inactivation of bacterial spores by uv-c light

6
Inactivation of bacterial spores by UV-C light E. Gayán, I. Álvarez, S. Condón Food Technology, Faculty of Veterinary of Zaragoza, Spain abstract article info Article history: Received 6 March 2013 Accepted 15 April 2013 Editor Proof Receive Date 15 May 2013 Keywords: Ultraviolet light Sterilization Combined processes Non-thermal technologies Bacterial spores This investigation evaluates the ability of UV light to inactivate bacterial spores of interest for food preservation. It also evaluates the effect of the absorptivity of the treatment medium on UV lethality and the effect of the simul- taneous or sequential combination of UV light and mild heating. A UV dose of 23.72 J/mL reached 2.25, 2.93, 3.24, 3.85, and 4.05 Log 10 reductions of Bacillus coagulans, Bacillus cereus, Alicyclobacillus acidocaldarius, Bacillus licheniformis, and Geobacillus stearothermophilus spores, respectively. The most UV-sensitive species (Bacillus stearothermophilus) was the most heat-resistant species. The shoulder phase of UV survival curves of the most resistant B. coagulans increased linearly with the absorptivity of the treatment medium whereas the inactivation rate decreased exponentially. The UV inactivation of B. coagulans increased synergistically when increasing the treatment temperature from 25 to 60 °C. A previous heat treatment (60 °C for 3.58 min) did not affect UV sensitivity of B. coagulans, but prior UV exposure (27.10 J/mL) sensitized bacterial spores to subsequent heat treatments. Industrial Relevance: This investigation demonstrated the ability of UV light to inactivate bacterial spores during food preservation. UV technology, unlike other nonthermal technologies, is a real alternative to current heat sterilization treatments. For UV sterilization of liquid foods with a high absorption coefcient, the efcacy of UV light can be enhanced when combined with heat. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Current consumer demand for minimally processed food has led to considerable efforts to develop alternative non-thermal processes for microbial inactivation. Most of these new technologies, such as Pulsed Electric Fields (PEF), and High Hydrostatic Pressure (HHP), are able to inactivate bacterial vegetative forms efciently but fail to inactivate bacterial spores. Their application in the food industry is focused on pas- teurization processes to control pathogenic microorganisms (NACMCF, 2006), but they are unsuitable to replace thermal sterilization treat- ments. Ultraviolet (UV) irradiation is another non-thermal technology that has been traditionally used to disinfect air, water, and surfaces (Bintsis, Litopoulou-Tzanetaki, & Robinson, 2000). However, it has gained considerable interest in the liquid food pasteurization eld because it is easy to use and lethal to most types of microorganisms. It does not generate chemical residue, and it is a dry cold process that can be effective at a low cost in comparison with other pasteurization methods (Guerrero-Beltrán & Barbosa-Cánovas, 2004; Tran & Farid, 2004). Unlike other non-thermal technologies, UV treatments are potentially capable of inactivating bacterial spores (Hijnen, Beerendonk, & Medema, 2006). When bacterial spores are treated with UV-C radiation (200280 nm), which is considered the most germicidal wavelength range, DNA absorbs photons and induces the formation of bipyrimidine dimmersespecially spore photoproducts (SP) (Moeller et al., 2007). These tran- siently block DNA transcription and replication, leading to cell death (Friedberg et al., 2006). The literature uses data obtained primarily from Bacillus subtilis, one of the challenging microorganisms used in biodosimetry assays to evaluate water treatment plant performance (USEPA, 2003). However, there is little additional information about UV inactivation of different bacterial spores of interest for food preserva- tion. The effect of important environmental factors on UV resistance of bacterial spores, such as the absorption coefcient of the treatment media, has not been investigated so far. The main drawback of UV processing is its low penetration capacity in liquid foods with a high absorption coefcient (Koutchma, Forney, & Moraru, 2009). It is foreseeable that this limitation increases with UV resistance of the target microorganism. Most authors agree that bacteri- al spores are more UV-tolerant than vegetative cells (Setlow, 2006) due to altered DNA conformation (A-form) caused by the presence of small acid-soluble proteins (SASP) binding to the DNA; an efcient DNA repair mechanisms specic to SP; the accumulation of a high percentage of dipicolinic acid in the dormant spore core; and the presence of a thick spore protein coating. It is expected that the inactivation of bacterial spores in media of high absorptivity will be difcult. UV light can be combined with other novel processing techniques or milder conventional preservation methods in a so-called hurdleInnovative Food Science and Emerging Technologies 19 (2013) 140145 Corresponding author at: Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, C/Miguel Servet 177, CP 50013, Zaragoza, Spain. Tel.: +34 976 76 26 38; fax: +34 976 76 15 90. E-mail address: [email protected] (S. Condón). 1466-8564/$ see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ifset.2013.04.007 Contents lists available at SciVerse ScienceDirect Innovative Food Science and Emerging Technologies journal homepage: www.elsevier.com/locate/ifset

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Innovative Food Science and Emerging Technologies 19 (2013) 140–145

Contents lists available at SciVerse ScienceDirect

Innovative Food Science and Emerging Technologies

j ourna l homepage: www.e lsev ie r .com/ locate / i fse t

Inactivation of bacterial spores by UV-C light

E. Gayán, I. Álvarez, S. Condón ⁎Food Technology, Faculty of Veterinary of Zaragoza, Spain

⁎ Corresponding author at: Tecnología de los AlimeUniversidad de Zaragoza, C/Miguel Servet 177, CP 500976 76 26 38; fax: +34 976 76 15 90.

E-mail address: [email protected] (S. Condón).

1466-8564/$ – see front matter © 2013 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.ifset.2013.04.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 March 2013Accepted 15 April 2013

Editor Proof Receive Date 15 May 2013

Keywords:Ultraviolet lightSterilizationCombined processesNon-thermal technologiesBacterial spores

This investigation evaluates the ability of UV light to inactivate bacterial spores of interest for food preservation. Italso evaluates the effect of the absorptivity of the treatment medium on UV lethality and the effect of the simul-taneous or sequential combination of UV light andmild heating. A UV dose of 23.72 J/mL reached 2.25, 2.93, 3.24,3.85, and 4.05 Log10 reductions of Bacillus coagulans, Bacillus cereus, Alicyclobacillus acidocaldarius, Bacilluslicheniformis, and Geobacillus stearothermophilus spores, respectively. The most UV-sensitive species (Bacillusstearothermophilus) was the most heat-resistant species. The shoulder phase of UV survival curves of the mostresistant B. coagulans increased linearlywith the absorptivity of the treatment mediumwhereas the inactivationrate decreased exponentially. The UV inactivation of B. coagulans increased synergistically when increasingthe treatment temperature from 25 to 60 °C. A previous heat treatment (60 °C for 3.58 min) did not affect UVsensitivity of B. coagulans, but prior UV exposure (27.10 J/mL) sensitized bacterial spores to subsequent heattreatments.Industrial Relevance: This investigation demonstrated the ability of UV light to inactivate bacterial spores duringfood preservation. UV technology, unlike other nonthermal technologies, is a real alternative to current heatsterilization treatments. For UV sterilization of liquid foods with a high absorption coefficient, the efficacy ofUV light can be enhanced when combined with heat.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Current consumer demand for minimally processed food has led toconsiderable efforts to develop alternative non-thermal processes formicrobial inactivation. Most of these new technologies, such as PulsedElectric Fields (PEF), and High Hydrostatic Pressure (HHP), are able toinactivate bacterial vegetative forms efficiently but fail to inactivatebacterial spores. Their application in the food industry is focused on pas-teurization processes to control pathogenic microorganisms (NACMCF,2006), but they are unsuitable to replace thermal sterilization treat-ments. Ultraviolet (UV) irradiation is another non-thermal technologythat has been traditionally used to disinfect air, water, and surfaces(Bintsis, Litopoulou-Tzanetaki, & Robinson, 2000). However, it hasgained considerable interest in the liquid food pasteurization fieldbecause it is easy to use and lethal to most types of microorganisms. Itdoes not generate chemical residue, and it is a dry cold process thatcan be effective at a low cost in comparison with other pasteurizationmethods (Guerrero-Beltrán & Barbosa-Cánovas, 2004; Tran & Farid,2004). Unlike other non-thermal technologies, UV treatments arepotentially capable of inactivating bacterial spores (Hijnen, Beerendonk,& Medema, 2006).

ntos, Facultad de Veterinaria,13, Zaragoza, Spain. Tel.: +34

rights reserved.

Whenbacterial spores are treatedwithUV-C radiation (200–280 nm),which is considered the most germicidal wavelength range, DNAabsorbs photons and induces the formation of bipyrimidine dimmers—especially spore photoproducts (SP) (Moeller et al., 2007). These tran-siently block DNA transcription and replication, leading to cell death(Friedberg et al., 2006). The literature uses data obtained primarilyfrom Bacillus subtilis, one of the challenging microorganisms used inbiodosimetry assays to evaluate water treatment plant performance(USEPA, 2003). However, there is little additional information aboutUV inactivation of different bacterial spores of interest for food preserva-tion. The effect of important environmental factors on UV resistance ofbacterial spores, such as the absorption coefficient of the treatmentmedia, has not been investigated so far.

The main drawback of UV processing is its low penetration capacityin liquid foods with a high absorption coefficient (Koutchma, Forney, &Moraru, 2009). It is foreseeable that this limitation increases with UVresistance of the targetmicroorganism.Most authors agree that bacteri-al spores are more UV-tolerant than vegetative cells (Setlow, 2006) dueto altered DNA conformation (A-form) caused by the presence of smallacid-soluble proteins (SASP) binding to the DNA; an efficient DNArepairmechanisms specific to SP; the accumulation of a high percentageof dipicolinic acid in the dormant spore core; and the presence of a thickspore protein coating. It is expected that the inactivation of bacterialspores in media of high absorptivity will be difficult.

UV light can be combined with other novel processing techniquesor milder conventional preservation methods in a so-called ‘hurdle’

141E. Gayán et al. / Innovative Food Science and Emerging Technologies 19 (2013) 140–145

approach to guarantee acceptable inactivation of microorganisms ofconcern (Maktabi, Watson, & Partson, 2011; Walkling-Ribeiro et al.,2008). To improve the UV lethal effect, UV light has been combinedwith chemical agents, mild heating, or other novel technologies. Thesporicidal effect of UV-C light increases in combination with hydrogenperoxide and ozone (Gardner & Shama, 1998; Jung, Oh, & Kang, 2008),and this has been the basis for the design of sterilization processes offood packaging materials. Unfortunately, this strategy cannot beused for food preservation. Infrared radiation and conductive heatingincrease the lethal effect of UV light (Hamanaka et al., 2011), likelydue to thermal effects. Recently, it has been demonstrated that thelethal effect of UV light on Escherichia coli and Salmonella entericasubsp. enterica serovar Typhimurium increases synergistically with atemperature between 40 and 60 °C (Gayán, Monfort, Álvarez, &Condón, 2011; Gayán, Serrano, Raso, Álvarez, & Condón, 2012). To thebest of our knowledge, there is no data in the literature regarding theeffect of UV light combined with mild heat treatments (UV-H treat-ments) on the inactivation of bacterial spores.

The goal of this study was to determine the ability of UV light toinactivate different spore-forming bacteria (Bacillus coagulans, Bacilluslicheniformis, Geobacillus stearothermophilus, Bacillus cereus, andAlicyclobacillus acidocaldarius) of interest in food preservation. Theeffect of the applied dose and absorptivity of the treatment mediumon UV lethality was studied using most UV resistant species. Finally,the lethal effect of the simultaneous or sequential combination of UVlight with mild heating was explored.

2. Material and methods

2.1. Bacterial spore production

The strains used in this investigation: B. coagulans (STCC 4522)B. licheniformis (STCC 4523), G. stearothermophilus (STCC 12980),B. cereus (STCC 9818), and A. acidocaldarius (STCC 5137) were providedby the Spanish Type Culture Collection (STCC). The bacterial sporesweremaintained frozen at−80 °C in cryovials. To obtain the vegetativecultures, we inoculated a loopful of growth in nutrient agar (Biolife,Milano, Italy) with 0.6% (w/v) yeast extract (Biolife) (NAYE) into a50 mL-flask of nutrient broth (Biolife) with 0.6% (w/v) yeast extract(Biolife) (NBYE); this was incubated at the required temperatures in ashaking incubator for 24 h. Sporulationswere carried out in Petri dishesof NAYE with 3 ppm (w/v) of manganese sulfate (Carlo Erba, Milan,Italy). The agar surface was inoculated with a 24 h culture in liquidmedia and incubated at the corresponding temperature for 5 days.After incubation, spores were collected by flooding the agar surfacewith sterile distilled water. After harvesting, spores were washed fivetimes by centrifugation at 2500 ×g for 20 min at 4 °C (Jouan, mod. CR4.11, Saint-Herblain, France) and re-suspended in sterile distilledwater. The spore suspensions were stored at 0–5 °C until used. Incuba-tion temperatures for growth and sporulationwere 35 °C for B. coagulans,B. licheniformis, and B. cereus; and 55 °C for G. stearothermophilus andA. acidocaldarius.

2.2. UV equipment and treatments

UV treatments were carried out using the equipment previouslydescribed by Gayán et al. (2011). It consisted of 8 individual annularthin film flow-through reactors connected sequentially. Each reactorconsisted of a low pressure UV-C lamp (TUV 8WT5, Philips, USA)with 8 W of input power, enclosed in a quartz sleeve to prevent directcontact with the treatment medium, and fixed at the axis of an outerglass with a sampling valve placed at the end. The entire system wassubmerged in a 90 L water bath heated by the circulating water of aperipheral thermostatic bath (Huber, mod. Kattebad K12, Offenburg,Germany). The equipment included a peristaltic pump (Ismatec,mod. ISM 10785, Glattbrugg, Switzerland) and a heating/cooling coil

exchanger prior to the entrance of the first UV reactor and placed inthe water bath. Two thermocouples (Almeco, mod. ZA 020-FS,Bernburg, Germany) fitted to the input of the first and the outlet of thelast reactor allowed the control of the treatment medium temperature.

The treatment medium was added with the spore suspensions toachieve 104–106 CFU/mL and pumped through the installation at8.5 L/h. When the treatment conditions were stabilized, sampleswere withdrawn through the sampling valve of each reactor, andeither 0.1 mL or 1 mL was immediately pour plated in the recoverymedia. A McIlvaine citrate–phosphate buffer with pH 7.0 at differentabsorption coefficients (from 3.9 to 20.0 cm−1; turbidity b 5 NTU)wasprepared by adding different quantities of tartrazine (Sigma-Aldrich, St.Louis, USA). The absorbance of treatment media was measured at254 nm using a Unicam UV500 spectrophotometer (Unicam Limited,Cambridge, UK). Sample solutions were diluted and evaluated usingquartz cuvettes (Hellma, Müllheim, Germany) with path lengths of 1,2, and 10 mm. The absorption coefficient of each sample was deter-mined from the slope of the absorbance versus path length, correctingfor the dilution factor. Turbidity was measured using a HI 83749 neph-elometer (Hanna Instrument, Szeged, Hungary).

2.3. Heat treatments

Heat treatments were carried out in a specially designedresistometer described by Condón, Arrizubieta, and Sala (1993). Thisinstrument consists of a 350 mL vessel with an electrical heater forthermostation, an agitation device to ensure inoculum distributionand temperature homogeneity, a pressurization system, and ports forinjecting the microbial suspension and for sampling. Once the presettemperature attained stability (T ± 0.05 °C), 0.2 mL of an adequatelydiluted microbial cell suspension were inoculated into the correspond-ing treatment medium. After inoculation, 0.2 mL samples were collect-ed at different heating times and immediately pour plated.

2.4. Incubation of treated samples and survival counting

Bacterial spores were recovered in NAYE, and plates were incubatedfor 48 h at the corresponding temperature for each microorganism.Longer incubation times did not change the profile of survival curves(data not shown). After incubation, colony forming units (CFU) werecounted with an improved Image Analyzer Automatic Colony Counter(Protos, Synoptics, Cambridge, UK), as described elsewhere (Condón,Oria, & Sala, 1987).

2.5. Curve fitting and statistical analysis

Survival curves to UV treatments were obtained by plotting the log-arithm of the survival fraction versus treatment doses expressed inenergy consumption unit (J/mL), according to Gayán et al. (2011), andto heat versus time inmin. To fit survival curves and calculate resistanceparameters, the Geeraerd and Van Impe inactivation model-fitting tool(GInaFiT) was used (Geeraerd, Valdramidis, & Van Impe, 2005). As UVsurvival curves obtained in this investigation did not show tails butrather shoulders, the log-linear regression plus shoulder model(Geeraerd, Van Impe, & Herremans, 2000) was used with the followingequation:

Nd ¼ N0e−k

maxdeKmaxSl

1þ eKmaxSl−1� �

e−kmaxd

!ð1Þ

where Nd represents the number of survivors, N0 the initial count, d theapplied dose, Sl the shoulder length, and Kmax the first-order inactiva-tion constant. This model describes the survival curves through twoparameters: the shoulder length (Sl), defined as lag phase before the ex-ponential inactivation begins, and the inactivation rate (Kmax), defined

0 5 10 15 20 25 30-5

-4

-3

-2

-1

0

Dose (J/mL)

Lo

g10

Nd/N

0

Fig. 1. Survival curves of Bacillus coagulans (●), Bacillus licheniformis (○), Bacillus cereus(■), Alicyclobacillus acidocaldarius (▲), and Bacillus stearothermophilus (□) spores to UVlight. Treatments were carried out at room temperature in citrate–phosphate bufferwith an absorption coefficient of 11.1 cm−1.

142 E. Gayán et al. / Innovative Food Science and Emerging Technologies 19 (2013) 140–145

as the slope of the exponential portion of the survival curve. Therefore,the traditional decimal reduction time value (D) can be calculated fromthe Kmax parameter by the equation: D = 2.303 / Kmax. For comparisonpurposes, the GInaFiT software also provides the parameter 4D, definedas the treatment dose necessary to inactivate 99.99% of the microbialpopulation. Heat survival curves that showed a log-linear profile werefitted by the Bigelow and Esty (1920) model (Eq. (2)) and wereincorporated into the GInaFiT software using the following equation:

Nt ¼ N0e−K maxt : ð2Þ

All microbial resistance determinations were performed at least 3times on different working days. The error bars in the figures corre-spond to the mean standard deviation. Statistical analyses, t-test(p = 0.05), and ANOVA tests (p = 0.05) were carried out using theGraphPad PRISM 5.0 software (GraphPad Software, Inc., San Diego,USA), and differences were considered significant when p ≤ 0.05.

3. Results

Survival curves of the five spore suspensions to UV light wereassessed in a buffer with an absorption coefficient of 11.1 cm−1

(Fig. 1). As observed, UV susceptibility of bacterial spores variedbetween species: A dose of 23.72 J/mL reached 2.25 ± 0.07, 2.93 ±0.09, 3.24 ± 0.38, 3.85 ± 0.5, and 4.05 ± 0.30 Log10 cycles of inacti-vation of B. coagulans, B. cereus, A. acidocaldarius, B. licheniformis, andG. stearothermophilus spores, respectively. UV survival curves showed

Table 1UV-resistance parameters (Sl and Kmax) obtained from the fitting of Geeraerd et al.'s modetemperature and at 60 °C in citrate–phosphate buffers with different absorption coefficient

Spore α (cm−1) Temperature (°C) Sl (

Alicyclobacillus acidocaldarius 11.1 25 7Geobacillus stearothermophilus 11.1 25 4Bacillus cereus 11.1 25 6Bacillus licheniformis 11.1 25 5Bacillus coagulans 11.1 25 8

60 53.9 25 16.4 25 38.8 25 6

60 513.9 25 12

60 617.0 25 13

60 720.0 25 15

Values in parentheses represent the standard deviation of the mean.Letters a, b and c indicate statistically significant differences (p ≤ 0.05) among resistance p

a significant shoulder, followed by an exponential order of death. Sur-vival curves were fitted with Geeraerd et al.'s equation (Eq. (1)), andresistance parameters (Sl and Kmax) and 4D values for comparison arecompiled in Table 1. Table 1 also includes the coefficient of determi-nation (R2) and the root mean square error (RMSE) to illustrate thegoodness of the fit. No statistically significant differences (p > 0.05)were found among Kmax values, but the shoulder length widelychanged among species: B. coagulans exhibited the highest Sl, whichwas twice the Sl of the most sensitive, G. stearothermophilus.

To study the effect of the absorptivity of the treatment medium onUV lethality at room temperature, survival curves of the most resistantspores, B. coagulans, were obtained in buffers with an absorption coeffi-cient ranging from 3.9 to 20.0 cm−1. UV resistance parameters werecalculated from the fit of experimental data with Geeraerd et al.'s equa-tion; this data is included in Table 1. As observed, the lethal effect ofUV light decreased by increasing the absorption coefficient. Less UVeffectiveness was due to both an increase in the shoulder length and adecrease in the inactivation rate. Plotting the Log10 Kmax against theabsorption coefficient (α) (Fig. 2), an exponential relationship wasdeduced (Log10 Kmax = −0.060 α + 0.208; R2 = 0.996), and Kmax de-creased ten-fold by increasing the absorption coefficient by 16.8 ±0.8 cm−1. But there was a linear relationship (Sl = 0.909 α − 1.896;R2 = 0.986) between shoulder length and absorptivity (Fig. 3).

To evaluate the lethal effect of UV light combined with mild heattreatments (UV-H treatments), B. coagulans spores were treated byUV light at 60 °C in buffers with different absorption coefficients(8.8–17.0 cm−1). Resulting survival curves are plotted in Fig. 4, andtheir kinetics parameters are included in Table 1. The UV inactivationthat applied a dose of 27.10 J/mL at 60 °C in buffers of 17.0, 13.9, 11.1,and 8.8 cm−1 increased 0.82, 1.19, 1.87, and more than 2 Log10 cycles,respectively, compared to treatments at 25 °C (Fig. 4). InactivationUV-H parameters (Table 1) showed that shoulder phases were shorterand inactivation rates were higher than those obtained at 25 °C. Therelationship between the absorption coefficient and Kmax values(Log10 Kmax = −0.065; α + 0.423; R2 = 0.999) is illustrated inFig. 2; the same along with Sl (Sl = 0.245; α + 3.125; R2 = 0.996)are illustrated in Fig. 3. The statistical analysis demonstrated that the ef-fect of the absorptivity on Kmax was the same (p > 0.05) at both treat-ment temperatures. But the effect of the absorptivity on Sl wassignificantly higher at 20 °C than at 60 °C. Because the lethal effect ofheat at 60 °C for 3.58 min (exposure time for a dose of 27.10 J/mL)was negligible—less than 0.08 ± 0.02 Log10 cycles of inactivation—asynergistic lethal effect was deduced from the simultaneous UV-Hcombination.

To explore the mechanism responsible for enhanced inactivationof B. coagulans with the combined process, both technologies were

l to survival curves of different bacterial spores. Treatments were carried out at rooms (α).

J/mL) Kmax (mL/J) 4D (J/mL) R2 RMSE

.71 (0.30)a 0.40 (0.07)a 31.25 (4.29) 0.986 0.170

.78 (0.74)b 0.47 (0.05)a 24.53 (1.09) 0.990 0.187

.22 (1.55)c 0.38 (0.03)a 30.74 (0.58) 0.997 0.084

.36 (1.66)b 0.47 (0.07)a 25.69 (1.48) 0.994 0.124

.33 (0.73)a 0.35 (0.04)a 35.30 (3.02) 0.993 0.099

.78 (0.48) 0.50 (0.06) 24.80 (1.34) 0.990 0.114

.25 (0.35) 0.92 (0.12) 11.84 (1.04) 0.997 0.206

.85 (0.05) 0.69 (0.06) 15.41 (0.76) 0.987 0.298

.01 (0.64) 0.51 (0.06) 23.94 (1.39) 0.994 0.151

.31 (0.41) 0.71 (0.05) 1.18 (0.72) 0.993 0.206

.02 (0.76) 0.22 (0.01) – 0.987 0.071

.59 (0.20) 0.32 (0.03) – 0.989 0.209

.29 (1.29) 0.15 (0.02) – 0.975 0.074

.28 (0.19) 0.21 (0.01) – 0.981 0.099

.66 (1.21) 0.11 (0.06) – 0.968 0.049

arameters of different spore suspensions treated by UV light.

0 5 10 15 20 25-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

Km

ax (

mL

/J)

Fig. 2. Relationship between the absorption coefficient (α) of the treatment mediumand the inactivation rate (Log10 Kmax) of Bacillus coagulans spores to UV light at 25(●) and 60 °C (○).

0 5 10 15 20 25 30-6

-5

-4

-3

-2

-1

0

Dose (J/mL)

Lo

g10

Nd/N

0

Fig. 4. Survival curves of Bacillus coagulans to UV light at 25 °C (solid symbols andlines) and at 60 °C (opened symbols and dotted lines) in citrate–phosphate buffersof 8.8 (●), 11.1 (■), 13.9 (▲), and 17.0 (♦) cm−1 absorption coefficients.

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4-4

-3

-2

-1

0

Time (min)

Lo

g10

Nt/N

0

A

-1

0B

143E. Gayán et al. / Innovative Food Science and Emerging Technologies 19 (2013) 140–145

applied sequentially in the two possible orders: a heat treatment at60 °C for 3.58 min followed by a UV treatment of 27.10 J/mL and aUV treatment of the same intensity followed by thermal sterilizationat different temperatures, using a buffer with an absorption coeffi-cient of 17.0 cm−1. The combination of heat followed by a UV treat-ment did not change significantly (p > 0.05) the UV inactivation(1.26 Log10 cycles). On the other hand, UV treatment prior to thermaltreatments improved heat lethality. Fig. 5 shows heat survival curvesof B. coagulans at 110, 105, and 100 °C, as well as those preceded bythe UV treatment. Heat inactivation curves of B. coagulans at 110and 105 °C showed a log-linear profile, and first-order kinetics wereapplied (Eq. (2)). However, survival curves at 100 °C displayed aninitial shoulder and were described by Geeraerd et al.'s equation inthe time term. The decimal reduction time value (DT time necessaryto reduce 90% of the microbial population at a treatment temperatureT) of heat treatments at 110, 105, and 100 °C were reduced 0.13, 0.59,and 5.31 min, respectively, after applying a UV irradiation treatment.However, the z value (number of degrees needed to decrease D valuesten-fold) from conventional heat treatments (z = 6.53 °C) did notchange significantly (p > 0.05) when they were preceded by the UVtreatment (z = 6.71 °C).

Lo

g10

Nt/N

0

0 1 2 3 4 5 6 7-4

-3

-2

4. Discussion

In this investigation, the UV-C resistance of different species'bacterial spores used in food preservation was tested in a “in flow”

device. The effect of the most important environmental factor—theabsorptivity of the treatment medium—on the UV inactivation kinet-ics of the most resistant species was studied, and the lethality of a

0 5 10 15 20 250

5

10

15

20

Sl (

J/m

L)

Fig. 3. Relationship between the absorption coefficient (α) of the treatment mediumand the shoulder length (Sl) of Bacillus coagulans survival curves to UV light at 25(●) and 60 °C (○).

Lo

g10

Nt/N

0

Time (min)

0 5 10 15 20 25 30-4

-3

-2

-1

0

Time (min)

C

Fig. 5. Survival curves of Bacillus coagulans spores to heat at 110 (A), 105, (B) and100 °C (C) with (opened symbols) and without (solid symbols) prior UV exposure(27.10 J/mL) in citrate–phosphate buffer of 17.0 cm−1 absorption coefficient.

144 E. Gayán et al. / Innovative Food Science and Emerging Technologies 19 (2013) 140–145

combined process of UV and heat, applied simultaneously or succes-sively, was explored.

UV survival curves of all species studied had shoulders (Fig. 1).Deviations from linearity are attributed to a non-uniform dose distri-bution inside UV reactors (Koutchma, Keller, Chirtel, & Parisi, 2004).However, this is not the case in our device, as we previously demon-strated (Gayán et al., 2011, 2012). This profile has also been related invegetative cells with damage and repair mechanisms (López-Malo &Palou, 2005). UV light creates mutated bases that compromise cellfunctionality, but bacteria have developed DNA repair mechanismsto restore DNA structure and functionality (Friedberg et al., 2006).This phenomenon is reflected in the shape of the inactivation curves.After the initial shoulder, the maximum DNA repair capability issurpassed, and survivors decline exponentially (López-Malo & Palou,2005). It is not expected that DNA repair mechanisms act immediatelyafter UV treatments because bacterial spores are metabolically inactivedue to the low water activity of the protoplast. However, spore repairsystems could operate during spore germination, resulting in a lagphase before the inactivation began (Mamane-Gravetz & Linden,2005). It is interesting that our data shows that Kmax values are thesame for all species tested and that differences in UV resistance isdue to changes in Sl. Therefore, the different UV spore resistance ofG. stearothermophilus b B. licheniformis b B. cereus b A. acidocaldarius bB. coagulanswas likely due to the different efficacies of their repairmech-anisms. Other authors have found that B. subtilis was more UV sensitivethan B. cereus (Rossitto et al., 2012) and G. stearothermophilus (Chaine,Levy, Lacour, Riedel, & Carlin, 2012). These disagreements could be dueto UV resistance variability among strains or to methodological differ-ences. Because the design of UV equipment, processing parameters, andoptical properties of the liquid play an important role in UV germicidalefficacy (Keyser, Muller, Cilliers, Nel, & Gouws, 2008; Koutchma et al.,2009), the comparisons of UV resistance data obtained by differentauthors are risky. Our results were obtained in the same conditions,so they can be directly compared. Table 1 shows that UV resistance (4Dvalues) of the most sensitive G. stearothermophilus was 30% lower thanthat of the most UV-resistant B. coagulans. This is a small differencewhen comparedwith the high heat resistance variations. Table 2 includesthe decimal reduction times at 111 °C (D111 °C) and z values of the strainsused in this investigation. As observed, D111 °C values can vary 338-fold.On the other hand, the most UV-sensitive species (B. stearothermophilus)is the most heat-resistant species. Therefore, the mechanisms of sporeinactivation using both technologies are quite different. Practically,the results indicate the advantage of UV treatments over current heatsterilization: The stability ensured by heat treatments depends on themicrobial species contaminating the raw material, but the stabilityensured by UV treatments is similar even if the contaminating bacterialspore changes.

Setlow (2001) estimated that the UV resistance of bacterial sporesof the Bacillus species is roughly 5–50 times higher than that of vege-tative cells. Results obtained in our laboratory with the samemethod-ology demonstrated that the UV resistance of spores is also greaterthan that of vegetative cells previously investigated but to a lesserextent. For instance, the estimated dose necessary to inactivate 99.99%of the initial population of the most resistant spore, B. coagulans, istwice that necessary for the most UV-tolerant strains of E. coli (Gayánet al., 2011) and Salmonella Typhimurium (Gayán et al., 2012). The

Table 2Heat resistance parameters (decimal reduction times and z values) of bacterial spores used

Spore D111 °C

Bacillus licheniformis (STCC 4523) 0.11Bacillus coagulans (STCC 4522) 1.7Geobacillus stearothermophilus (STCC 12980) 37.2Bacillus cereus (STCC 9818) 0.27Alicyclobacillus acidocaldarius (STCC 5137) 2.6

difference in UV resistance between bacterial spores and vegetativecells is small when compared to the difference in heat resistance(DT multiply approximately 1012-fold). The higher heat resistance ofbacterial spores correlates with the low water activity of the protoplast(Setlow, 2006), and it has been demonstrated that low water activitydoes not protect bacterial cells from UV light (Gayán et al., 2011,2012). This could explain our results.

Table 1 data shows that higher absorption coefficients increaseshoulder length (Sl) and decrease inactivation rate (Kmax). Theseresults are consistent with Beer–Lambert–Bougerts Law, which statesthat the amount of light that penetrates a solution decreases whenthe absorbance of the solution increases. Plotting Kmax against theabsorption coefficient shows that there is an exponential relationshipbetween both variables (Fig. 2). While working with vegetative cellsin the same experimental conditions, Gayán et al. (2011, 2012) alsofound an exponential relationship. The regression line that relatesLog10 Kmax and the absorption coefficient showed that the inactiva-tion rate (Kmax) of B. coagulans decreases ten times by increasingthe absorption coefficient 16.8 ± 0.8 cm−1. This value does notsignificantly differ (p > 0.05) from that obtained for E. coli (15.9 ±1.0 cm−1) (Gayán et al., 2011) and Salmonella Typhimurium(18.9 ± 2.8 cm−1) (Gayán et al., 2012). These results indicated thatthe effect of the absorptivity has a physical basis and is not relatedto the biological behavior of microorganisms. Contrary to the inacti-vation rate, shoulder length (Sl) increases linearly with absorptivity(Fig. 3). There is currently no data in the literature to which similarbehavior can be compared.

The difficulty in UV light treatment achieving the necessary micro-bial reduction (due to the low penetration capacity of UV photons inmedia with high absorptivity) has prompted development of hurdlestrategies that combine UV light with other novel processing tech-niques or milder conventional preservation methods. It has beendemonstrated that the combination of mild heat with non-thermaltechnologies such as PEF and US at low intensity results in an equiv-alent or even higher degree of microbial inactivation (Heinz, Toepfl, &Knorr, 2003; Raso, Pagan, Condón, & Sala, 1998). However, data onthe combination of UV light and mild heat treatments is scarce.Gayán et al. (2011, 2012) demonstrated that the lethality of UVlight on vegetative cells increases synergistically from 40 to 60 °C.Nevertheless, there is no data in the literature on the effect of thecombined process on bacterial spores. Our results demonstrated thatthe UV inactivation (27.10 J/mL) of the most resistant B. coagulansspores increases 10 to 100 times by increasing the temperature from25 to 60 °C. The magnitude of the effect was the same at 55 and 65 °C(data not shown). The comparison of UV resistance parameters(Table 1) obtained at both temperatures demonstrated that the shoul-der length decreases and the inactivation rate increases with tempera-ture. The effect of media absorptivity on Kmax values (Fig. 2) is thesame at both temperatures, but the differences in shoulder lengthincrease with the absorption coefficient (Fig. 3). Therefore, the syner-gism of the combined UV-H treatment is greater in media with higherabsorptivity—when it might be more useful for UV food sterilization.

To explore themechanism of the synergistic lethal effect of the com-bined UV-H treatment, we applied both technologies sequentially intwo possible orders. Results demonstrated that a previous treatmentat 60 °C for 3.58 min does not sensitize B. coagulans to a subsequent

in this investigation.

z Reference

6.8 Palop, Raso, Pagan, Condón, and Sala (1996)8.9 Palop, Raso, Pagan, Condón, and Sala (1999)7.6 López, González, Condón, and Bernardo (1996)7.7 González, López, Martínez, Bernardo, and González (1999)6.7 Palop, Álvarez, Raso, and Condón (2000)

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UV treatment (27.10 J/mL). This fact discarded the possibility that thesublethal heat treatment could activate dormant spores potentiatingthe germination, thereby sensitizing them to UV light, as has been sug-gested for the combination of high pressures and mild temperatures(Lovdal, Hovda, Granum, & Rosnes, 2011). In contrast, the applicationof a prior UV treatment (27.10 J/mL) sensitized bacterial spores to asubsequent heat treatment (Fig. 5). The magnitude of the sensitizingeffect was the same at 100, 105, and 110 °C (31.71, 30.72, and 38.01%,respectively), which means that it was independent of the treatmenttemperature. In other words, the UV treatment does not affect z values.This behavior was similar to that observed for vegetative cells. Thesequential application of heat (55 °C for 3.58 min) and UV light(27.10 J/mL) on Salmonella Typhimurium showed an additive lethaleffect whereas in the inverse order, the thermotolerance of Salmonelladecreased synergistically (Gayán et al., 2012). The magnitude of thesynergistic lethal effect on Salmonellawas independent of the treatmenttemperature and did not affect the z value. So the mechanism of thesynergistic lethal effect of the combined treatment on bacterial sporeinactivation could be related to that of vegetative bacteria. Gayán et al.(2012) suggested that the mechanism of the synergistic effect of UVlight and heat on Salmonella is related to a sensitization of cell envelopesor the cell's inability to repair these structures. The most synergisticbenefits of the UV light in combination with chemical oxidant com-pounds to inactivate spores have been observed when both agents areapplied simultaneously (compared to the two sequential orders),suggesting that the oxidant agent could destroy the surface componentof the cell and assist the UV transmission into inner cell componentsandDNA damage (Jung et al., 2008). Overall, this is an interesting aspectthat deserves further investigation.

5. Conclusion

Unlike most non-thermal technologies, UV light is a real alternativeto current heat sterilization treatments aimed at the inactivation of bac-terial spores. The most UV-sensitive species (B. stearothermophilus) isthe most heat-resistant species. The main limitation of UV bacterialinactivation is the absorption coefficient of the treatment medium,but this limitation is of similar importance in the pasteurization (inacti-vation of vegetative cells) and sterilization (inactivation of bacterialspores) processes. For UV sterilization of liquid foods with high absorp-tion coefficients, the efficacy of UV light can be enhanced when com-bined with heat.

Acknowledgments

This study has been carried out with financial support fromthe Ministerio de Ciencia e Innovación de España, EU-FEDER(CIT020000-2009-40) and the Departamento de Ciencia, Tecnología yUniversidad del Gobierno de Aragón. E. G. gratefully acknowledges thefinancial support for her doctoral studies from the Ministerio deEducación y Ciencia de España.

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