher the incubation temperature is, the lower the number of bacteria survives. However, contrary to these observations, the incubation temperature positively affected the reactivation of E. coli O157:H7 following exposure to the UV lights at same wavelengths in bovine milk in the present study. Notably, the highest reactivation of E. coli O157:H7 was observed when the UV treated samples were

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incubated at 37  C, particularly in that following exposure to the UV light at 222 nm with 1.33 Log reactivation after 6 h incubation, which is still lower than that of non-UV light treated cells with 1.58 Log reactivation. In other words, higher temperature stimulated the reactivation of UV light damaged cells in bovine milk, and such effect is more obvious in cells following exposure to the UV light at 222 nm than that at 254 nm. The most likely explanation is that bovine milk provided a nutrient-rich and neutral pH environment for bacteria surviving, repair and proliferation. With the increase of the incubation temperature, the protein or DNA repairing activities were stimulated. Therefore, exposure to the UV light at 254 nm followed by storage at refrigerated conditions would be a practical and effective way to control the safety of milk. 5. Conclusions The inactivation efficiency of the UV light at 254 nm was higher than that at 222 and 282 nm on E. coli O157:H7 in bovine milk. The reactivation ratio of E. coli O157:H7 following exposure to the UV light at 254 nm was lower than that at 222 and 282 nm in milk medium. The incubation temperature stimulated the reactivation of E. coli O157:H7 regardless of the UV light sources irradiation during dark incubation phase; however, the reactivation of this specific pathogenic bacteria following exposure to the UV light at 254 nm was the lowest compared to that following exposure at 222 and 282 nm at 37  C. Acknowledgments This research was supported by the Risk Mitigation program of Agriculture & Agri-Food Canada. F. Yin was a visiting graduate student financially supported by the China Scholarship Council through the MOE-AAFC Ph.D. Research Program, and is currently an NSERC Visiting Fellow to the Canadian Federal Government Laboratories (NSERC-VF). References Bandla, S., Choudhary, R., Watson, D.G., Haddock, J., 2012. Impact of UV-C processing of raw cow milk treated in a continuous flow coiled tube ultraviolet reactor. Agric. Eng. Int. CIGR J. 14, 86e93. Bernabucci, U., Basirico, L., Morera, P., 2013. Impact of hot environment on colstrum and milk composition. Cell. Mol. Biol. 59, 67e83. Bintsis, T., Litopoulou-Tzanetaki, E., Robinson, R.K., 2000. Existing and potential applications of ultraviolet light in the food industry-a critical review. J. Sci. Food Agric. 80, 637e645. Chondrogianni, N., Petropoulos, I., Grimm, S., Georgila, K., Catalgol, B., Friguet, B., Grune, T., Gonos, E.S., 2014. Protein damage, repair and proteolysis. Mol. Asp. Med. 35, 1e71. Choudhary, R., Bandla, S., 2012. Ultraviolet pasteurization for food industry. Int. J. Food Sci. Nutr. Eng. 2, 12e15. Christen, L., Lai, C.T., Hartmann, B., Hartmann, P.E., Geddes, D.T., 2013a. Ultraviolet-C irradiation: a novel pasteurization method for donor human milk. PLoS One 8, e68120. Christen, L., Lai, C.T., Hartmann, B., Hartmann, P.E., Geddes, D.T., 2013b. The effect of UV-C pasteurization on bacteriostatic properties and immunological proteins of donor human milk. PLoS One 8, e85867. Centers for Disease Control and Prevention, 2007. Escherichia coli O157:H7 infection associated with drinking raw milk-Washington and Oregon. NovembereDecember 2005 MMWR 56, 165e167. Costa, V., Quintanilha, A., Moradas-Ferreira, P., 2007. Protein oxidation, repair mechanisms and proteolysis in Saccharomyces cerevisiae. IUBMB life 59, 293e298. Denny, J., Bhat, M., Eckmann, K., 2008. Outbreak of Escherichia coli O157: H7 associated with raw milk consumption in the Pacific Northwest. Foodborne Pathog. Dis. 5, 321e328. Eischeid, A.C., Linden, K.G., 2011. Molecular indications of protein damage in adenoviruses after UV disinfection. Appl. Environ. Microbiol. 77, 1145e1147. Falguera, V., Pag an, J., Garza, S., Garvín, A., Ibarz, A., 2011. Ultraviolet processing of liquid food: a review: Part 2: effects on microorganisms and on food components and properties. Food Res. Int. 44, 1580e1588.

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Inactivation and potential reactivation of pathogenic Escherichia coli O157:H7 in bovine milk exposed to three monochromatic ultraviolet UVC lights.

The ultraviolet (UVC) light irradiation has been recently studied as a novel non-thermal treatment method for milk. However, the potential reactivatio...
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