Environmental and biological factors influencing the UV-C resistance of Listeria monocytogenes

Food Microbiology 46 (2015) 246e253

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Environmental and biological factors influencing the UV-C resistance of Listeria monocytogenes

n, M.J. Serrano, R. Paga
n, I. Alvarez,
n* E. Gaya S. Condo Tecnología de los Alimentos, Universidad de Zaragoza, Facultad de Veterinaria, C/ Miguel Servet 177, CP 50013 Zaragoza, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 November 2012 Received in revised form 22 July 2014 Accepted 17 August 2014 Available online 27 August 2014

In this investigation, the effect of microbiological factors (strain, growth phase, exposition to sublethal stresses, and photorepair ability), treatment medium characteristics (pH, water activity, and absorption coefficient), and processing parameters (dose and temperature) on the UV resistance of Listeria monocytogenes was studied. The dose to inactivate 99.99% of the initial population of the five strains tested ranged from 21.84 J/mL (STCC 5672) to 14.66 J/mL (STCC 4031). The UV inactivation of the most resistant strain did not change in different growth phases and after exposure to sublethal heat, acid, basic, and oxidative shocks. The pH and water activity of the treatment medium did not affect the UV resistance of L. monocytogenes, whereas the inactivation rate decreased exponentially with the absorption coefficient. The lethal effect of UV radiation increased synergistically with temperature between 50 and 60
C (UV-H treatment). A UV-H treatment of 27.10 J/mL at 55
C reached 2.99 and 3.69 Log10 inactivation cycles of L. monocytogenes in orange juice and vegetable broth, and more than 5 Log10 cycles in apple juice and chicken broth. This synergistic effect opens the possibility to design UV combined processes for the pasteurization of liquid foods with high absorptivity. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Ultraviolet radiation Food pasteurization Listeria monocytogenes Combined processes Absorption coefficient

1. Introduction Heat treatment is generally the most used preservation technique for microbial inactivation since it results in the death of both vegetative cells and bacterial spores. However, some detrimental effects on sensorial and nutritional characteristics of foods often accompany thermal treatments. Thus, the use of ultraviolet (UV)based technologies as substitute for thermal pasteurization of liquid foods, especially for fruit juices, is gaining in popularity. UV radiation is able to inactivate a wide range of spoilage and pathogenic microorganisms with minimal changes in the nutritional and sensorial quality of foods, and it requires low energy consumption compared to other non-thermal pasteurization processes (Geveke,
n and Barbosa-Ca
novas, 2004). However, to 2005; Guerrero-Beltra transfer UV technology to the food industry, it is necessary to improve the knowledge on UV resistance of foodborne pathogens of concern and the effect of different environmental and processing factors on their sensitivity. Short-wave UV or UV-C radiation (200e280 nm) is considered the most germicidal UV region. UV-C photons are absorbed by the

* Corresponding author. Tel.: þ34 976 76 15 81; fax: þ34 976 76 15 90.
n). E-mail address: [email protected] (S. Condo http://dx.doi.org/10.1016/j.fm.2014.08.011 0740-0020/© 2014 Elsevier Ltd. All rights reserved.

nitrogenous bases of microbial DNA causing the formation of crosslinking photoproducts that inhibit transcription and replication,
pez-Malo and Palou, 2005). To and eventually lead to cell death (Lo cope with DNA damage, microorganisms have developed different DNA repair mechanisms that include photorepair or photoreactivation and light-independent or dark repair systems (Sinha and H€ ader, 2002). Photorepair ability, which consists in reversing DNA lesions by photolyase enzymes using the energy of visible light, is the most studied pathway due to its importance in the UV disinfection of water (Hallmich and Gehr, 2010). Although the germicidal effect of UV radiation has been widely studied, the UV resistance of different species of bacterial foodborne pathogens and their intraspecific variability have been scarcely studied (Koutchma et al., 2009). Furthermore, it is difficult to compare published data on microbial inactivation by UV radiation because conformation and geometry of UV equipment, flow pattern, and optical properties of the liquid play an important role in UV lethal efficacy (Müller et al., 2011). In addition, knowledge on the effect of physiological state of cells on UV resistance, such as growth phase and environmental stress history prior to treatment, is still limited. The physicochemical characteristics of the treatment medium also affect the bactericidal efficacy of UV treatments. The UV transmittance of a liquid mainly depends on the absorptivity of the


n et al. / Food Microbiology 46 (2015) 246e253 E. Gaya

medium and the present amount of suspended solids which scatter UV photons (Koutchma et al., 2004). Additional critical factors are the UV dose distribution inside the reactor (Koutchma et al., 2009) and the treatment temperature. The combination of UV radiation and mild heat has been reported to increase the UV inactivation of Escherichia coli and Salmonella enterica in laboratory media (Gay
an et al., 2011, 2012). The United States Food and Drug Administration has identified as pertinent bacterial pathogens for juice safety E. coli O157:H7 and S. enterica because of their historical association with foodborne outbreaks (U.S. FDA, 2001). Listeria monocytogenes has been also proposed by the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) due to its ubiquity and the threat that it poses to pregnant women. The ability of UV radiation to inactivate L. monocytogenes has been demonstrated in milk (Lu et al., 2011), fruit juice (Gabriel and Nakano, 2009), meat products (Sommers et al., 2010), and vegetable surfaces (Chun et al., 2010). However, the influence of microbial and environmental factors on the UV resistance of L. monocytogenes in liquids remains unknown. The objective of this work was to investigate the effect of microbiological factors (strain, growth phase, exposition to sublethal stresses, and photorepair ability), treatment medium characteristics (pH, water activity, and absorptivity), and processing parameters (UV dose and temperature) on the UV resistance of L. monocytogenes. The inactivation of L. monocytogenes in different liquid foods (orange juice, apple juice, vegetable broth, and chicken broth) by UV treatment was also explored. 2. Materials and methods 2.1. Bacterial culture and media The strains of L. monocytogenes STCC 4301, 4302, 5366, 932, and 5672 were provided by the Spanish Type Culture Collection (STCC). The strain of L. monocytogenes EGD-e and its isogenic deletion mutant DsigBdchromosomal deletion of region 930.725 bp 931.393 bp (Chatterjee et al., 2006)dwere used to study the role of the RNA polymerase sigma-B factor (sB) on UV resistance. The DsigB strain was kindly provided by Prof. Chakraborty (Institute for Medical Microbiology, Giessen, Germany). The bacterial cultures were maintained at 80
C in tryptone soy broth (TSB; Biolife, Milan, Italy) with 25% glycerol added. A broth subculture was prepared by inoculating 10 mL of TSB (Biolife) supplemented with 0.6% (w/v) yeast extract (Biolife) (TSBYE) with a single colony from tryptone soy agar (Biolife) supplemented with 0.6% (w/v) yeast extract (TSAYE). The subculture was incubated at 35
C for 6 h in a shaking incubator. With this subculture, a 250 mL Erlenmeyer flask containing 50 mL of TSBYE was inoculated to a concentration of 104 CFU/mL. Then, the culture flask was incubated with stirring at 35
C until the desired growth phase was reached: earlyexponential phase (containing approximately 105 CFU/mL; 6 h of incubation), mid-exponential phase (containing approximately 107 CFU/mL; 8 h of incubation), early-stationary phase (24 h of incubation), and late-stationary phase (72 h of incubation). 2.2. Treatment media and analytical measurements McIlvaine citrate-phosphate buffers (Dawson et al., 1974) of different pH (3.0, 4.0, 5.0, 6.0, and 7.0), water activities (aw; 0.94, 0.96, 0.98, and >0.99), and absorption coefficients (a; from 6.12 to 22.77 cm1) were used as treatment media. Citrate-phosphate buffers of different water activities and absorption coefficients were prepared by adding different quantities of glycerol (Panreac, Barcelona, Spain) and tartrazine (SigmaeAldrich, St. Louis MO, ~ oz y USA), respectively. As food matrices, apple juice (Antonio Mun

247

Cia, Spain; pH ¼ 3.21, a ¼ 25.54 cm1, turbidity ¼ 3.34 NTU), orange juice (Dafsa, Spain; pH ¼ 3.57, a ¼ 91.10 cm1, turbidity ¼ 4460 NTU), vegetable broth (Interal, Spain; pH ¼ 5.81, a ¼ 29.56 cm1, turbidity ¼ 2340 NTU), and chicken broth (Interal; pH ¼ 5.20, a ¼ 23.63 cm1, turbidity ¼ 4310 NTU) were used. The absorption coefficient (254 nm) of treatment media was measured using a Unicam UV500 spectrophotometer (Unicam Limited, Cambridge, UK) by following the procedure described by Koutchma et al. (2009). Sample solutions were diluted and evaluated using quartz cuvettes (Hellma, Müllheim, Germany) with path lengths of 1, 2, and 10 mm. The absorption coefficient of the sample solution was determined from the slope of the absorbance versus the path length. Turbidity was measured using an HI 83749 nephelometer (Hanna Instrument, Szeged, Hungary). The pH was adjusted using a pH meter BASIC 20 (Crison Instrument, Barcelona, Spain). Water activity was measured at room temperature with a specially designed instrument (Water Activity System mod. CX-1, Decagon Devices, Pullman, WA, USA). 2.3. Adaptation to sublethal stresses Prior to UV treatment, cells in the early-stationary phase of growth were exposed to different sublethal stresses (heat, acid, basic, and oxidative shocks) which exerted the highest increase in the homologous resistance of L. monocytogenes (data not shown). For stress adaptation, 1 mL of the bacterial suspension was resuspended in 9 mL of TSBYE either acidified (pH 4.5) with hydrochloric acid (Panreac) or alkalinized (pH 9.0) with sodium hydroxide (Panreac), or prewarmed at 48
C (pH 7.0). Cells were maintained for 1 h under acid and heat stress conditions and 2 h under the basic shock. For oxidative stress, bacteria were suspended in TriseHCl buffer (pH 7.0) with hydrogen peroxide (SigmaeAldrich) added (5 mM) and incubated for 2 h. During acid, alkaline, and hydrogen peroxide adaptation the temperature of the medium was kept at 25
C. 2.4. UV treatments UV treatments were carried out in the equipment previously
n et al. (2011). The system consisted of eight described by Gaya individual annular thin film flow-through reactors connected sequentially and fed by a peristaltic pump (ISM 10785, Ismatec, Glattbrugg, Switzerland). Each reactor consisted of a low-pressure mercury vapor lamp (8 W input power; TUV 8WT5, Philips, USA), that emitted 85% of energy at a wavelength of 254 nm, fixed at the axis of an outer glass tube (25 mm of inner diameter) and enclosed by a quartz tube (20 mm of outer diameter) to prevent the lamp's direct contact with the treatment medium. In the annular gap (2.5 mm), a stainless steel coil spring was installed in order to improve the flow's turbulence. Outside and inside coil diameters of the spring were 23 and 25 mm, respectively, and its length and pitch were 270 and 10 mm, respectively. To conduct UV-H treatments, the entire unit was submerged in a 90 L water bath heated by the circulating water of a peripheral thermostatic bath (Kattebad K12, Huber, Offenburg, Germany). A heating/cooling coil exchanger was placed before the inlet of the first reactor. Two thermocouples (639K, Crison Thermometer, Barcelona, Spain) that were fitted to the input of the first and the outlet of the last reactor allowed for the control of the temperature. The treatment medium was inoculated with the bacterial suspension to achieve approximately 107 CFU/mL and pumped through the equipment at a flow rate of 8.5 L/h. At this flow rate, the ratio between the mean residence time obtained from the residence time distribution curve (t) and the theoretical residence time calculated by the flow rate and the volume reactor (bt ¼ V=Q ) was

248


n et al. / Food Microbiology 46 (2015) 246e253 E. Gaya

close to the unit (0.944), indicating that the flow approached to a
n et al., 2011). When the treatment conditurbulent regime (Gaya tions were stabilized, samples were withdrawn through the sampling valves at each reactor's outlet, and 0.1 mL or 1 mL of the appropriate dilution was immediately pour-plated in the recovery media. Although most authors express the intensity of UV treatments by the delivered dose in terms of fluence rate per surface unit (Oteiza et al., 2010; Quintero-Ramos et al., 2004), for our purpose, the energy consumption per unit volume (mJ/L) was used because it allowed for comparison of the energetic efficiency of our system with other UV equipment and with other technologies. Treatment doses were calculated by the total input power of UV lamps and the flow rate, as the theoretical retention time in a reactor at 8.5 L/h was close to that determined experimentally from the residence time distribution of different volume fractions. The UV dose actually delivered to the treatment medium was estimated by the chemical actinometer iodide-iodate following the indications of Rahn et al. (2003). The actinometer buffer was pumped through the installation at the treatment flow rate and the increase in absorbance (352 nm) was determined at the outlet of each reactor
n et al., 2011). From these data, the photon flux (254 nm) (Gaya received per volume unit and the corresponding effective dose was estimated. Thus, from the energy consumed in each reactor (3.39 mJ/L), 0.49 mJ/L were delivered in form of UV photons. 2.5. Heat treatments Heat treatments were carried out in a specially designed resis
n et al. tometer (thermoresistometer TR-SC) described by Condo (1993). Briefly, this consisted of a 400-mL vessel equipped with an electrical heater for thermostation, an agitation device, and ports for injecting the microbial suspension and for sample extraction. Once the preset temperature had attained stability (T ± 0.05
C), 0.2 mL of an adequately diluted microbial suspension was inoculated into the corresponding treatment medium (350 mL). After inoculation, samples of 0.2 mL were collected at different heating times (intervals between 10 s and 1 min) and immediately pour-plated.

Automatic Colony Counter (Protos, Synoptics, Cambridge, UK), as
n et al. (1987) detailed. It was considered as detection limit Condo 30 cfu/plate. 2.7. Photoreactivation experiments For photoreactivation tests, a volume of 20 mL of different dilutions of each sample was spread-plated in TSAYE and incubated under daylight in a dispositive formed by four fluorescent luminaires (T16 13 W/827-EVG, Osram, Munich, Germany), which
n emitted light in the 360 nme700 nm wavelength range (Gaya et al., 2011). Plates were exposed under a mean illuminance (visible light radiometer FL A603 VL4, Ahlborn, Holzkirchen, Germany) of 11.15 klux for 60 min at room temperature. Longer exposure times showed no improvement in counts. In each experiment, a duplicate TSAYE plate of each UV-treated sample was maintained in a dark box under the same conditions. After daylight exposure, Petri dishes were incubated for 24 h at 35
C in dark conditions. 2.8. Curve fitting and statistical analysis Survival curves to UV treatments were obtained by plotting the logarithm of the survival fraction (Log10 N/N0) versus treatment doses expressed in energy consumption. When UV radiation was combined with mild heat, the intensity of treatments was also expressed in time unit (min), as in the survival curves for heat treatments. To fit survival curves and to calculate kinetics parameters, the GInaFiT model-fitting tool (KU Leuven, Leuven, Belgium) was used. Because our survival curves showed ‘shoulders’ and no ‘tails’, the log-linear regression plus shoulder model of Geeraerd et al. (2000) was used (Equation 1). This model describes the survival curves through two parameters: the shoulder length (Sl), defined as the dose or time before the exponential inactivation begins, and the inactivation rate (Kmax), which corresponds to the slope of the exponential portion of the survival curve. The GInaFiT software also provides the 4D value, defined as the treatment dose or time that inactivates 99.99% of the microbial population, which was used for comparisons.

0

2.6. Incubation of treated samples and survival counting

Sl Kmax @

The recovery medium used was TSAYE. Where indicated, cells were also recovered in TSAYE supplemented with the maximum non-inhibitory concentration (MNIC) of sodium chloride (TSAYESC) (Panreac). The lack of tolerance to the presence of sodium chloride can be attributed to damage to the functionality and/or integrity of the cytoplasmic membrane (Mackey, 2000). The MNIC of sodium chloride used (4.5e5.5% w/v depending on the strain) was chosen by plating non-treated cells in TSAYE with various concentrations of the chemical (% w/v), and it was defined as the sodium chloride concentration which caused a decrease in counts less than 25% (data not shown). In addition, samples were also plated in TSAYE supplemented with 0.1% (w/v) of sodium pyruvate (Panreac) (TSAYE-P). Sodium pyruvate removes peroxides and improves recovery of oxidative stressed cells (Mackey, 2000). TSAYE-P plates were incubated under anaerobic conditions (5% hydrogen, 10% carbon dioxide, and 85% nitrogen) in a variable-atmosphere incubator (MACS VA500, Don Whitley Scientific Limited, Shipley, UK). Samples recovered in the non-selective medium with and without sodium pyruvate added were incubated for 48 h at 35
C, and those recovered in the selective medium for 72 h at 35
C. Previous experiments demonstrated that longer incubation times did not change the survival counts. After incubation, colony forming units (CFU) were counted with an improved Image Analyzer

Nt ¼ N0 e

Sl

eKmax

1

A   Sl t 1 þ eKmax 1 eKmax

(1)

Statistical analyses, t-test and ANOVA tests, were carried out using the GraphPad PRISM 5.0 software (GraphPad Software, Inc., San Diego, CA, USA), and differences were considered to be significant for p  0.05. All microbial resistance determinations were performed at least three times on different working days. The error bars in the figures correspond to the mean standard deviation. 3. Results and discussion 3.1. Biological factors influencing UV resistance of L. monocytogenes Fig. 1 shows the survival curves of five strains of L. monocytogenes in the early-stationary phase of growth treated by UV radiation at room temperature (25
C) in a reference treatment medium: citrate-phosphate buffer of pH 7.0 with 0.25 g/L of tartrazine added (absorption coefficient of 11.04 cm1). Typically, microbial inactivation curves by UV radiation display a sigmoidal profile characterized by an initial lag phase, an exponential death phase, and a tailing phase towards the end of the treatment (Sastry et al., 2000). However, some authors have described UV inactivation as a first-order kinetics (Franz et al., 2009; Oteiza et al., 2010).


n et al. / Food Microbiology 46 (2015) 246e253 E. Gaya

Fig. 1. Survival curves of L. monocytogenes STCC 5672 (C), 5366 (:), 4032 (-), 932 (B), and STCC 4031 (D) treated by UV radiation in citrate-phosphate buffer of pH 7.0 with an absorption coefficient of 11.04 cm1. Cells were recovered in a non-selective medium (TSAYE).

In this work, survival curves showed a lag or shoulder phase that can be attributed to the multi-hit nature of UV inactivation. UV-C photons absorbed by microbial DNA cause mutations that compromise cell viability (Sinha and H€ ader, 2002). However, bacteria have developed different strategies to cope with DNA damage and restore its functionally, which results in shoulders phases of UV inactivation curves. When DNA repair mechanisms are surpassed, minimal additional UV exposure would be lethal for microorgan
pez-Malo and isms and survivor numbers decline exponentially (Lo Palou, 2005). Tailing phases, which have been related to different UV resistance of subpopulations, cellular aggregates, and/or non-

249

uniform dose distribution inside the reactor (Koutchma et al., 2004), were not observed in this study. To compare UV resistance, survival curves were described by the log-linear regression plus shoulder model proposed by Geeraerd et al. (2000). Table 1 shows the averages and standard deviations of the kinetics parameters obtained after fitting survival curves of the five strains of L. monocytogenes tested in the reference conditions, as well as the coefficient of determination (R2) and the root mean square error (RMSE) of the fits. As it is shown in Table 1, UV resistance of L. monocytogenes varied widely among strains: a treatment of 20.32 J/mL reached 3.82 ± 0.02, 4.76 ± 0.12, 4.98 ± 0.04, 5.34 ± 0.16, and more than 6 Log10 reductions of L. monocytogenes STCC 5672, STCC 4032, STCC 5366, STCC 932, and STCC 4031, respectively (Fig. 1). The dose to inactivate 99.99% of the initial population (4D value) ranged from 21.84 J/mL for the most UV resistant strain (STCC 5672) to 14.66 J/mL for the most sensitive one (STCC 4031) (Table 1). Although there are some studies on the ability of UV radiation to inactivate L. monocytogenes in laboratory media and in food matrices, it is difficult to compare UV resistance data because the design of UV equipment, processing parameters, and optical properties of the liquid change UV germicidal efficacy (Müller et al., 2011). Furthermore, to the best of our knowledge, the variability in UV resistance among L. monocytogenes strains has not been exhaustively studied. However, results included in Table 1 can be directly compared with UV resistance data previously obtained in our installation in the same treatment medium for other bacterial species. The 4D value estimated for the most resistant L. monocytogenes strain (STCC 5672; 4D ¼ 21.84 ± 0.77 J/mL) was significantly higher than that previously obtained for the most resistant strain of E. coli (4D ¼ 16.60 ± 0.48 J/mL) and S. enterica

Table 1 Kinetics parameters (Sl, Kmax, and 4D) obtained after fitting the log-linear with shoulder model of Geeraerd et al. (2000) to the survival curves of five strains of L. monocytogenes treated by UV radiation (citrate-phosphate buffer of pH 7.0 with an absorption coefficient of 11.04 cm1) in different growth phases and recovered in different media. Strain

Growth phase

Recovery medium

Sl (J/mL)

L. monocytogenes STCC 5672

Early-stationary

TSAYE TSAYE-SC TSAYE-P TSAYE-visible light TSAYE TSAYE-SC TSAYE-P TSAYE TSAYE-SC TSAYE-P TSAYE TSAYE-SC TSAYE-P TSAYE TSAYE-SC TSAYE-P TSAYE TSAYE-SC TSAYE-P TSAYE TSAYE-SC TSAYE-P TSAYE TSAYE-SC TSAYE-P

8.24 7.58 7.28 9.42 7.59 8.91 8.63 8.00 8.55 8.97 8.31 9.13 8.47 4.53 3.59 3.71 6.50 6.32 6.64 5.92 5.34 6.51 5.71 5.76 6.14

Early-exponential

Mid-exponential

Late-stationary

L. monocytogenes STCC 4031

Early-stationary

L. monocytogenes STCC 4032

Early-stationary

L. monocytogenes STCC 5366

Early-stationary

L. monocytogenes STCC 932

Early-stationary

(0.14)aA (0.51) (0.97) (0.46)* (1.63)A (1.56) (0.56) (0.84)A (1.17) (1.24) (1.12)A (0.82) (1.23) (0.11)b (0.87) (0.44)* (0.49)c (1.21) (0.55) (0.17)c (0.87) (0.73) (0.84)c (1.36) (1.62)

Kmax (mL/J) 0.71 0.78 0.74 0.72 0.84 0.87 0.98 0.94 0.98 0.87 0.87 0.89 0.82 0.92 0.86 0.96 0.75 0.73 0.71 0.72 0.71 0.75 1.36 1.10 1.07

(0.05)aA (0.04) (0.07) (0.06) (0.19)A (0.10) (0.20) (0.13)A (0.04) (0.09) (0.07)A (0.08) (0.12) (0.02)b (0.03) (0.05) (0.07)a (0.15) (0.02) (0.01)a (0.03) (0.03) (0.16)c (0.14) (0.15)

4D (J/mL) 21.84 19.88 19.86 22.55 20.07 18.05 18.15 21.04 20.69 21.00 20.24 19.65 19.90 14.66 14.80 15.75 18.97 18.28 19.54 18.86 18.37 18.96 17.98 18.37 16.84

(0.77)aA (0.84) (0.59) (1.35) (1.93)A (0.24) (0.54) (0.94)A (0.57) (0.33) (1.20)A (0.53) (0.36) (0.10)b (0.28) (0.23) (0.75)c (0.96) (0.43) (0.17)c (0.32) (0.26) (0.25)c (0.47) (0.74)

R2

RMSE

0.983 0.993 0.978 0.983 0.986 0.974 0.981 0.993 0.946 0.982 0.993 0.991 0.990 0.988 0.996 0.980 0.995 0.977 0.998 0.993 0.996 0.996 0.984 0.996 0.971

0.331 0.232 0.429 0.355 0.270 0.486 0.344 0.199 0.168 0.285 0.208 0.526 0.365 0.355 0.203 0.455 0.207 0.486 0.136 0.264 0.210 0.190 0.144 0.210 0.409

Values in parentheses represent the standard deviations of means of three replicates. TSAYE, tryptic soy agar supplemented with yeast extract (0.6%); TSAYE-SC, TSAYE with sodium chloride added (4.5e5.5%); TSAYE-P, TSA with sodium pyruvate added (0.1%); TSAYE-visible light, TSAYE with a prior photoreactivation step (11.13 klux for 60 min). Lowercase letters indicate statistically significant differences (p  0.05) among kinetics parameters of different strains in the early-stationary phase of growth recovered in TSAYE. Uppercase letters indicate statistically significant differences (p  0.05) among kinetics parameters of different growth phases of the same strain recovered in TSAYE. Asterisk indicates statistically significant differences (p  0.05) between kinetics parameters of the same microorganism in the same growth phase obtained in TSAYE, TSAYESC, and TSAYE-P.

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n et al., 2011, 2012). Several authors (4D ¼ 18.03 ± 0.13 J/mL) (Gaya have reported the higher UV resistance of L. monocytogenes in comparison to other foodborne pathogens in milk (Lu et al., 2011), fruit juice (Gabriel and Nakano, 2009), and solid surfaces (Rowan et al., 1999). This fact has been attributed to the thicker peptidoglycan cell wall and higher chromosome condensation of Grampositive bacteria in comparison to Gram-negatives (Beauchamp and Lacroix, 2011), as well as to the higher efficient DNA repair systems of L. monocytogenes in comparison to E. coli (Cheigh et al., 2012). The detection of sublethal damaged bacteria following exposure to UV radiation is critical since injured cells could recover and return to normal physiology and pathogenicity under suitable conditions (Wuytack et al., 2003). However, there is so far little information available on the sublethal damage induced by UV treatment in foodborne pathogens. In our study, the comparison of survivors recovered in the non-selective (TSAYE) and the selective medium with sodium chloride added (TSAYE-SC) showed that UV treatment did not damage the functionality and integrity of L. monocytogenes cytoplasmic membrane in any of the strains investigated (Table 1). Similarly, Pataro et al. (2011) found no appreciable sublethal damage in Listeria innocua after being treated by pulsed UV light using selective growth media technique. Counts obtained in non-selective medium with 0.1% of sodium pyruvate added (TSAYE-P) showed that UV treatment also did not cause oxidative stress (Table 1). Exposure to visible light of UV-irradiated cells slightly increased the recovery of survivors of the most resistant strain of L. monocytogenes. As shown in Table 1, the 4D value for L. monocytogenes STCC 5672 increased from 21.84 ± 0.77 to 22.55 ± 1.35 J/mL after photoreactivation. This increase in UV resistance was due to the lengthening of the shoulder length (Sl), whereas the inactivation rate (Kmax) barely changed. This evidences that the shoulders of our inactivation curves were related to the action of DNA repair mechanisms. The photoreactivation ability of L. monocytogenes was lower than that we reported for E. coli under
n et al., 2011). the same experimental conditions (Gaya Table 1 includes UV kinetics parameters of L. monocytogenes STCC 5672 in the early-exponential, mid-exponential, earlystationary, and late-stationary phase of growth. In general, stationary phase cells of L. monocytogenes are more resistant to thermal and non-thermal preservation techniques than exponentially
growing cells (Alvarez et al., 2002; Mackey et al., 1995). Surprisingly, no significant differences were found between the UV resistance of exponential and stationary phase cells of L. monocytogenes STCC 5672. In contrast, several authors have reported the dependence of the UV resistance on the growth phase of E. coli (Bucheli
n et al., 2011) and S. enterica (Child et al., Witschel et al., 2010; Gaya
n et al., 2012). The higher UV resistance of stationary 2002; Gaya phase cells of Gram-negative bacteria has been attributed to the transcription of the general stress sigma factor RpoS. Similarly, Gram-positive bacteria possess the alternative sigma B factor (sB) which is considered by many researchers as functionally homologous to the RpoS factor (Gertz et al., 2000). In fact, it has been demonstrated that the enhanced resistance of stationary phase cells of L. monocytogenes to other preservation techniques are induced by the activation of sB (Becker et al., 1998; Somolinos et al., 2010). Therefore, our results suggest that the expression of the alternative sB factor would not be involved in the UV resistance of L. monocytogenes. As in the case of early-stationary phase cells, neither signs of cytoplasmic membrane injury nor oxidative damage was found after UV treatment in any of the tested cell ages (Table 1). Exposure of microorganisms to sublethal environmental stresses during food processing may trigger adaptive responses that enhance their resistance to subsequent applications of the

same stress (homologous response) or to other stresses of a different nature (heterologous response) (Van Schaik and Abee, 2005; Wesche et al., 2009). The stress adaptation of foodborne pathogens and its consequent cross-protection to novel food processing technologies, especially to UV radiation, has been scarcely studied. Table 2 shows the UV kinetics parameters of L. monocytogenes STCC 5672 after being exposed to heat, acid, basic, and oxidative sublethal shocks. As observed, the prior exposition of L. monocytogenes to any of the adverse conditions did not increase its UV resistance. Data available on the UV resistance of stressed L. monocytogenes cells are limited and contradictory. Bradley et al. (2012) also reported that the exposure of L. monocytogenes to sublethal acid and heat conditions resulted in similar or increased sensitivity to pulsed UV light treatments. By contrast, McKinney et al. (2009) found that the adaptation of L. monocytogenes to acidic environments provided cross-protection against UV exposure, while heat shocks made L. monocytogenes more vulnerable. Alternative sB factor regulon is regarded as the main regulator of adaptive response of L. monocytogenes to multiple adverse conditions, in addition to its role during the transition from exponential growth to stationary phase (Van Schaik and Abee, 2005). Therefore, the lack of environmental stress response was consistent with data obtained when the effect of growth phase was studied (Table 1), suggesting again that the activation of stress promoters did not affect UV resistance. To confirm the irrelevant role of the sB factor on UV resistance, survival curves of L. monocytogenes EGD-e and its isogenic deletion mutant DsigB in the mid-exponential and earlystationary phase of growth were obtained (Table 3). No significant differences were found between 4D values of the wild-type and mutant strain in either stationary or exponential phase of growth, evidencing that the UV resistance of L. monocytogenes was not related to the sB factor. Our results agree with those obtained by Wassmann et al. (2011) where the resistance of stationary phase cells of a Bacillus subtilis wild-type strain was almost identical to that of its isogenic DsigB mutant. 3.2. Effect of environmental factors on the UV inactivation of L. monocytogenes It is well known that the physicochemical characteristics of the treatment medium can affect the bactericidal efficacy of most food preservation techniques. In general, the pH and water activity of the treatment medium are the most influential factors (Gould, 1992). Table 4 includes kinetics parameters of L. monocytogenes STCC 5672 treated by UV radiation in media of different pH and water activities. Both physicochemical properties had little effect on the UV sensitivity of L. monocytogenes between pH 3.0 to 7.0 and water activities between 0.94 and >0.99. Either the interaction between pH and water activity in extreme conditions did not change the UV Table 2 Kinetics parameters (Sl, Kmax, and 4D) obtained after fitting the log-linear with shoulder model of Geeraerd et al. (2000) to the survival curves of L. monocytogenes STCC 5672 treated by UV radiation (citrate-phosphate buffer of pH 7.0 with an absorption coefficient of 11.04 cm1) after the exposition to heat, acid, basic, and oxidative shocks. Cells were recovered in a non-selective medium (TSAYE). Stress

Sl (J/mL)

Kmax (mL/J)

4D (J/mL)

R2

RMSE

Control Heat shock Acid shock Basic shock Oxidative shock

8.24 8.34 7.32 7.11 8.27

0.71 0.74 0.78 0.83 0.78

21.84 20.79 18.97 18.31 18.94

0.983 0.985 0.983 0.998 0.975

0.331 0.335 0.397 0.136 0.440

(0.14) (1.20) (1.02) (0.43)* (0.48)

(0.05) (0.07) (0.06) (0.07) (0.10)

(0.77) (1.27) (1.15) (0.93)* (1.26)

Values in parentheses represent the standard deviations of means of three replicates. Asterisk indicates significant differences (p  0.05) among kinetics parameters of different stressed cells and non-adapted (control) cells.


n et al. / Food Microbiology 46 (2015) 246e253 E. Gaya

251

Table 3 Kinetics parameters (Sl, Kmax, and 4D) obtained after fitting the log-linear with shoulder model of Geeraerd et al. (2000) to the survival curves of L. monocytogenes EGD-e (wild-type; WT) and its isogenic DsigB mutant treated by UV radiation (citrate-phosphate buffer of pH 7.0 with an absorption coefficient of 11.04 cm1) in the early-stationary and mid-exponential phase of growth. Cells were recovered in a non-selective medium (TSAYE). Strain Growth phase

Sl (J/mL)

Kmax (mL/J) 4D (J/mL)

R2

RMSE

WT

5.55 4.60 5.18 4.47

0.88 0.79 0.85 0.80

0.992 0.995 0.987 0.988

0.277 0.201 0.367 0.307

DsigB

Early-stationary Mid-exponential Early-stationary Mid-exponential

(0.69) (0.36) (1.06) (0.62)

(0.08) (0.15) (0.12) (0.07)

16.59 16.45 16.32 16.21

(0.52) (0.36) (0.83) (0.59)

Values in parentheses represent the standard deviations of means of three replicates.

resistance of L. monocytogenes. These results are in agreement with previously published data on the UV inactivation of other bacterial
n et al., species in media with different pH and water activities (Gaya 2011, 2012; Murakami et al., 2006). Optical properties exert a major influence in UV germicidal effect in liquid media due to absorption and scattering phenomena (Koutchma et al., 2009). However, the contribution of each individual factor has been scarcely investigated. In this study, we focused on the effect of the treatment medium's absorbance for the inactivation of L. monocytogenes STCC 5672, while other physicochemical parameters (pH 7.0, aw > 0.99) and turbidity (5.92 NTU) were held constant. Table 4 shows the UV kinetics parameters of L. monocytogenes in media with absorption coefficients ranging from 6.12 to 22.77 cm1. As shown in Table 4, the shoulder length (Sl) and 4D value increased with the absorption coefficient, whereas the inactivation rate (Kmax) decreased. These results are consistent with the BeereLamberteBougerts Law which states that the amount of UV photons that penetrates through a solution decreases with increases in the absorbance of the liquid. Plotting Kmax values against the absorption coefficient, an exponential relationship between both variables was observed (Fig. 2). Nevertheless, other authors (Koutchma et al., 2004; Oteiza et al., 2005) have deduced a linear relationship between UV inactivation and the absorption coefficient working with media of a narrow range of absorptivities. From the regression line that related Log10 Kmax and

Fig. 2. Relationship between treatment medium's absorption coefficient (a) and the inactivation rate (Log10 Kmax) obtained after fitting the log-linear with shoulder model of Geeraerd et al. (2000) to the survival curves of L. monocytogenes STCC 5672 treated by UV radiation in citrate-phosphate buffers (pH 7.0, aw > 0.99) of different absorptivities. Cells were recovered in a non-selective medium (TSAYE).

Table 4 Kinetics parameters (Sl, Kmax, and 4D) obtained after fitting the log-linear with shoulder model of Geeraerd et al. (2000) to the survival curves of L. monocytogenes STCC 5672 treated by UV radiation in citrate-phosphate buffer of different pH, water activities (aw), and absorption coefficients (a). Cells were recovered in a nonselective medium (TSAYE).

3.3. UV-H treatments

pH

aw

a (cm1) Sl (J/mL)

Kmax (mL/J)

4D (J/mL)

R2

RMSE

3.0 4.0 5.0 6.0 7.0 7.0 7.0 7.0 3.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0

0.99 0.99 0.99 0.99 0.99 0.98 0.96 0.94 0.94 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99

11.04 11.04 11.04 11.04 11.04 11.04 11.04 11.04 11.04 6.12 8.89 12.94 14.61 17.02 18.91 19.89 22.77

0.71 0.71 0.79 0.70 0.71 0.74 0.71 0.65 0.71 1.65 1.04 0.56 0.34 0.24 0.22 0.17 0.10

19.98 20.12 20.00 21.58 21.84 20.61 21.00 20.51 20.01 10.81 13.55 24.94 e e e e e

0.980 0.990 0.984 0.989 0.983 0.979 0.991 0.995 0.996 0.993 0.990 0.992 0.985 0.994 0.996 0.989 0.985

0.121 0.247 0.381 0.277 0.331 0.433 0.223 0.172 0.169 0.315 0.372 0.172 0.147 0.071 0.046 0.058 0.240

8.64 7.65 8.18 8.21 8.24 8.12 7.82 7.49 8.10 2.82 6.01 8.98 9.12 9.49 9.06 10.10 11.24

(0.73) (0.53) (0.37) (0.29) (0.14) (0.77) (0.32) (0.99) (0.11) (0.48) (0.35) (1.50) (0.54) (1.22) (0.89) (1.74) (3.50)

(0.2) (0.07) (0.02) (0.09) (0.05) (0.04) (0.08) (0.09) (0.03) (0.34) (0.01) (0.05) (0.03) (0.02) (0.01) (0.02) (0.02)

(0.62) (0.13) (0.41) (0.62) (0.77) (0.62) (0.21) (0.95) (0.66) (0.68) (0.31) (0.54)

Values in parentheses represent the standard deviations of means of three replicates.

the absorption coefficient (Log10 Kmax ¼ 0.0722a þ 0.652, R2 ¼ 0.991) was deduced that the inactivation rate decreased ten times by increasing the medium's absorption coefficient by 14.1 ± 2.0 cm1. This value does not significantly differ from that obtained for E. coli (15.9 ± 1.0 cm1) and S. enterica
n et al., (18.9 ± 2.8 cm1) in the same experimental conditions (Gaya 2011, 2012). This result indicates that the effect of medium's absorptivity on UV resistance is based on physical aspects, the number of photons that reach the microbial DNA, and therefore it does not depend on the biological response of microorganisms. As Table 4 illustrates, UV treatment scarcely reduced L. monocytogenes population in media of high absorption coefficients. Thus, applying the maximum dose possible in a single pass through our installation (27.10 J/mL) only 0.84 ± 0.03 Log10 inactivation cycles were achieved in the medium of absorption coefficient of 22.77 cm1. Consequently, UV treatment of liquid foods such as fruit juices, whose absorption coefficient are close to these values (Koutchma et al., 2009), will be far from achieving the 5 Log10 reductions required by the U.S. FDA (2001). This makes it difficult to transfer UV technology to the food industry.

We previously demonstrated that the combination of heat treatment at mild temperatures with UV radiation (UV-H treatment) enhances synergistically the UV inactivation of E. coli and
n et al., 2011, 2012). This hurS. enterica in laboratory media (Gaya dles approach opens the possibility to design UV-H combined processes for the pasteurization of liquid foods with high absorptivity. Nowadays, there are no reported data on the inactivation of L. monocytogenes by UV-H treatments. Therefore, the effect of mild temperatures on the UV resistance of this species was studied. For this purpose, L. monocytogenes STCC 5672 was UV treated at temperatures ranging from 50 to 60
C in citrate-phosphate buffer of pH 7.0 with an absorption coefficient of 22.77 cm1. As observed in Fig. 3, the UV inactivation of L. monocytogenes hardly changed when the temperature was raised up to 50
C, but above this temperature UV lethality drastically increased: a UV treatment of 27.10 J/mL (3.58 min) at 50.0, 52.5, 55.0, 57.5, and 60.0
C reduced the survival counts by 1.34 ± 0.06, 2.26 ± 0.23, 3.27 ± 0.32, 4.41 ± 0.55, and more than 6 Log10 cycles, respectively. Previously, it was reported that more than 5 Log10 reductions of the most UV resistant strain of E. coli in the same treatment conditions was achieved with a UV

252


n et al. / Food Microbiology 46 (2015) 246e253 E. Gaya

Fig. 3. Survival curves of L. monocytogenes STCC 5672 treated by UV radiation at different temperatures d25.0 (C), 50.0 (-), 52.5 (:), 55.0 (B), 57.5 ( ), and 60.0
C (D) din citrate-phosphate buffer (pH 7.0, aw > 0.99) with an absorption coefficient of 22.77 cm1. Cells were recovered in a non-selective medium (TSAYE).

▫


n et al., 2011). However, for treatment of 27.10 J/mL at 55.0
C (Gaya L. monocytogenes inactivation, the temperature should be raised up to 60
C to achieve the same goal. These differences might be explained by the higher heat and UV resistance of L. monocytogenes. To determine the contribution of thermal effects to the inactivation of UV-H treatments, heat resistance of L. monocytogenes was determined in the same treatment medium. Heat treatments at 50.0, 52.5, and 55.0
C for the same time than UV treatment (3.58 min) showed a negligible lethal effect (<0.3 Log10 inactivation cycles), whereas the combination of UV and heat reached 1.34 ± 0.06, 2.26 ± 0.23, 3.27 ± 0.32 Log10 reductions, respectively. Thus, the inactivation of L. monocytogenes by UV-H treatments resulted from a synergistic lethal effect between both technologies, since the lethal effect of the combined treatment was higher than the sum of the inactivation obtained by heat and UV treatment (27.10 J/mL) separately. At temperatures higher than 55.0
C, the lethality of UV-H treatment was further increased, as the heat inactivation rate did. Fig. 4 displays the relationship between temperature and the Log10 Kmax values for heat and UV-H treatments, expressed in time units (min1). As observed, the relationship between temperature and the inactivation rate for heat treatments was exponential, from which was estimated a z value of 6.1 ± 0.3
C. Similar z values have been obtained for L. monocytogenes by other authors (Monfort et al., 2012; Pag
an et al., 1998). By contrast, the inactivation rate of UV-H treatment

Fig. 4. Relationship between temperature and the inactivation rate (Log10 Kmax) obtained after fitting the log-linear with shoulder model of Geeraerd et al. (2000) to the survival curves of L. monocytogenes STCC 5672 treated by UV-H (C) and heat alone (B) in citrate-phosphate buffer (pH 7.0, aw > 0.99) with an absorption coefficient of 22.77 cm1. Cells were recovered in a non-selective medium (TSAYE).

hardly changed with temperature up to 50.0
C. Over this value, the Kmax value increased ten times by increasing the temperature by 13.5 ± 1.9
C. As consequence, the UV inactivation was less thermodependent than heat inactivation, so that the UV-H synergistic effect tended to disappear with temperature increases. This reveals that the optimization of UV-H treatment temperature is essential to exploit the potential of the combination of both technologies. To evaluate if the synergistic UV-H inactivation also occurred in food matrices, L. monocytogenes STCC 5672 was treated by the combined treatment in orange juice, apple juice, vegetable broth, and chicken broth. Fig. 5 shows the Log10 reductions of L. monocytogenes by a UV-H treatment of 27.10 J/mL (3.58 min) at 55.0
C, as well as by the corresponding heat and UV treatment at 25
C. The UV-H treatment reduced by 2.99 ± 0.14, 5.63 ± 0.52, 3.69 ± 0.19, and 5.03 ± 0.32 Log10 cycles the L. monocytogenes population inoculated in orange juice, apple juice, vegetable broth, and chicken broth, respectively. Again, the lethal effect of the combined treatment was higher than the sum of heat and UV inactivation: 1.12 ± 0.27, 2.72 ± 0.52, 1.84 ± 0.22, and 1.98 ± 0.36 additional Log10 cycles of inactivation were achieved in orange juice, apple juice, vegetable broth, and chicken broth, respectively (Fig. 5). Thus, synergistic lethal effect of UV-H combination also occurred in foods, although the magnitude of the synergy varied markedly among food products. These differences may be explained by the different physicochemical characteristics (pH, absorption coefficient, and turbidity) of foods that determine UV and heat resistance. This is an interesting aspect that deserves further investigation. 4. Conclusions In this investigation, the UV inactivation of different strains of L. monocytogenes was characterized, which reflected the wide intraspecific variability in resistance of this species. The most UV resistant strain of L. monocytogenes was more resistant than UV tolerant strains of other emerging foodborne bacteria such as E. coli and S. enterica. Therefore, L. monocytogenes should be considered as a pertinent pathogen for processing foods susceptible to L. monocytogenes contamination and/or growth. Growth phase and sublethal shocks did not change the UV resistance of L. monocytogenes, which advantages UV technology over other thermal and non-thermal processing technologies. This agrees with the irrelevant role of sB factor on the UV resistance of L. monocytogenes, which has been described for the first time in this work.

Fig. 5. Inactivation of L. monocytogenes STCC 5672 by UV (27.10 J/mL or 3.58 min at room temperature; black bars), heat (3.58 min at 55.0
C; white bars), and UV-H treatment (27.10 J/mL or 3.58 min; gray bars) in different food matrices: orange juice (OJ), apple juice (AJ), vegetable broth (VB), and chicken broth (CB). Cells were recovered in a non-selective medium (TSAYE).


n et al. / Food Microbiology 46 (2015) 246e253 E. Gaya

Contrary to most non-thermal technologies, UV resistance of L. monocytogenes was not affected by the pH and water activity of the treatment medium. However, the UV inactivation was drastically impaired by increases of absorption coefficient's medium, indicating the difficulty of processing liquid foods, such as fruit juice, by UV radiation. However, the UV inactivation of L. monocytogenes can be enhanced synergistically increasing the treatment temperature between 50 and 60
C. Also synergistic UVH inactivation of L. monocytogenes appears in fruit juices and broths, which opens the possibility of designing combined treatments to pasteurize these products at mild temperatures. Acknowledgments This study has been carried out with financial support from the
n, EU-FEDER (CIT020000-2009Ministerio de Ciencia e Innovacio 40) and the Departamento de Ciencia, Tecnología y Universidad del
n (UNIZAR-229307). E. G. and M. J. S. gratefully Gobierno de Arago acknowledge the financial support for their doctoral studies from
n y Ciencia. the Ministerio de Educacio References

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