International Journal of Food Microbiology 201 (2015) 7–16

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Previous physicochemical stress exposures influence subsequent resistance of Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes to ultraviolet-C in coconut liquid endosperm beverage Alonzo A. Gabriel ⁎ Department of Food Science and Nutrition, College of Home Economics, Alonso Hall, A. Ma. Regidor Street, University of the Philippines Diliman, 1101 Quezon City, Philippines

a r t i c l e

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Article history: Received 16 September 2014 Received in revised form 29 January 2015 Accepted 2 February 2015 Available online 8 February 2015 Keywords: Coconut beverage Non-thermal food processing Pasteurization Stress adaptation Ultraviolet radiation

a b s t r a c t This study investigated the influences of prior exposures to common physicochemical stresses encountered by microorganisms in food and food processing ecologies such as acidity, desiccation, and their combinations, on their subsequent susceptibility towards UV-C treatment in coconut liquid endosperm beverage. Cocktails of Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes were separately subjected to gradually acidifying environment (final pH 4.46), exposed to abrupt desiccation by suspension in saturated NaCl solution (aw = 0.85) for 4, 8, and 24 h, and sequential acidic and desiccated stresses before suspending in the coconut beverage for UV-C challenge. The exposure times (D) and UV-C energy dose values (DUV-C) necessary to reduce 90% of the population of the different test organisms varied with previous exposures to different sublethal stresses, indicating possible influence of implicit microbial factors towards resistance to UV-C. All tested individual and combined stresses resulted in increased resistance, albeit some were not statistically significant. Non-stressed cells had D values of 3.2–3.5 s, and corresponding DUV-C values of 8.4–9.1 mJ/cm2. Cells exposed to previous acid stress had D values of 4.1–4.8 s and corresponding DUV-C values of 10.7–12.5 mJ/cm2. Prior exposure to desiccation resulted in D values of 5.6–7.9 s and DUV-C values of 14.7–20.6 mJ/cm2, while exposure to combined acid and desiccation stresses resulted in D values of 6.1–8.1 s and DUV-C values of 15.9–21.0 mJ/cm2. The D and DUV-C values of S. enterica after previous exposure to sequential acid (24 h) and desiccation (24 h) stresses were found significantly greatest, making the organism and physiological state an appropriate reference organism for the establishment of UV-C pasteurization process for the beverage. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Young coconut (Cocos nucifera L.) liquid endosperm beverage is a naturally sterile drink commonly consumed as a refreshing beverage in many coconut-producing regions of the world (Fonseca et al., 2009; Rolle, 2007; Walter et al., 2009a,b). Coconut water is most commonly consumed directly from the drupe since the endosperm remains sterile while still in an undamaged fruit. The endosperm becomes prone to microbial contaminations and physicochemical degradation when exposed to external environment (Awua et al., 2011; Rolle, 2007). The most alarming form of microbial contamination takes place when disease-causing microorganisms are introduced to the product that may eventually lead to outbreaks of infections when contaminated products are consumed. In reported outbreaks of diseases involving fruit beverages, Salmonella spp. immediately follows Escherichia coli O157:H7 and enterotoxigenic E. coli as most common causative agents ⁎ Department of Food Science and Nutrition, Teodora Alonso Hall, College of Home Economics, A. Ma. Regidor Street, University of the Philippines, Diliman Campus, 1101 Quezon City, Philippines. Tel./fax: +63 981 8500 loc 3408. E-mail address: [email protected].

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.02.003 0168-1605/© 2015 Elsevier B.V. All rights reserved.

(Federal Register, 2001; Harris et al., 2003). In the CDC reports summarized by Vojdani et al. (2008), the 5 outbreaks caused by Salmonella spp. accounted for 52% of the reported illnesses and 63% of the reported hospitalizations. Apple juice products were reported vehicles in 10 of the 21 outbreaks and 8 outbreaks were reported to be due to orange juice consumption. Pineapple juice, fruit juice and a mix of juices (apple, orange, grape and pineapple mix) were implicated in the remaining 3 outbreaks. Outbreaks of other microorganisms such as the spore-forming Bacillus cereus and the parasitic Cryptosporidium parvum have similarly been linked to unpasteurized juice consumption (Federal Register, 2001). Listeria monocytogenes has also been proposed by the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) to be a pertinent microbial hazard due to its ubiquity and the threat it poses to pregnant consumers (Mazzotta, 2001). Despite the absence of reports of outbreaks due to coconut beverage consumption, the highly manual nature of beverage preparation and the low acid pH and high water activity of the beverage make the commodity a potential vector of diseases. The ability of cells to resist suboptimal environments such as the inherent acidity of juices or heat treatment may be attributed to their ability to induce adaptive mechanisms upon sublethal stress exposures.

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Such mechanisms include genetic and physiologic changes that enhance survivability (Foster and Hall, 1990; Goodson and Rowbury, 1989; Linton et al., 1996). Adaptive mechanisms can be homologous, where a cell develops resistance towards a stress factor after previous exposure to the same stressful condition (Tsakalidou and Papadimitriou, 2011). On the other hand, heterologous adaptation takes place when a microorganism develops resistance towards a stress factor after previous exposure to a different stressful condition (Buchanan and Edelson, 1999). Most studies on the effects of previous stress exposures on the subsequent microbial resistance towards processing conditions focus on thermal processing. There is limited information on the effects of stress adaptation on UV-C-mediated microbial inactivation. The United States Food and Drug Administration (FDA) and Department of Agriculture (USDA) have evaluated the utility of UV as a food processing technology and approved its use as an alternative treatment to heat processing of fruit juice products (Federal Register, 2000). Ultraviolet-C (UV-C) is a germicidal spectrum of ultraviolet light having a wavelength range of 100–280 nm, which can readily be absorbed by and cause physicochemical changes in deoxyribonucleic acid (DNA) (Donahue and Bushway, 2004). However, traditional low-pressure mercury UV lamps with a predominant emission spectrum of 254 nm are commonly used for food and contact surface applications (Koutchma, 2014). Exposure of DNA to UV-C can therefore lead to genetic material alteration; particularly strand breakage and thymine dimerization that results in impaired reproductive mechanisms (Billmeyer, 1997; Donahue and Bushway, 2004). The germicidal efficacy of UV-C processing has been reported to be affected by a number of foodrelated intrinsic, and process-related extrinsic factors such as absorptivity, soluble solids, insoluble suspended matter, flow characteristics, and volume (Guerrero-Beltran and Barbosa-Canovas, 2004). There are very few studies, and differing reports on the effects of implicit adaptive mechanisms of foodborne microorganisms towards UV-C after previous exposures to stress-inducing environments of food and food processing ecologies. Gayán et al. (2015) explained that in order for the UV-C technology to be accepted by and transferred to the industry, the need to further understand UV resistance of pertinent foodborne pathogens; as well as the dependence of resistance to microbial adaptation must be addressed. This study therefore established the effects of previous exposures to some environmental stressors such as acidity and desiccation, and their combinations on the subsequent UV-C resistance of some foodborne pathogens such as E. coli O157:H7, Salmonella enterica, and L. monocytogenes in coconut liquid endosperm beverage. These physicochemical stresses are normally encountered by cells in their environments, which can influence their survival in the kill step applied during processing. The results obtained in this work can be used in the identification of appropriate target microorganisms for the establishment of safe UV-C pasteurization process schedule for coconut beverages and similar food commodities. 2. Materials and methods 2.1. Preparation of beverage from young and mature coconut liquid endosperms The coconut beverage used as suspending medium in the UV-C challenge studies was previously developed from a mixture of young and mature coconut liquid endosperms. Briefly, young (6–9 months old) and mature (10–12 months old) coconuts were purchased from Barangay Krus na Ligas, Quezon City. Liquid endosperm samples from the coconuts were obtained by removing the exocarp and mesocarp until the endocarp is revealed to allow perforation for the liquid endosperm to be aspirated out of the drupes. Liquid endosperm samples were then subsequently transferred into sterile glass bottles and transported to the laboratory for beverage preparation composed of a young-to-mature coconut endosperm v/v ratio of 80:20. This formulation

is based on the Philippine National Standard (BAFPS-DA, 2006) for coconut beverage composed of an 80:20 v/v ratio of young liquid endosperm and water. Sweetness and tartness of the liquid endosperm mixture were then respectively adjusted with sucrose to 8 °Bx, and malic acid to 0.13% v/v titratable acidity. The mixture had a pH of 5.31. The prepared beverage was first filtered through 2 layers of Whatman No. 1 (11 μm, Whatman, UK) filter paper; with the filtrate kept at chilled conditions using an iced water bath. A subsequent filter-sterilization was conducted using a microfiltration setup composed of a coarse fritted glass support base, a glass funnel, an anodized aluminum clamp, a silicone stopper, and a filtering flask with a sidearm for pump hose connection through with a 0.45 μm pore-sized membrane filter (Advantec, Tokyo Roshi Kaisha Ltd., Japan). Aliquots of 9.9 mL of the filter-sterilized liquid endosperm beverage were aseptically pipetted into sterile 25-mm (internal diameter) test tubes and kept at refrigerated storage for not more than 2 days until UV-C challenge studies. 2.2. Microbial cultures Five strains of E. coli O157:H7, three strains of S. enterica, and two strains of L. monocytogenes were used in the inactivation studies. The tested E. coli O157:H7 strains included the HCIPH 96055 from the Hiroshima City Institute of Public Health, Hiroshima, Japan and the strains MY-29, DT-66, MN-28, and CR-3 from the National Food Research Institute in Tsukuba, Japan. The L. monocytogenes strains included 4b (HCIPH AS-1) and 1/2c (HCIPH M24-1). Tested Salmonella spp. included serovars Enteritidis (HCIPH B11), and Infantis (C-269) and Montevideo from the Laboratory of Food Microbiology and Hygiene of Hiroshima University. All the organisms used in the study were gifts from Dr. Hiroyuki Nakano of the Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science of the Hiroshima University. Working cultures were prepared by aseptically transferring stabs of each of the refrigerated stock culture into individual 1-mL sterile nutrient broths (NB, HiMedia, India) and incubated at 37 °C for 18–24 h. Individual loopfuls from the activated culture strains were then transferred into nutrient agar (NA, HiMedia, India) slants before incubation at 37 °C for another 18–24 h. The slants were then kept at refrigerated temperature (4 °C) until further use in the experiments. Fresh working slants were prepared every two weeks following the aforementioned protocols. 2.3. Inoculum preparation Cells obtained from the refrigerated working slants were activated by transferring into sterile 10 mL NB tubes and incubated at 37 °C for 18–24 h. Loops of each of the strains were then enriched by transferring into 10 mL NB and incubated again for another 18–24 h at the same temperature. Only the cells that were subjected to these activation and enrichment steps were used in the UV-C challenge studies. To prepare the composited inoculum, aliquots of 1 mL from each of the enriched strains were combined according to species in a sterile test tube, and vortexed (Scientific Products, USA) for 1 min. Aliquots of 1 mL of each of the composited cells were then transferred into 2.0mL microcentrifuge tubes and then harvested by centrifugation (Cole Parmer, USA) at 6000 rpm for 15–20 min. The supernatant was drawn out using a pipettor and the resulting pellet was resuspended in 1 mL filter-sterilized liquid endosperm beverage through vortex-mixing for at least 1 min. The cells were acclimatized in the liquid endosperm suspension for not longer than 20 min at ambient temperature prior to UV-C inactivation. 2.4. Acid stress exposure To determine the effect of previous acid stress exposure on the subsequent UV-C inactivation rates of the test pathogens in the coconut

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beverage, cells obtained from the first 24-h culture were transferred into NB supplemented with 1% w/v glucose (Sigma-Aldrich, USA). Inoculated glucose-supplemented NB (NBG) tubes were then incubated at 37 °C for 18–24 h, during which the cells were gradually exposed to increasingly acidifying environment due to organic acids produced by their sugar metabolism (Gabriel and Nakano, 2011). Prior to UV-C challenge, composited inoculum of each test pathogen species was then prepared following previously described protocols.

2.5. Desiccation stress exposure The study also determined the effects of prior desiccation stress exposure on the subsequent UV-C inactivation rates of the test pathogens. Cells from the second 24-h NB were composited according to species and harvested by spinning, following previously described protocols. Pelleted cocktails of strains were subjected to desiccation stress by resuspending the pellets in sterile 23% v/v NaCl solution (aw = 0.85) for 4, 8, and 24 h at 37 °C. Prior to UV-C challenge, the stressed cells were harvested after each predetermined exposure time by spinning, resuspended, and acclimatized in the coconut beverage following previously described protocols.

Fig. 1. Emission spectra of the 15 W UV-C lamp source showing predominant emission wavelength at 254 nm, at a 14.0 cm source-to-detector distance.

2.8. Survivor enumerations and D and DUV-C value calculations 2.6. Combined acid and desiccation stress exposure To determine the effects of previous exposures to both acid and desiccation stresses on the subsequent UV-C inactivation rates of all the test pathogens, cells were first subjected to gradually acidifying stress following previously described protocols. Cells exposed to 24-h acid stress were harvested and exposed to desiccation stress (aw = 0.85) for 4, 8, and 24 h following previously described protocols. Prior to UV-C challenge, stressed cells were harvested, resuspended, and acclimatized in the coconut beverage following previously described protocols.

2.7. UV-C exposures in coconut beverage suspending medium Aliquots of 9.9 mL of the filter-sterilized coconut liquid endosperm beverage were pipetted into 55-mm plastic Petri dishes (at a 0.6 cm liquid sample thickness). Aliquots of 0.1 mL were then obtained from the suspension of each test organisms previously acclimatized in the coconut liquid endosperm mix; and inoculated into the beverage in Petri dishes to introduce an initial inoculum population of approximately 6.0 log CFU/mL. The inoculated beverages were then subjected to UV-C radiation without stirring using a fabricated UV-C box with a 15-W mercury vapor lamp UV-C light source (Toshiba Lighting and Technology Corp., Tokyo, Japan), at a lamp-to-sample surface distance of 14 cm. The UV-C lamp in the fabricated UV-C box used in this study was subjected to optical emission spectroscopy that characterized predominant radiation emission of 254 nm at the treatment distance of 14 cm (Fig. 1). Measurements were done using a Spectrometer (Ocean Optics, Inc., FL., USA) with a dispersion of 0.2467 nm per pixel and an optical resolution of 1.0855 nm in the range of 200–1100 nm. Furthermore, the coconut beverage was subjected to optical characterizations at 254 nm using an SQ-4802 UV/VIS Double Beam Spectrophotometer (Unico, New Jersey, USA). The UV-C absorbance and transmittance of non-inoculated and inoculated samples in quartz cuvettes were previously determined at a path length of 1 cm, and demonstrated that the beverage inoculated with the different test organisms had similar UV-C absorbance/transmittance characteristics (Table 1). Furthermore, the extinction coefficient (ε) of the tested coconut liquid endosperm beverage was determined to be 0.13 cm−1. The ε value was determined from the relationship of beverage dilution and UV-C (254 nm) absorbance (data not presented).

The inoculated samples were exposed to UV-C at pre-determined time intervals, after which subjected to survivor enumerations. A freshly inoculated plate was used per exposure time to control the effect of volume change on the efficacy of the UV-C against the test organisms. Surviving cells were enumerated from each of the exposed inoculated coconut beverage by obtaining 1 mL aliquot from each Petri dish, and subjecting it to serial, 10-fold dilution with 0.1% Peptone water (HiMedia, Mumbai, India). Appropriate dilutions were then spreadplated onto pre-solidified NA plates, and incubated at 37 °C for 24 h. Colonies that emerged were enumerated and survivor populations were reported as log CFU/mL. The inactivation parameter, death rate (kD), was determined by plotting the enumerated populations per microorganism, per physiological state, using the DMFit Version 2.1 (Institute of Food Research, Reading, UK). The decimal reduction time (D value, sec) was then calculated from the negative inverse of the kD values, and is defined as the length of exposure to UV-C that will result in 90% (1 log cycle) reduction in the inoculated initial microbial population. The calculated D values were then used to determine the UV-C energy dose necessary to reduce the initial populations by 1 log cycle (DUV-C values, mJ/cm2). In the determinations of the DUV-C values, the D values were multiplied with the UV-C irradiance at the surface of the coconut beverage of 2.60 mW/cm2, which was calculated from the

Table 1 Ultraviolet-C absorption and transmittance of non-inoculated and inoculated coconut beverage. Coconut beverage samples1 Non-inoculated beverage Non-filtered coconut beverage Filter-sterilized beverage Inoculated beverage3 Escherichia coli O157:H7 Salmonella enterica Listeria monocytogenes a, b, c, d

Absorbance2

% transmittance

1.43 ± 0.00a 1.35 ± 0.00b

3.74 ± 0.00d 4.47 ± 0.00a

1.36 ± 0.00c 1.37 ± 0.00d 1.36 ± 0.00c

4.31 ± 0.03b 4.23 ± 0.03c 4.32 ± 0.03b

Absorbance and transmittance values followed by the same letters are not significantly different, p N 0.05. 1 Tested coconut beverage was formulated from an 80:20 v/v ratio of young and mature coconut liquid endosperms, sweetened to 8 °Bx, acidified to 0.13% malic acid, pH = 5.3. For filter sterilization, the formulated beverage was passed through a 0.45 μm pore-sized membrane filter. 2 Absorbance was measured at λ = 254 nm, path length = 1 cm. 3 The beverage was inoculated with control, non-stressed cells.

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details provided in the manufacturer's specifications of the UV-C lamp used in the study. 2.9. Statistical analyses Data obtained from inactivation studies were subjected to Analysis of Variance (ANOVA) using the General Linear Model (PROC GLM) procedure of the Statistical Analysis Systems (SAS Institute, Cary, NC) to test for significant differences (p b 0.05). The Duncan's Multiple Range Test (DMRT) was used in the post-hoc analyses when significant differences existed. 3. Results and discussion 3.1. UV-C inactivation of test pathogens with various physiological states: screening for and selection of an appropriate challenge organism A UV-C processing technique should be able to process high volumes of liquid food to be economically feasible and indeed, non-uniformity in dosage is a concern that needs to be addressed, particularly in laminarflowing or non-stirred, UV-absorbing liquid food products. This study tried to address this gap by keeping the treated volume as small as possible, and by minimizing the lamp-to-treated surface distance. Furthermore, this study only aimed to identify an appropriate target pathogen and physiological state that should be used as reference organism for downstream operations such as establishment and evaluation of a UV-C process protocol and schedule for the test food commodity. To maximize the efficacy of a UV-C process against a reference organism, exposures to radiation of turbulent-flowing or thin film liquid food systems may be explored as similarly previously reported by a number of studies (Franz et al., 2009; Gabriel, 2015a; Müller et al., 2011, 2013; Orlowska et al., 2014; Simmons et al., 2012). In order to establish a UV-C process schedule that aims for safety against potential contaminating microorganisms, the study determined and compared the UV-C inactivation rates of pathogens including E. coli O157:H7, S. enterica, and L. monocytogenes. Furthermore, the study also determined the effects of different types of physicochemical stresses that the test microorganisms encounter in food and food processing ecologies. These physicochemical stresses are commonly encountered by cells in their environments prior to processing, which can influence their susceptibility or resistance to the kill step applied during processing. Cells may be exposed to gradual acidification when microorganisms grow on a fermenting substrate. Furthermore, exposure to desiccated environments may happen with the evaporation of moisture from the cell environments. Representative inactivation curves of the different test organisms after various physiological stress exposures are summarized in Figs. 2–4. Results showed that all test microorganisms, regardless of physiological state, exhibited logarithmic linear inactivation behavior in UV-Cradiated coconut beverage suspending medium; with coefficients of determination (R2) values ≤ 0.90 and as much as 0.99. Gabriel (2012a) similarly reported the logarithmic linear inactivation behavior of individual and composited inoculum of E. coli O157:H7 in UV-C-treated clear apple juice. The D values calculated from the inactivation curves are summarized in Table 1. These inactivation kinetic parameters are averages of 4 values obtained from independently run challenge studies, and are measures of the exposure times to UV-C that result in 90% reduction in the population. Results showed that among non-stressed test microorganisms, L. monocytogenes exhibited the greatest resistance towards UV-C, while E. coli O157:H7 was most susceptible to inactivation. It should however be noted that the differences in the susceptibilities of the tested non-stressed microorganisms were not significantly different (p N 0.05). This observed trend is similar to that reported by Gabriel and Nakano (2009) where L. monocytogenes exhibited significantly

greater resistance towards UV-C than E. coli O157:H7 and S. enterica when suspended in phosphate-buffered saline (PBS) and apple juice. Exposure to the gradually acidifying NBG environment for 24 h prior to UV-C inactivation resulted in the increase in the resistance of all test organisms although significant increase was only observed for S. enterica, with a D-value of 4.4 s. Acid-stressed E. coli O157:H7 and L. monocytogenes respectively exhibited the greatest and least resistance towards UV-C (Table 2). It should however be noted that the UV-C resistances of the test organisms were still not significantly different from each other after acid stress exposure. Further increase in the UV-C resistance of all test organisms was observed after exposure to desiccation stress. After 4 h of desiccation stress exposure, all test microorganisms had significantly greater subsequent UV-C resistance than those observed after prior exposure to acid stress, or in organisms that were not stressed at all. S. enterica exhibited the greatest D value of 7.9 s, followed closely by E. coli O157:H7 and L. monocytogenes. The D values of 4-h desiccation-stressed organisms were not significantly different from each other. Furthermore in all organisms tested, a decrease in the UV-C resistance was observed with increasing prior desiccation exposure time. Compared to the 4-h desiccation exposure, both S. enterica and L. monocytogenes significantly became more susceptible to UV-C after 24-h desiccation stress exposure. Finally, the study also tested the influence of prior exposure to combinations of acid and desiccation stresses on the subsequent UV-C resistances of the test organisms. All organisms were first subjected to the 24-h gradually acidifying environment prior to 4-, 8- or 24-h exposure to desiccation stress. The cells subjected to the stresses were then subsequently exposed to UV-C. Unlike the results reported for desiccation stress no distinct pattern for the effect of prior combined stress exposures on the subsequent UV-C resistance was observed. For E. coli O157:H7, the observed D values after 4, 8, and 24 h of desiccation stress exposure of previously acid-stressed cells were not significantly different from each other. It should also be noted that these D values were not significantly different from those observed in cells that were subjected to desiccation stress alone. For E. coli O157:H7, the largest D value (7.1 s) observed was that of cells subjected to 24 h gradually acidifying stress, followed by additional 8 h of desiccation stress exposure. Similarly, no distinct trend for the effect of combined acid and desiccation stresses was observed for L. monocytogenes. Moreover for this organism, no significant difference in the D values was observed between those subjected to desiccation and combined acid and desiccation stresses. Finally among the test organisms, direct relationship between desiccation stress exposure time after acid stress exposure and UV-C resistance was only observed in S. enterica. For this organism, the highest D value was observed at 8.1 s, after exposure of acidstressed cells to additional 24 h desiccation stress. This D value was however not significantly different from cells exposed to other combination stresses, or those cells that exhibited greatest resistance to UV-C after exposure to desiccation stress alone. The D values (Table 1) established per organism and physiological state was also translated into UV-C dose necessary to reduce the initial populations by 1 log cycle (DUV-C). The manufacturer's specifications of the UV-C lamp used in this study stipulated an irradiance value of 0.05 mW/cm2 at a lamp-to-surface distance of 100 cm. Irradiance is a measure of power arriving per unit area of the treated surface (Koutchma, 2014). The irradiance value of 2.60 mW/cm2 at the lampto-surface distance of 14 cm applied in the treatment of the coconut beverage in this study was determined using the inverse square relationship between irradiance from a point source and distance (Ryer, 1997). The calculated surface irradiance was then multiplied by the respective D values in Table 1 to determine the DUV-C values or UV-C fluence presented in Fig. 5. Results showed that among non-stressed, control organisms, E. coli O157:H7 had a D UV-C of 8.4 mJ/cm2. Those of S. enterica and L. monocytogenes were not significantly different from E. coli at 8.6 and 9.1 mJ/cm 2 , respectively. As previously noted, a general

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Fig. 2. Representative inactivation curves of E. coli O157:H7 with varying physiological states in UV-C treated coconut beverage (8 °Bx, 0.13% malic acid). (a) Non-stressed, 24 h in NB; (b) acid-stressed, 24 h in NB + 1% glucose, NBG, gradual acidification to final pH = 4.46; (c) desiccation stressed, 24 h in NB + 4 h in aw = 0.85; (d) desiccation stressed, 24 h in NB + 8 h in aw = 0.85; (e) desiccation stressed, 24 h in NB + 24 h in aw = 0.85; (f) acid + desiccation stressed, 24 h in NBG + 4 h in aw = 0.85; (g) acid + desiccation stressed, 24 h in NBG + 8 h in aw = 0.85; (h) acid + desiccation stressed, 24 h in NBG + 24 h in aw = 0.85.

increase in the DUV-C was observed with stress exposure in all organisms. The maximum DUV-C observed in E. coli O157:H7 was 18.4 mJ/cm2, which was observed in cells previously exposed to a combination of

24 h acid stress and 8 h desiccation stress. L. monocytogenes had the highest DUV-C after exposure to desiccation stress for 4 h equal to 18.0 mJ/cm2. Among the tested organism, S. enterica exhibited the

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Fig. 3. Representative inactivation curves of S. enterica with varying physiological states in UV-C treated coconut beverage (8 °Bx, 0.13% malic acid). (a) Non-stressed, 24 h in NB; (b) acidstressed, 24 h in NB + 1% glucose, NBG, gradual acidification to final pH = 4.46; (c) desiccation stressed, 24 h in NB + 4 h in aw = 0.85; (d) desiccation stressed, 24 h in NB + 8 h in aw = 0.85; (e) desiccation stressed, 24 h in NB + 24 h in aw = 0.85; (f) acid + desiccation stressed, 24 h in NBG + 4 h in aw = 0.85; (g) acid + desiccation stressed, 24 h in NBG + 8 h in aw = 0.85; (h) acid + desiccation stressed, 24 h in NBG + 24 h in aw = 0.85.

highest UV-C resistance after exposure to the combined 24-h acid and 24-h desiccation stresses, which resulted in a DUV-C value of 21.0 mJ/cm2.

There are only a few studies that report the UV-C inactivation of foodborne pathogens in coconut based beverages, hence comparison with literature values will be difficult. However, comparing with

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Fig. 4. Representative inactivation curves of L. monocytogenes with varying physiological states in UV-C treated coconut beverage (8 °Bx, 0.13% malic acid). (a) Non-stressed, 24 h in NB; (b) acid-stressed, 24 h in NB + 1% glucose, NBG, gradual acidification to final pH = 4.46; (c) desiccation stressed, 24 h in NB + 4 h in aw = 0.85; (d) desiccation stressed, 24 h in NB + 8 h in aw = 0.85; (e) desiccation stressed, 24 h in NB + 24 h in aw = 0.85; (f) acid + desiccation stressed, 24 h in NBG + 4 h in aw = 0.85; (g) acid + desiccation stressed, 24 h in NBG + 8 h in aw = 0.85; (h) acid + desiccation stressed, 24 h in NBG + 24 h in aw = 0.85.

available data, all DUV-C values established in this current study were lower than that reported by Koutchma (2014) for E. coli O157:H7 in apple juice at 186.1 mJ/cm2. In the same study, the DUV-C value of

E. coli O157:H7 in malate buffer of 2.5 mJ/cm2 is lower but closer of those of non-stress cells in the test coconut beverage. Moreover, the DUV-C values in water enumerated by Hijnen et al. (2006) for a number

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Table 2 Ultraviolet-C resistance of different test organisms with different physiological states in coconut beverage. Test organism

No stress3

D values (s)1 of differently stressed organisms in coconut beverage2 Acid stress4

Desiccation stress5 4h

E. coli O157:H7 S. enterica L. monocytogenes

c, x

3.2 ± 0.3 3.3 ± 0.0d, x 3.5 ± 0.0c, x

bc, x

4.8 ± 0.6 4.4 ± 0.0c, x 4.1 ± 0.8c, x

Desiccation stress after 24-h acid stress6 8h

a, x

7.0 ± 0.7 7.9 ± 0.8a, x 6.9 ± 0.5a, x

24 h ab, x

6.2 ± 1.1 6.8 ± 0.4ab, x 5.8 ± 0.3ab, x

4h ab, x

5.9 ± 0.7 6.1 ± 0.1b, x 5.6 ± 0.2b, x

8h a, xy

6.7 ± 0.2 7.4 ± 0.7a, x 6.2 ± 0.5ab, y

24 h a, x

7.1 ± 1.2 8.0 ± 0.6a, x 6.5 ± 0.1ab, x

6.2 ± 0.3ab, x 8.1 ± 0.2a, x 6.1 ± 0.8ab, x

a, b, c

Values on the same row followed by the same letter are not significantly different (p N 0.05). Values on the same column followed by the same letter are not significantly different (p N 0.05). 1 D-values are averages of at least 4 values obtained from independently run experiments. D-value is the length of time of UV-C exposure in the coconut beverage suspension that shall result in 1 log (90%) reduction in the initial microbial population. 2 Tested coconut beverage was formulated from an 80:20 v/v ratio of young and mature coconut liquid endosperms, sweetened to 8 °Bx, acidified to 0.13% malic acid, pH = 5.3. UV-C exposures of non-stirred samples were done at a lamp-to-surface distance of 14 cm and a sample thickness of 0.6 cm. 3 Non-stressed organisms were propagated in nutrient broth (pH = 7.08, aw = 0.99) for 24 h prior to UV-C challenge. 4 Acid-stressed organisms were subjected to gradual acidification in nutrient broth supplemented with 0.1% glucose (final pH = 4.46, aw = 0.99) for 24 h prior to UV-C challenge. 5 Desiccation-stressed organisms were suspended in saturated NaCl solution (pH = 7.03, aw = 0.85) for 4, 8, or 24 h prior to UV-C challenge. 6 Acid- and desiccation-stressed organisms were first subjected to 24-h gradual acidification in NBG, prior to 4, 8, or 24-h suspension in saturated NaCl solution, and eventual UV-C challenge. x, y

of vegetative cells of bacteria were close to those of the non-stressed cells challenged in this study. The UV-C mediated 1 log reduction of Streptococcus faecalis, Shigella sonnei, Salmonella Typhi, E. coli O157:H7, E. coli, Yersenia enterocolitica, Shigella dysenteriae, and Vibrio cholera required DUV-C of 9.0, 6.0, 6.0, 5.0, 5.0, 3.0, 3.0 and 2.0 mJ/cm2, respectively. Variations in the previously reported and observed DUV-C values are due to intrinsic food properties such as dissolved and suspended solids, and optical properties, extrinsic process parameters, and microbial implicit characteristics. The lethal effect of UV-C radiation process results from the crosslinking of neighboring pyrimidine bases in the same DNA strand that hampers replication (Sizer and Balasubramaniam, 1999). The efficacy of UV-C in inactivating microorganisms have been reported to be affected by the nature of the medium to which cells are suspended such as suspending medium absorptivity, soluble solid content, and amount of suspended matter in the medium (Turtoi and Borda, 2013). Such dependence has been demonstrated in a previous work reported by Gabriel

and Nakano (2009) that demonstrated significant differences in the susceptibility of pathogens such as E. coli O157:H7, S. enterica, and L. monocytogenes in PBS and clear apple juice. Like previously reported works, this study was also able to demonstrate that the UV-C susceptibility is also organism-specific. When suspended in the same medium such as PBS and apple juice, E. coli O157:H7, S. enterica, and L. monocytogenes exhibited significantly different inactivation rates (Gabriel and Nakano, 2009). Guerrero-Beltrán and Barboza-Cánovas (2005) also demonstrated differences in UV-C susceptibilities of L. monocytogenes and E. coli in apple juice. The variation in the D values of the same organism in different suspending medium is attributable to the physicochemical attributes of the medium, including optical characteristics. However, the variation in the D values of different organisms in the same suspending medium is definitely due to implicit factor, or innate susceptibility or resistance of the test species. Determination and comparison of the relative susceptibilities towards processing techniques of microorganisms that could potentially

Fig. 5. UV-C dose necessary to reduce differently stressed test organisms by 1 log cycle (90% reduction). Values were determined by multiplying the decimal reduction times (sec) with the UV-C irradiance at a 14 cm treatment distance equal to 2.60 mW/cm2. Treated sample had a thickness of 0.6 cm. The letters labeling the bars represent organism-specific Duncan's Multiple Range Test grouping across stress exposures.

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contaminate food products are important as process lethality standards apply to the most resistant challenge species (Federal Register, 2001). This study particularly tested and compared the UV-C resistance of E. coli O157:H7, S. enterica, and L. monocytogenes as these three microorganisms have been identified in the Federal Register (2001) to be pertinent sentinel organisms for fruit juice safety. The identification of these organisms was based on their historical association with fruit beverage products, as well as the possibility of these organisms to be associated with this commodity in future outbreaks. Aside from the dependence of UV-C D values on the species of the test organisms, this study was also able to demonstrate that susceptibility is also physiology-dependent. That is, the susceptibility or resistance of a particular organism towards UV-C is also affected by physicochemical conditions that cells encounter in food and food processing ecologies prior to exposure to UV-C. In a model illustrated by Gabriel (2015b), healthy or unstressed cells may encounter physicochemical stresses from intrinsic food properties and extrinsic food processing ecological conditions. These stresses may individually or interactively influence the subsequent microbial responses to food processing techniques that could lead to survival and eventual outbreaks of disease when contaminated products are consumed. The effects of prior stress exposures on the subsequent resistance and susceptibility of microorganisms to thermal treatment are well established (Gabriel and Nakano, 2011; Gabriel, 2012b). A number of studies have reported that subjection of pathogens to gradually acidifying environment during propagation has led to cross protection from thermal inactivation in fruit juices, milk, and chicken broth (Buchanan and Edelson, 1999; Gabriel and Nakano, 2011; Mazzotta, 2001; Ryu and Beuchat, 1998; Sharma et al., 2005). Furthermore, simultaneous exposure of E. coli O157:H7 in acid and desiccation stresses have been previously demonstrated by Gabriel and Nakano (2010) to result in cells with greater resistance towards thermal treatment. However for the influences of prior exposures to intrinsic and extrinsic stress factors on the subsequent UV-C, there is still a dearth of information. In a recent comprehensive exposition on the application of UV for preservation and shelf life extension of fluid foods (Koutchma, 2014), there was no mention on the consideration of target organism physiology and its effect on UV resistance for establishment of process schedules for specific food systems; except for adapting target cells to the acidic fruit environment if inactivation studies shall be conducted in such a food system. Aside from this, no definite effects of prior stress exposures on subsequent UV inactivation were discussed. The results reported by Bradley et al. (2012) are some of the few observations made with regard to the effect of prior exposures to mild food processing stresses on the pulsed UV inactivation of different morphotypes of L. monocytogenes. In their work, they reported that prior exposure to 7.5% (w/v) NaCl for 1 h, pH 5.5 for 1 h, or heating at 48 °C for 1 h resulted in the inability of the test organisms to adapt to normally lethal levels of pulsed UV radiation. Gayán et al. (2015) also recently reported the non-significant influences of prior exposures to sublethal heat (48 °C, 1 h, pH 7.0), acidity (HCl-acidified tryptone soy broth yeast extract, TSBYE broth to pH 4.5, 1 h), and alkalinity (NaOHalkalinized TSBYE to pH 9.0, 1 h) on subsequent UV-mediated inactivation of L. monocytogenes. These results do not agree with those observed in this current study, demonstrating significant increase in the UV-C resistance of all test organisms after exposures to almost all physicochemical stresses. The variation may be attributed to the differences in the stress-exposure protocols used. On the other hand, the results reported by Bernbom et al. (2011) are in accordance with this current study's observations. In their work, L. monocytogenes exhibited enhanced UV-C survival in the presence of 5% NaCl in the suspending medium. Bernbom et al. (2011) explained that it is not known whether the observed enhancement is due to physiological changes in the cells, a physical protection in the food matrix, or a combination. The results obtained in this work provide evidence of physiological changes in the cells upon exposure to desiccation that

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resulted in enhanced survival towards UV-C exposures. Based on the results obtained in this current work (Table 1), the study deemed the tested cocktail of S. enterica cells previously exposed to 24-h gradually acidifying environment, followed by 24-h desiccation stress as appropriate reference microorganism for the establishment of UV-C process schedule for the developed coconut beverage. Inactivation of these cells will similarly ensure mortality of less resistant pathogen- and physiological types. To sum up, this work presents new evidences that foodborne pathogens that could potentially be introduced to beverages that will be processed with UV-C radiation may exert enhanced resistance to the kill step if the cells are previously exposed to pertinent stress-inducing physicochemical conditions. The heterologous adaptations to UV-C after exposures to acidity, desiccation, and various combinations of both signify the importance of considering such physiological states in the establishment of pasteurization process schedule to better control safety of appropriate commodities.

Acknowledgments The author acknowledges the Office of the Chancellor of the University of the Philippines Diliman, through the Office of the Vice Chancellor for Research and Development, for funding support through the PhD Incentive Grant (PN 121229). The author also recognizes the assistance rendered by Michael Louie C. Aguila, Maria Elizabeth A. Rosero, Kimberly Anne M. Tupe, and Amabelle B. Malaluan in conducting the challenge studies. The assistance extended by the Plasma Physics Laboratory of the National Institute of Physics, UP Diliman headed by Dr. Henry Ramos is also being recognized.

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Previous physicochemical stress exposures influence subsequent resistance of Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes to ultraviolet-C in coconut liquid endosperm beverage.

This study investigated the influences of prior exposures to common physicochemical stresses encountered by microorganisms in food and food processing...
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