Food Microbiology 46 (2015) 329e335

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Inactivation and potential reactivation of pathogenic Escherichia coli O157:H7 in apple juice following ultraviolet light exposure at three monochromatic wavelengths Fugui Yin a, b, Yan Zhu b, Tatiana Koutchma b, *, Joshua Gong b a

Key Laboratory of Agri-Ecological Processes in the Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, 410125, People's Republic of China Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, N1G 5C9, Canada

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 February 2014 Received in revised form 27 May 2014 Accepted 17 August 2014 Available online 9 September 2014

Ultraviolet (UV) light irradiation at 254 nm is considered as a novel non-thermal method for decontamination of foodborne pathogenic bacteria. However, lower penetration depth of UV light at 254 nm in apple juice resulted in higher UV dose consumption during apple juice decontamination. In addition, no studies are available on the reactivation of pathogens following exposure to UV light in drinks and beverages. Two novel monochromatic UV light sources (l ¼ 222 and 282 nm) have been developed for bacterial disinfection. However, the inactivation of pathogenic Escherichia coli O157:H7 following exposure to these UV wavelengths is still unclear. Therefore, the present study was conducted to determine the inactivation and reactivation potential of pathogenic E. coli O157:H7 in apple juice following exposure to UV light at three monochromatic wavelengths: Far UV (l ¼ 222 nm), Far UVþ (l ¼ 282 nm) and UVC light (l ¼ 254 nm). The results showed that E. coli O157:H7 is acid-resistant, and up to 99.50% of cells survived in apple juice when incubated at 20  C for 24 h. Inactivation of E. coli O157:H7 following exposure to Far UV light (2.81 Log reduction) was higher (P < 0.05) than the inactivation caused by UVC light (1.95 Log reduction) and Far UVþ light (1.83 Log reduction) at the similar levels of UV fluence of 75 mJ/cm2. No any reactivation potential was observed for E. coli O157:H7 in dark incubation phases after exposure to UV light as determined by the regular plating method. In addition, the exposure to Far UV light at 222 nm followed by incubating at 37  C significantly decreased (P < 0.05) the survival of E. coli O157:H7 during dark incubation phase compared to that of UVC and Far UVþ light. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Far UV light UVC light Far UVþ light Inactivation Reactivation Escherichia coli O157:H7 Apple juice

1. Introduction Escherichia coli (E. coli) O157:H7, a cause of hemorrhagic colitis, is often associated with devastating or life-threatening systemic manifestations (Oteiza et al., 2005; Zhang et al., 2007). Several foodborne disease outbreaks attributed to unpasteurized apple juices contaminated with E. coli O157:H7 have demonstrated that unpasteurized juice can be a vehicle for foodborne diseases (CDC, 1996; Cody et al., 1999). Traditionally, thermal processing has been used for juice pasteurization (Mohideen, 2011). However, because heating may cause substantial changes in nutritional composition and flavor of the juice, there is a growing need worldwide to explore non-thermal alternative strategies for mild

* Corresponding author. Tel.: þ1 226 217 8123; fax: þ1 226 217 8181. E-mail addresses: [email protected], [email protected] (T. Koutchma). http://dx.doi.org/10.1016/j.fm.2014.08.015 0740-0020/© 2014 Elsevier Ltd. All rights reserved.

decontamination of the foodborne pathogenic bacteria in the food industry (Koutchma et al., 2007; Koutchma, 2009). Ultraviolet (UV) light is considered a promising and viable treatment technology as it can kill microorganisms by various means. UV light irradiation can cause deoxyribonucleic acid (DNA) damage and DNA pyrimidine base mispairing, with greater effect at wavelengths between 250 and 260 nm (Oteiza et al., 2005). The UV light wavelength of 253.7 nm is one of the most efficient in terms of germicidal effect since photons are absorbed particularly well by the DNA of microorganisms at this specific wavelength (Koutchma, 2009). UV light also destroys the integrity of the outer membrane and protein molecules of microorganisms (Schenk et al., 2011), and thus affects the physiochemical and metabolic processes critical to n and Mackey, 2000). In 2000, the US Food and Drug survival (Paga Administration (FDA) approved UV-light as alternative treatment to thermal pasteurization of fresh juice products (US FDA, 2000). Notably, several studies have been conducted to determine the disinfection efficiency of UV light at 254 nm in semi-opaque and

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opaque fluids, for example juice (Keyser et al., 2008; Fredericks et al., 2011) and milk (Matak et al., 2005). Additionally, the effect on the quality parameters was also evaluated (Pala and Toklucu, 2011, 2013). However, because the penetration depth of UV light at 254 nm is lower (only 0.036 cm) in apple juice, higher UV dose for 254 nm lamps is required in order to achieve a satisfying level of microbial reduction (Koutchma, 2014). Despite the benefits of UV non-thermal treatment for food decontamination, the potential reactivation of pathogenic bacteria after exposure to UV light should not be ignored. Bacteria generally possess molecular mechanisms to compensate for the damaging effects of UV light radiation on DNA (Zimmer and Slawson, 2002). The formation of a pyrimidine dimer can be reversed through two major mechanisms for UV light-induced DNA damage correction: photo-reactivation and nucleotide excision reactivation (dark reactivation) (Thoma, 1999; Ura et al., 2001). Photo-reactivation is light dependent, which requires specific wavelengths of light ranging from 300 to 500 nm to complete the reactivation process (Salcedo et al., 2007). Unlike photoreactivation, dark reactivation is a multistep and lightindependent process. Two different dark-activated repair mechanisms in response to UV light-induced damage for bacteria exist: base excision repair and nucleotide excision repair (Sinha and €der, 2002; Rastogi et al., 2010). Notably, all mechanisms are Ha associated with the recA gene pathway (Jungfer et al., 2007). Because of its key position in many molecular bacterial regulation processes, the recA protein exists widely and is highly conserved in a range of microorganisms (Bichara et al., 2007; Chau et al., 2008), including E. coli O157:H7 (Moore et al., 2008). Both the photo and dark reactivation of pathogenic bacteria following exposure to UV light at 254 nm have been well studied in water (Jungfer et al., 2007); however, no studies are available on the reactivation of pathogens following exposure to UV light in drinks and beverages. Recently, two novel monochromatic UV light sources with the wavelength of 222 nm (Far UV) and 282 nm (Far UVþ) have been developed for bacterial disinfection (Pennell et al., 2008). The UV light at the wavelength of 222 nm seems specific for destroying the bacterial outer membrane and protein molecules (Edward Neister, 2010). However, the disinfection efficiency of these two novel UV light sources in drinks and beverages is unclear and needs to be tested. The apple juice is detained for a long time for production, distribution and selling prior to reaching the consumer, particularly, the storage time at refrigerated conditions at 4  C in the dark or at the ambient temperature on the shelf at 20  C with light. During this period, UV light treated microorganisms may undergo photo and dark reactivation to potentially re-grow within the system. However, no reports are available on the photo or dark reactivation of pathogenic organisms following exposure to Far UV and Far UVþ lights in apple juice. In this regard, the objectives of this study were to compare the inactivation efficacies of monochromatic UV lights with the wavelengths of 222, 254 and 282 nm on pathogenic E. coli O157:H7, and to examine the potential reactivation of this pathogenic strain in dark incubation phases following exposure to these three UV light sources in apple juice. 2. Materials and methods 2.1. Tested microorganisms E. coli O157:H7 was chosen for this study as its photo and dark reactivation characteristics are well studied after exposure to UV light, with the wavelength of 254 nm, in drinking water, and it is commonly used as an indicator in disinfection studies (Zimmer and

Slawson, 2002; Quek and Hu, 2008). In the present study, the pure cultured E. coli O157:H7 strain EDL 933 stock was obtained from the microbial collection of Dr. Magdalena Kostrzynska (Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada). 2.2. Test medium The test medium was pasteurised apple juice (Allen's, Lassonde Industries Inc., Rougemont, QC, Canada) with vitamin C concentration to supply 100% of the recommended human daily value, and pH value of 3.5, and purchased from a local super market and stored at 20  C for 24 h before performing the tests. 2.3. Bacterial survival To ensure sufficient activation of the cell, the frozen pure E. coli O157:H7 EDL 933 stock was inoculated onto two Tryptic soy agar (TSA, Difco, Sparks, NV, USA) plates, and incubated under optimal condition at 37  C for 18 h. After incubation, three single colonies were picked from the plates, inoculated into each of three culture tubes (Simport, Beloeil, QC, Canada) filled with 3 mL of Tryptic soy broth (TSB, Difco, Sparks, NV, USA), incubated at 37  C in a Forma bench-top orbital shaker (Thermo Scientific Inc., Marietta, GA, USA) at 200 rpm for 20 h. The culture in stationary phase was used to represent not only the growth phase most typically observed in the environment but also the most resistant stage of growth (Mofidi et al., 2002). After that, the E. coli O157:H7 concentration was determined by referring to the standard curve of the OD600 values and bacterial colony-forming units (CFU) that had been established previously in our laboratory. From the culture 300 mL was collected to measure the optical densities (OD) at 600 nm through a serial 2  dilution. Soon after, a 2 mL suspension of E. coli O157:H7 was centrifuged (Thermo-Fisher 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 II, VWR Canada, Mississauga, ON, Canada) and centrifuged again at 400  g for 10 min, and then aseptically drawn off the supernatant. The above procedures were repeated twice. The pellet was re-suspended in 178 mL apple juice to obtain an E. coli O157:H7 concentration of 2  107 CFU/mL. The sample was kept at 20  C for 30 min to acclimatize the bacteria to the new environment. Next, a 2 mL sample was collected, serially diluted with 0.1% peptone water, plated automatically by an Eddy Jet Spiral Plater (Neutec group Inc. Farmingdale, NY, USA) on TSA plates in triplicate and incubated aerobically at 37  C for 20 h to determine the initial cell concentration. The plates with appropriate dilutions were selected for CFU counting and expressed as Log CFU/mL. Another 16 mL sample was collected and divided equally into 4 culture tubes (4 mL sample per tube) and incubated at 20  C for 24 h without agitation. After incubation, the bacterial concentration was determined as described above. Each trial was repeated 4 times. The survival ratio of E. coli O157:H7 in the apple juice was calculated according to the Equation (1):

% survival ¼

N24h  100% N0:5h

(1)

where the N24h represents the E. coli O157:H7 concentration in the apple juice after 24 h incubation at 20  C (Log CFU/mL), and N0.5h represents the E. coli O157:H7 concentration in the apple juice after 0.5 h incubation at 20  C to acclimatize bacteria to new environment.

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331

2.4. Characterization of the UV light absorption in apple juice The absorbance of apple juice is a function of the UV light wavelength and its knowledge is needed to evaluate the UV fluence. The absorption spectrum of apple juice in the light wavelength range from 200 to 350 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 path lengths of 0.02 cm. The regression curve slope obtained by plotting absorbance versus sample concentration was considered the absorption coefficient. The absorption coefficients for UV wavelengths of 222, 254 and 282 nm with versus path length of 1 cm were determined according to Equation (2).

Absorptioni ð1cmÞ

Absorptionið0:02cmÞ ¼ 0:02ðversus path lengthÞ

dL

L

UV lamp

d

H

Petri dish x

y

(2)

z

d dE(x,y,z,l)

Fig. 1. Geometric structure of triple wavelengths UV reactor.

where Absorption i (1 cm) represents the absorption coefficient of apple juice in response to different UV light sources for path length of 1 cm; Absorption i (0.02cm) represents the absorption coefficient of apple juice with different UV light sources measured at the path length of 0.02 cm; i represents the UV light wavelength of either 222, 254 or 282 nm, respectively. 2.5. Triple UV light wavelength unit experimental setup and determination of UV fluence rate

H ¼ Eavg $t

Three monochromatic UV lamps with light wavelengths of 222 (Far UV), 254 (UVC) and 282 (Far UVþ) nm were fixed on the top of the batch unit and used as UV light sources in the present study. The Far UV and Far UVþ lamps are novel mercury-free UV sources developed by Healthy Environment Innovations LLC. (Dover, NH, USA). Each lamp consisted of quartz tube filled with proper rare gas-halide mixture. The Far UV lamp was filled with KrCl* mixture and emitted light at 222 nm, while Far UVþ lamp filled with XeBr* and emitted light at 282 nm. A low pressure mercury lamp (15 W, GE/Hitachi, Wilmington, NC, USA) was used as UV light source for the wavelength of 254 nm. The length for Far UV, UVC and Far UVþ lamps are 20, 29 and 20 cm, respectively. To stabilize the temperature and prevent overheating, the Far UV and Far UVþ lamps were air-cooled during their operation. The sample was 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. The distances from the juice sample surfaces to the Far UV, UVC and Far UVþ lamps were 5, 7.5 and 5 cm, respectively. UV fluence (H) was calculated taking into account the geometry of the UV light experimental setup (Fig. 1). Briefly, each UV lamp was considered as a composition of continuous point light sources. At any small volume in the liquid sample with a coordinate (x, y, z), which receives the UV light exposure from the point on the lamp at a distance (l) from the centre of the lamp, the UV fluence rate (E) can be calculated using Equation (3) which is deduced from the equation proposed with a consideration of reflectance factor (Jacobm and Dranoff, 1970).

 E x; y; z; l



  P 0 ¼ $ 1  Re $10ad 4pnd2

light refraction in the apple juice was ignored in the present study. The details on determination of these parameters were previously described (Zhu et al., 2014). The average UV fluence rate (Eavg) was calculated from serial fluence rates of small volume with equal increments along the axis of x, y and z. The UV fluence can be determined using Equation (4), where t (s) is UV light exposure time.

(3)

In Equation (3), P is the output power of the UV lamp; n is the number of proposed point light sources; a is the UV absorption coefficient of apple juice; d is the distance between the point of the UV light source and the small volume (x, y, z); d' is the part of d in the apple juice; Re is the reflectance coefficient at the interface between air and apple juice. Notably, to simplify the model, the UV

(4)

2.6. Inactivation efficiency of UV light on E. coli O157:H7 in apple juice The inactivation efficiency of Far UV, UVC and Far UVþ lights on E. coli O157:H7 in apple juice were determined according to the following protocols. Briefly, the preparation of apple juice with an approximate concentration of E. coli O157:H7 of 7.3 Log CFU/mL was described above. Thereafter, 20 mL of the suspension (apple juice þ E. coli O157:H7) was transferred into a sterile Petri dish (f ¼ 5.5 cm), stirred and placed directly below the UV lamps for the treatment. Before cells exposure to UV light was conducted, the UVC lamp was allowed to stabilize by turning on and preheated for at least 30 min. The temperature was kept at 20  C during UV light exposure. Each UV light treatment condition was conducted in triplicate and the whole trial was also repeated twice. After UV light treatment was completed, the samples were collected and the surviving populations of E. coli O157:H7 were enumerated by a regular plating technology as described above. Two samples collected prior to treatment served as controls. The UV inactivation efficiency of E. coli O157:H7 was expressed as the Log reduction of the bacterial concentration after exposure to UV lights. 2.7. Reactivation of E. coli O157:H7 under dark incubation The procedure for UV light treatment was similar to that described above. To get an approximate 2 Log reduction of E. coli O157:H7 after exposure to each UV light treatment, the irradiation time for Far UV, UVC and Far UVþ lights were 156, 102 and 74 s, respectively, as calculated according to the inactivation efficiency of each UV source. After UV irradiation treatment was completed, the samples were immediately collected into 7 culture tubes, with 1.5 mL sample per tube. Each tube was covered well with aluminum foil and incubated in Forma bench-top orbital shakers (Thermo Scientific Inc.), shaken at 200 rpm at either 4, 20 or 37  C for their

F. Yin et al. / Food Microbiology 46 (2015) 329e335

assigned incubation time (0e6 h). The UV irradiation free sample with an E. coli O157:H7 concentration of 5.3 Log CFU/mL (C-105) was also prepared as positive controls. At each incubation time point, the surviving populations of E. coli O157:H7 were enumerated by serial dilution and platting on TSA plates with the Eddy Jet Spiral Plater (Neutec group Inc). The plates were incubated aerobically at 37  C for 18 h and the CFU counting was determined using plates with appropriate dilutions. The bacterial concentration was expressed as Log CFU/mL apple juice. Due to the fact that the application of the same UV fluence from the same UV source does not exactly lead to 2 Log reduction in bacteria counts, the following Equation (5) was applied to calculate the bacterial reactivation ratio at incubation time point (Quek and Hu, 2008).

1.2 1.0

UV absorbance

332

0.8 Far UV 30.25/cm

222 =

0.6

UVC 23.70/cm

254 =

Far UV+ 16.15/cm

0.4

282 =

0.2 0.0

Nt  N0 % reactivation ¼  100% Ninitial  N0

(5)

where Nt is the concentration of E. coli O157:H7 at incubation time t after exposure to UV light (Log CFU/mL), N0 is the concentration of E. coli O157:H7 immediately after exposure to UV light (Log CFU/ mL), and Ninitial is the initial concentration of E. coli O157:H7 before exposure to UV light (Log CFU/mL). 2.8. Statistical analysis All experiments were performed at least in duplicate. The data on E. coli O157:H7 concentration in apple juice incubated darkly at each time point were summarized and means were calculated with Excel 2010 (Microsoft, USA). The data on E. coli O157:H7 inactivation efficiency under each UV light with different UV fluences was analyzed as a split-plot design for repeated measures using the GLM procedure of SAS 9.13 (SAS Institute, Inc., Cary, USA). The statistical model included the effect of treatment (UV light source) as the main plot (tested by the E. coli O157:H7 concentration within treatment variance) and the effects of UV fluence and the UV light source  UV fluence interaction as the subplot. The comparisons among UV light source within 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 during dark incubation phase at same incubation time but different UV light source irradiation were also analyzed as a splitplot design for repeated measures. The statistical model included the effect of treatment (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 within 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 dark reactivation of E. coli O157:H7 under same UV light source irradiation with different incubation time were analyzed by one-way ANOVA using the GLM procedure of SAS (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. Effect of apple juice media on survival of E. coli O157:H7 E. coli O157:H7 EDL 933 is an acid-resistant strain, approximately 99.50% (SD ¼ 0.36) of the cells survived in apple juice (pH ¼ 3.5) even when incubated at 20  C for 24 h. In other words, E. coli O157:H7 concentration was reduced only approximately 0.002 Log CFU/mL when healthy cells were incubated in environmental conditions with a low pH.

200

220

240

260

280

300

320

340

UV light wavelength (nm) Fig. 2. The UV absorbance in apple juice with 0.2 mg/mL vitamin C under different light wavelengths. The apple juice (Allen's, vitamin C added up to 100% of daily value, pH ¼ 3.5) was pasteurized and commercially available from local super market. The data was collected by using a demountable quartz cuvette with path length of 0.02 cm.

3.2. UV light absorbance of apple juice and UVexposures at three monochromatic wavelengths The absorption coefficients of apple juice (a) for Far UV, UVC and Far UVþ lights were a 222 ¼ 30.25/cm, a 254 ¼ 23.70/cm and a 282 ¼ 16.15/cm, respectively (Fig. 2). The average fluence rates for Far UV, UVC and Far UVþ lights were calculated as 0.17, 0.21 and 0.40 mW/cm2, respectively, for the apple juice sample with the thickness of 0.25 cm. The calculated UV exposure times for Far UV, UVC and Far UVþ lights to achieve similar UV fluence are summarized in Table 1. It can be seen that with the increase in the UV wavelength, the much shorter exposures times were required to achieve similar values of the UV fluences due to the differences in the absorption coefficients of apple juice and penetration depth of UV photons in Far UV, UV-C and Far UVþ ranges.

3.3. Inactivation of E. coli O157:H7 by UV exposures at three wavelengths Generally, the inactivation of E. coli O157:H7 was affected (P < 0.05) by both UV light wavelength and UV fluence. Consequently, comparisons of the means among UV fluence within UV light wavelengths were made. The reduction of E. coli O157:H7 following exposure to Far UV light at 222 nm (0.55 Log) was higher (P < 0.05) than that following Far UVþ light (0.34 Log) at 282 nm at the fluence of 5 mJ/cm2. With the increase of UV fluence up to 75 mJ/cm2, the reduction of E. coli O157:H7 following exposure to Far UV light (2.81 log) was significantly higher (P < 0.05) than that following UVC light (1.95 Log) and Far UVþ light (1.83 Log), respectively (Fig. 3).

Table 1 Calculated exposure time (s) to achieve similar fluences at three UV light wavelengths in apple juice. UV sources

UV fluence (mJ/cm2) 5

25

75

Far UV, 222 nm UVC, 254 nm Far UVþ, 282 nm

29 24 13

147 119 63

441 357 188

Note: The volume of the sample for UV irradiation treatment was 20 mL. The distance between far UV, UVC and Far UVþ lamps and the sample surfaces were 5, 7.5 and 5 cm, respectively.

F. Yin et al. / Food Microbiology 46 (2015) 329e335

333

3.5

Log reduction (Log CFU/mL)

3

a

2.5 b

2

b

1.5

1 a 0.5

b

ab

0 5

25 UV dose (mJ/cm2)

75

Fig. 3. Effect of UV light wavelength on the inactivation of Escherichia coli O157:H7 in apple juice. ( ), Far UV; ( ), UVC; ( ), Far UVþ; a, b mean values under the same UV fluence, with unlike letters were significantly different (P < 0.05, n ¼ 12). The concentration of E. coli O157:H7 in the apple juice before UV light treatments was 7.32 Log CFU/mL.

3.4.1. Effect of UV light wavelength The reactivation of E. coli O157:H7 in dark incubation phase following Far UV, UVC and Far UVþ lights treatments were affected (P < 0.05) by both the UV light wavelength and UV light wavelength  incubation time interaction. Consequently, comparisons of the means among UV light sources within same incubation time were made. At 4  C, the reactivation ratio of E. coli O157:H7 following exposure to Far UV light was lower (P < 0.05) than that following UVC and Far UVþ lights exposure and the control during 1e6 h incubation; that of E. coli O157:H7 subjected to either UVC or Far UVþ light treatment was lower (P < 0.05) than the control during 5e6 h, or 4e6 h incubation, respectively (Fig. 4). At 20  C, the reactivation ratio of E. coli O157:H7 following Far UV light exposure was lower (P < 0.05) than that following Far UVþ light exposure during 2e6 h incubation, and that exposed to Far UVþ light was also lower (P < 0.05) than that subjected to UVC light irradiation and the control during 2e6 h incubation, respectively (Fig. 5). At 37  C, the reactivation ratio of E. coli O157:H7 exposed to Far UV light was lower (P < 0.05) than that subjected to UVC and Far a

0

a

-10

a a

b

a a

b

a a a b

a a a b

-20

a

a

b a b c

b c d

% Log reactivation

-30 -40 -50

-60 -70

UVþ lights irradiation and the control during 1e6 h incubation, and that subjected to UVC and Far UVþ lights irradiation were lower (P < 0.05) than the control during 2e6 h incubation, respectively (Fig. 6). 3.4.2. Effect of incubation temperature The concentration of E. coli O157:H7 during dark incubation following Far UV, UVC and Far UVþ light disinfection was also affected (P < 0.05) by both the incubation temperature and incubation temperature  incubation time interaction. Consequently, comparisons of the means among incubation temperatures within incubation time were made (The means and standard errors of the data were the same as indicated in Figs. 4e6 but with different statistical model). For the E. coli O157:H7 following exposure to Far UV light, the reactivation ratio at 37  C was lower (P < 0.05) than that incubated at both 4 and 20  C during 1e6 h incubation. That incubated at 20  C was lower (P < 0.05) than incubated at 4  C during 2e6 h incubation, respectively. The lowest level (less than 2 Log) of E. coli O157:H7 subjected to Far UV light irradiation was observed at 37  C during 2e6 h incubation. For the E. coli O157:H7

% Log reactivation

3.4. Reactivation of E. coli O157:H7 in dark incubation phase

0

a

-10

b

-20

c

-30

a a

a a

a

b

b

c

c

c

-40

a b

a

b c

d d

-50 -60 -70 -80

-80

-90

-90

-100

-100 0

1

2

3

4

5

6

Incubation time (h) Fig. 4. Reactivation of Escherichia coli O157:H7 during dark incubation phase after exposure to different UV light irradiation in apple juice at 4  C. ( ), Far UV; ( ), UVC; ( ), Far UVþ; ( ), C-105. a, b, c, d mean values within the same incubation time, with unlike letters were significantly different (P < 0.05, n ¼ 8). The concentration of E. coli O157:H7 in the apple juice before UV light irradiation was 7.32 Log CFU/mL.

0

1

2

3

4

5

6

Incubation time (h) Fig. 5. Reactivation of Escherichia coli O157:H7 during dark incubation phase after exposure to different UV light irradiation in apple juice at 20  C. ( ), Far UV; ( ), UVC; ( ), Far UVþ; ( ), C-105. a, b, c, dmean values within the same incubation time, with unlike letters were significantly different (P < 0.05, n ¼ 8). The concentration of E. coli O157:H7 in the apple juice before UV light irradiation was 7.32 Log CFU/mL.

334

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a

0

a a

-20

% Log reactivation

-40

a b b

-60

a

a

a

b b

b

-80

a

b

b

b b

-100

b b

-120 -140 -160

c

c

c

c

c

3

4

5

6

-180 -200 0

1

2

Incubation time (h) Fig. 6. Reactivation of Escherichia coli O157:H7 during dark incubation phase after exposure to different UV light irradiation in apple juice at 37  C. ( ), Far UV; ( ), UVC; ( ), Far UVþ; ( ), C-105. a, b, c mean values within the same incubation time, with unlike letters were significantly different (P < 0.05, n ¼ 8). The concentration of E. coli O157:H7 in the apple juice before UV light irradiation was 7.32 Log CFU/mL.

following exposure to UVC light, the reactivation ratio of cultures incubated at 37  C was lower (P < 0.05) than that of cultures incubated at 20 and 4  C during 1e6 h incubation, and no statistical difference was observed between cultures incubated at 20 and 4  C. For the E. coli O157:H7 following exposure to Far UVþ light irradiation, the reactivation ratio of cultures incubated at 37  C was lower (P < 0.05) than that incubated at 20 and 4  C during 2e6 h, and 1e6 h incubation, and that incubated at 20  C was also lower (P < 0.05) than that incubated at 4  C during 2e6 h incubation, respectively. 4. Discussion The E. coli O157:H7 strain 933 used in the present study is acidresistant, and up to 99.50% survived in apple juice (pH ¼ 3.5) at 20  C for 24 h. Notably, previous reports also indicated that this strain is capable of survival in different fruit products with pH values ranging from 2.51 to 3.26 at 4  C for 30 days (Marques et al., 2001; Mutaku et al., 2005). Because the refrigerated temperature of 4  C and ambient temperature of 20  C are normally used for the storage for apple juice products, it is necessary to decontaminate the apple juice due to enhancement of product safety. The E. coli O157:H7 is UV light sensitive and can be inactivated by UV light (Yaun et al., 2003). In addition, it seems that the inactivation of E. coli O157:H7 is UV fluence-dependent; the higher the fluence is, the lower the pathogen survives (Zimmer-Thomas et al., 2007). Similar results were also observed for Far UV and Far UVþ light in the present study. Notably, the inactivation of Far UV light on E. coli O157:H7 was more efficient than that of UVC and Far UVþ lights at the same UV fluences, and similar result was also observed with other bacterial strains (Pennell et al., 2008). The reason could be that different UV light wavelength caused different damage on the bacteria and it can be speculated that the cell damage caused by Far UV light was even more serious than that of the Farþ and UVC lights; however, further evidences are needed. The current study has investigated the potential reactivation of E. coli O157:H7 during dark incubation phase following exposure to each of the Far UV, UVC and Far UVþ light by simulating the refrigerated temperature of 4  C and ambient temperature of 20  C for apple juice storage, as well as after human ingested at 37  C (the regular human body temperature). It was obvious that the UV light wavelength strongly affected the survival of E. coli O157:H7 due to the significant differences of reactivation ratios among treatments

regardless of the incubation temperature. Notably, no reactivation of E. coli O157:H7 was observed during the 6-h dark incubation. Similar results for E. coli O157:H7 following exposure to UVC light were also observed during the time of holding in buffer (saline) and dark incubation for 48 h (Sommer et al., 2000). The negative reactivation is also observed in other E. coli strains following UV irradiation, for example strains K12 and B (Kashimada et al., 1996). In addition, the positive reactivation in dark incubation phase was only observed in some particularly responsive E. coli strains (Harm, 1968), which seems not to be valid for E. coli species in general. Interestingly, the E. coli O157:H7 concentration was decreased as the incubation temperature increased regardless of the UV light sources. The lowest concentration of E. coli O157:H7 was observed in samples following Far UV irradiation at each incubation time point. In this regard, the inactivation efficiency of Far UV light is more significant than that of UVC and Far UVþ lights on food borne pathogens. The integrities of bacterial membrane or reporter protein activities could be very important for evaluation of the effect of UV light irradiation on food borne pathogens, because Far UV light seems specific for destroying bacterial outer membranes and protein molecules rather than DNA compared to the UVC light (Edward Neister, 2010; Abdallah et al., 2012). This could be true in the present study, particularly for Far UV light treated E. coli O157:H7 cells, because the damaged membrane would result in the apple juice penetrating the E. coli O157:H7 cells and breaking the balance of the buffer system of intracellular fluid and affect the physiochemical and metabolic process critical to survival. It has been reported that the incubation temperature strongly affected the reactivation of E. coli in photo or dark incubation phase n et al., 2011); however, only following exposure to UV light (Gaya limited report is available on the effect of temperature on the dark reactivation of bacteria after exposure to UV light in juices. Previous research indicated that dark reactivation of E. coli following exposure to UV light occurs to a considerably lower degree than that in photo-incubation phase. After the maximum survival is reached, a decay process is observed, with survival diminishing in a linear trend over time regardless of the incubation temperature ranging from 5 to 30  C during the dark incubation phase (Salcedo et al., 2007). In the present study, the incubation temperature negatively affected the reactivation ratio of E. coli O157:H7 regardless of the UV light sources. The bacterial survival decreased as the incubation temperature increased. Notably, the significant reduction of E. coli O157:H7 occurred when the UV treated samples were incubated at 37  C during the dark incubation phase, particularly in that following exposure to Far UV light with more than an extra 3 Log reduction (171% of Log reactivation) within 2 h incubation. In other words, the survival of E. coli O157:H7 decreased rapidly to a concentration less than 2 Log CFU/mL within 2 h of incubation and probably continued decreasing until all cells were dead during the dark incubation phase (the plating limitation in the present study is 2 Log CFU/mL, and the detection that all cells were inactivated during 3e6 h of incubation was therefore impossible). The reasonable explanation is that with the increase of the incubation temperature, the metabolic processes critical to survival in the injured cells were stimulated, and the death of E. coli O157:H7 cells were accelerated. Such observation was previously proposed as n and Mackey, 2000; Leistner, 2000). “metabolic exhaustion” (Paga Therefore, exposure to Far UV light followed by storage at medium temperature would be a practical and effective way to decontaminate food borne bacteria. 5. Conclusions The inactivation of E. coli O157:H7 following exposure to Far UV light at 222 nm was higher than that following the irradiation of

F. Yin et al. / Food Microbiology 46 (2015) 329e335

UVC at 254 nm and Far UVþ lights at 282 nm in apple juice. No reactivation was observed during dark incubation phase for E. coli O157:H7 following exposure to far UV, UVC and Far UVþ lights in apple juice medium; particularly, the significant decrease of E. coli O157:H7 concentration was observed following Far UV light exposure. The incubation temperature strongly affected the survival of E. coli O157:H7 during dark incubation phase; the higher the temperature is, the lower the bacterial survives. The 37  C incubation caused approximately an extra 3 Log reduction of E. coli O157:H7 following Far UV light exposure, which is more effective than that of UVC and Far UVþ light.

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).

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Inactivation and potential reactivation of pathogenic Escherichia coli O157:H7 in apple juice following ultraviolet light exposure at three monochromatic wavelengths.

Ultraviolet (UV) light irradiation at 254 nm is considered as a novel non-thermal method for decontamination of foodborne pathogenic bacteria. However...
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