International Journal of Food Microbiology 192 (2015) 72–76
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
The effect of carvacrol on enteric viruses C. Sánchez a, R. Aznar a,b, G. Sánchez a,b,⁎ a b
Departament of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, Burjassot, 46100 Valencia, Spain Departament of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), Av. Agustín Escardino, 7, Paterna, 46980 Valencia, Spain
a r t i c l e
i n f o
Article history: Received 16 July 2014 Received in revised form 18 September 2014 Accepted 27 September 2014 Available online xxxx Keywords: Norovirus Hepatitis A virus Carvacrol Natural compounds Fresh-cut vegetables
a b s t r a c t Carvacrol, a monoterpenic phenol, is said to have extensive antimicrobial activity in a wide range of food spoilage or pathogenic fungi, yeast and bacteria. The aim of this study was to assess its antiviral activity on norovirus surrogates, feline calicivirus (FCV), murine norovirus (MNV), and hepatitis A virus (HAV), as well as its potential in food applications. Initially, different concentrations of carvacrol (0.25, 0.5, 1%) were individually mixed with each virus at titers of ca. 6–7 log TCID50/ml and incubated 2 h at 37 °C. Carvacrol at 0.5% completely inactivated the two norovirus surrogates, whereas 1% concentration was required to achieve ca. 1 log reduction of HAV. In lettuce wash water, carvacrol efficacy on MNV was dependent on the chemical oxygen demand (COD), with no effect over 300 ppm. A 4 log reduction in FCV infectivity was observed when 0.5% carvacrol was used to sanitize lettuce wash water, regardless of COD. Carvacrol was also evaluated as a natural disinfectant of produce, showing 1% carvacrol reduced inoculated NoV surrogates titers in lettuce by 1 log after 30 min contact. These results represent a step forward in improving food safety by using carvacrol as an alternative natural additive to reduce viral contamination in the fresh vegetable industry. © 2014 Elsevier B.V. All rights reserved.
1. Introduction There is increasing awareness of the importance of foodborne diseases caused by enteric viruses; furthermore, several international organizations have detected an upward trend in their incidence. Epidemiological evidence indicates that enteric viruses, in particular human norovirus (NoV) and hepatitis A virus (HAV), are the leading causes of foodborne illnesses in developed countries mainly associated with the consumption of shellfish, soft fruits and leafy greens (Anonymous, 2013; EFSA, 2014a). In the EU, foodborne viruses were identified as the most frequently detected causative agent of foodborne outbreaks in vegetables in 2012, accounting for 25.6% of the reported cases (EFSA, 2014a). The transmission of enteric viruses associated with the consumption of leafy greens is regularly reported after contamination by virusinfected food handlers during harvesting, packaging, or food preparation or by polluted irrigation water. Therefore, it is important to find natural antiviral biopreservatives which are safe, environment friendly, and preferably inexpensive for use in the food industry. The growing demand for the use of natural additives has produced a substantial increase in the number of studies based on natural extracts such as essential oils or their main compounds in the last decade. They are categorized as Generally Recognised as Safe (GRAS), and are
therefore potential alternatives to chemical additives. Furthermore, the antibacterial, antifungal, insecticidal, antitoxigenic and antiparasitic activities of these compounds have been extensively reported (Burt, 2004). In contrast, reports on the antiviral effects of essential oils or their compounds are somewhat limited (Table 1). Carvacrol, a monoterpenic phenol, has emerged due to its wide spectrum activity extended to food spoilage or pathogenic fungi, yeast and bacteria (Nostro and Papalia, 2012). Carvacrol is the primary component of oregano essential oil and has been identified as a natural economical food preservative (Lu and Wu, 2010; Obaidat and Frank, 2009) with potential for incorporation in food packaging (Guarda et al., 2011). It has recently been reported that carvacrol could effectively reduce the infectivity of murine norovirus (MNV) (Gilling et al., 2014a), a human norovirus surrogate, and rotavirus (Pilau et al., 2011). However, the effectiveness of carvacrol against other foodborne viruses, as well as its efficacy in food applications has yet to be explored. In this study, the effect of carvacrol on the infectivity of HAV and two norovirus surrogates, MNV and feline calicivirus (FCV) has been assessed. We have also studied the efficacy of carvacrol in reducing viral loads in fresh-cut lettuce wash water and on a produce model. 2. Material and methods 2.1. Virus strains and cell lines
⁎ Corresponding author at: Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Avda. Agustín Escardino, 7, Paterna, Valencia, Spain. Tel.: +34 96 3900022; fax: +34 96 3939301. E-mail address: [email protected] (G. Sánchez).
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.028 0168-1605/© 2014 Elsevier B.V. All rights reserved.
The cytopathogenic MNV-1 strain (kindly provided by Prof. H. W. Virgin, Washington University School of Medicine, USA), the F9 strain of FCV (ATCC VR-782) and the HM-175 strain of HAV (ATCC VR-1402)
C. Sánchez et al. / International Journal of Food Microbiology 192 (2015) 72–76
73
Table 1 Efficacy of essential oils and their compounds on different enteric viruses. Tested virus
Experimental setup (time, temperature)
Inactivation (log reduction)
References
Essential oils Allspice (4%) Clove (1%) Hyssop (0.2%)
MNV FCV, MNV AdV-2, MNV
3.41 3.75; 0.67 No effect
Gilling et al. (2014b) Elizaquível et al. (2013) Kovac et al. (2012)
Lemongrass (4%) Marjoram (0.2%)
MNV AdV-2, MNV
24 h, 24 °C 2 h, 37 °C 2 and 24 h 4, 25 and 37 °C 24 h, 24 °C 2 and 24 h 4, 25 and 37 °C
2.74 No effect
Gilling et al. (2014b) Kovac et al. (2012)
Mexican oregano Oregano (2%) Oregano (4%) Tea tree (0.025%) Zataria (0.1%)
RV FCV, MNV MNV PV 1, Echo 9, Coxsackie B1, AdV-2 FCV, MNV
No effect 3.75; 1.62 1.10 No effect 4.51; 0.25
Pilau et al. (2011) Elizaquível et al. (2013) Gilling et al. (2014a) Garozzo et al. (2009) Elizaquível et al. (2013)
Main compounds Carvacrol (0.5%) Citral (4%)
MNV MNV
3.87 3.00
Gilling et al. (2014a) Gilling et al. (2014b)
2 h, 37 °C 6 h, 24 °C 1 h, 37 °C 2 h, 37 °C
3 h, 24 °C 24 h, 24 °C
were propagated and assayed in RAW 264.7 (kindly provided by Prof. H. W. Virgin), CRFK (ATCC CCL-94) and FRhK-4 cells (kindly provided by Prof. Albert Bosch, University of Barcelona), respectively. Semi-purified stocks were subsequently produced from the same cells by centrifugation of infected cell lysates at 660 ×g for 30 min. Infectious viruses were enumerated by determining the 50% tissue culture infectious dose (TCID50) with eight wells per dilution and 20 μl of inoculum per well.
FCV at ca. 106 TCID50/ml, and carvacrol was added to the inoculated water to obtain concentrations of 0.5%. Triplicate samples were incubated at 37 °C during 2 h with gentle agitation (150 rpm) in a water bath. Treated and non-treated samples were 10-fold serial diluted, and cell culture assays were performed the same day.
2.2. Cytotoxicity determination of carvacrol on cell lines
Determination of the virucidal activity of carvacrol wash was performed by adapting the procedure described by Su and D' Souza, 2013. Briefly, locally purchased fresh lettuce was cut in pieces of 3 × 3 cm and sterilized with UV light in a safety cabinet under laminar flow for 15 min prior to inoculation of the test viruses. A suspension of FCV or MNV diluted in PBS buffer (at 106 or 104 TCID50for both viruses) was seeded by distributing 30 μl over 3 spots onto the lettuce surface. Inoculated samples were air dried in a laminar flow hood for 45 min. Thereafter, 0.15 ml of water or a carvacrol solution at 0.5 or 1% was added for 30 min to inoculated lettuce samples. After treatments, viruses were eluted from the lettuce surface by pipetting with 0.85 ml DMEM containing 2% FCS, and samples were 10-fold serial diluted, and cell culture assays were performed the same day. Each treatment was done in triplicate.
Carvacrol (≥ 98% purity; Sigma Aldrich) at various concentrations were added to individual wells of confluent RAW 264.7, CRFK and FRhK-4 cells in 96-well plates and incubated 2 h under 5% CO2. Thereafter cells were added with 150 μl of supplemented with 2% of fecal calf serum (FCS) and incubated further for 2 to 15 days. Cytotoxicity effects were determined by visual inspection under the optical microscope. 2.3. Antiviral effects of carvacrol Carvacrol diluted in 50% ethanol was added to virus suspensions in DMEM with 2% FCS (ca. 6–7 log TCID50/ml) and further incubated at 37 °C (unless indicated) in a shaker 2 h. Then, infectious viruses were enumerated by cell culture assays as described above. Each treatment was done in triplicate. Positive controls were virus suspensions added with ethanol in amounts corresponding to the highest quantity present. For exposure time experiments, carvacrol at 0.5% was added to MNV and FCV suspensions at ca. 6–7 log TCID50/ml. Samples were immediately removed (t = 0) and at different time intervals (30, 60 and 120 min), and ten-fold diluted in DMEM with 2% FCS to neutralize the carvacrol action. Then, infectious viruses were enumerated by cell culture assays as described above. Experiments were performed in triplicates. Antiviral activity of carvacrol was estimated by comparing the number of infectious viruses on suspensions without carvacrol and on the carvacrol-treated virus suspensions. 2.4. Antiviral activity of carvacrol in fresh-cut lettuce wash water The process wash water with high chemical oxygen demand (COD) was prepared as described by López-Gálvez et al., 2011. Fresh-cut lettuce wash water was diluted with tap water to obtain a batch with 500 mg/l COD. The process wash water were further diluted with tap water to obtain COD values of ca. 200, 300, and 400 mg/l. Lettuce wash water with different COD values were inoculated with MNV and
2.5. Produce wash test using lettuce
2.6. Statistical analysis The significance of differences among the mean numbers of viruses determined after the various treatments was determined by Student's t test with a significance level of P b 0.05 (Microsoft Office Excel; Microsoft, Redmond, WA, USA). 3. Results 3.1. Determination of cytotoxicity of carvacrol on cell lines Carvacrol was found to be cytotoxic at concentrations that exceeded 1%, for the three cell lines. Thus, this value was the maximum concentration of carvacrol added to determine the antiviral effects of carvacrol against MNV, FCV and HAV. 3.2. Effect of carvacrol on the infectivity of norovirus surrogates and HAV Incubation of MNV and FCV with carvacrol at concentrations of 0.25, 0.5 and 1% for 2 h at 37 °C decreased the titer of the two norovirus surrogates (Table 2). Carvacrol at 0.5 and 1% reduced FCV and MNV titers to undetectable levels, while carvacrol at 0.25% reduced FCV by 3.4 log
74
C. Sánchez et al. / International Journal of Food Microbiology 192 (2015) 72–76
Table 2 Effect of carvacrol against feline calicivirus (FCV), murine norovirus (MNV) and Hepatitis A virus (HAV) after 2 h of incubation at 37 °C. FCV
MNV
Log10 TCID50/ml
Log10 TCID50/ml
HAV Log10 TCID50/ml
Treatmenta
Recovered titer
Reduction
Recovered titer
Reduction
Recovered titer
Reduction
Control 0.25% C 0.5% C 1% C
5.82 2.41 b1.32 b1.32
3.41 N4.53 N4.53
5.70 3.84 b2.13 b2.13
1.86 N3.57 N3.57
6.12 5.96 5.98 5.15
0.16 0.14 0.97
± ± ± ±
0.32A 0.07B 0.00C 0.00C
± ± ± ±
0.00A 1.29B 0.00C 0.00C
± ± ± ±
0.35A 0.12A 0.19A 0.14B
Within each column for each virus, different letters denote significant differences between treatments (P b 0.05). a Each treatment was done in triplicate.
TCID50/ml. On the other hand, the hardier surrogate virus, MNV, was reduced by 1.8 log TCID50/ml after treatment with carvacrol at 0.25%. For HAV, no differences in titers reduction were observed between HAV suspensions treated or non-treated with carvacrol at 0.25 and 0.5%, whereas ca. 1 log reduction was observed when treated with carvacrol at 1% (Table 2). Carvacrol treatments were also performed at low HAV titers (ca. 4 log TCID50/ml) but this viral load rendered the same results (data not shown). Since HAV titers were slightly affected by carvacrol treatment, further experiments focused only on the two norovirus surrogates. Fig. 1 shows the infectivity of MNV and FCV after treatment with carvacrol at 0.5% detected by cell culture at different exposure times. Significant reduction 3.1 log (P b 0.05) in MNV-1 infectivity was observed after 30 min treatment with carvacrol at 0.5%. Complete inactivation of MNV was achieved after 1 hour treatment whereas 30 min treatment was enough to fully inactivate FCV, the other NoV surrogate tested. The virus titer reduction was also found to be dependent on the temperature. As shown in Fig. 2, incubation of norovirus surrogates with 0.5% carvacrol showed titer reductions to undetectable levels of both MNV and FCV at 37 °C, whereas titer reductions of 0.75, and 2.19 log TCID50/ml in MNV and 2.31, and 3.19 log TCID50/ml in FCV were found at 24 and 4 °C respectively. Statistical analyses showed that, overall, 0.5% carvacrol treatment at 37 °C for 2 h caused greater reduction on MNV and FCV than room and refrigerated temperatures (P b 0.05). Moreover, higher inactivation rates were reported for 4 °C than 24 °C for both norovirus surrogates, but yet less than 37 °C. 3.3. Effect of carvacrol in water treatment using model lettuce wash water Reductions in the infectious titers of MNV and FCV inoculated in lettuce wash water with different COD values (200, 300 and 400 mg/l),
A
either with or without 0.5% carvacrol added as a sanitizer, are shown in Table 3. The antiviral effect of carvacrol at 0.5% was very much dependent of COD values for MNV, where no virus reduction was observed in lettuce wash water with COD values of 300 ppm or higher. For FCV, the inactivating effect of carvacrol was slightly decreased as the COD values increased. 3.4. Efficacy of carvacrol on produce The infectivity of NoV surrogates with or without treatment with carvacrol on lettuce samples is shown in Table 4. Sensorial parameters of lettuce were not affected (data not shown). When inoculated at high titers, no significant reduction (P b 0.05) of MNV and FCV infectivity was observed after washing of lettuce with 0.5% carvacrol, whereas ca. 1 log reduction was observed for both surrogates at 1% carvacrol. MNV at low titers was reduced by ca. 1.8 log after washing lettuce with 1% carvacrol, whereas for FCV, virus titers had fallen to below detectable limits when inoculated at low titers (Table 4). 4. Discussion Although our knowledge of the enteric virus inactivation by natural compounds is far from complete, a dramatic increase in publications evaluating the antiviral properties of different natural compounds (reviewed by Li et al., 2013) has been seen in recent years, including some potential applications in the food industry (Azizkhani et al., 2013; Li et al., 2012; Su and D' Souza, 2013). This may be partly due to increased awareness of the prevalence of enteric viruses in food products, likewise the increasing consumer for effective economic, safe, healthy natural products. This study clearly shows that carvacrol was effective in reducing the titers of norovirus surrogates in a dose-dependent manner, where
B
Fig. 1. Infectivity of murine norovirus (MNV) (A) and feline calicivirus (FCV) (B) detected by cell-culture either without (black bars) or with 0.5% carvacrol treatment (gray bars) at different interval times. Each column represents the average of triplicates. Dashed line depicts the detection limit. Star indicates the time that virus reached total inactivation.
C. Sánchez et al. / International Journal of Food Microbiology 192 (2015) 72–76
B
A 8
8
6
6
Log( TCID50/mL)
Log (TCID50/mL)
75
4
2
4
2
0
0 37
24
4
37
Temperature(°C)
24
4
Temperature(°C)
Fig. 2. Infectivity of murine norovirus (MNV) (A) and feline calicivirus (FCV) (B) detected by cell-culture either without (black bars) or with 0.5% carvacrol treatment (gray bars) at different temperatures. Each column represents the average of triplicates. Dashed line depicts the detection limit.
increasing concentrations of carvacrol showed increased reduction in viral titers. The efficacy of carvacrol has also recently been assessed on the MNV S7-PP3 strain. Gilling et al. (2014a) reported 1.77 log TCID50/ ml reduction after 30 min treatment with 0.5% carvacrol at 24 °C whereas in this study MNV-1 infectivity was reduced by 3.01 log at 37 °C for 30 min or 0.79 log at room temperature for 2 h. The differences reported may be due to strain variability and dissolvent used to prepare the carvacrol solution. Furthermore carvacrol treatment resulted in slight reductions on HAV infectivity with a maximum reduction of less than 1 log TCID50/ml at the maximum concentration tested. Overall, this study showed that carvacrol treatment caused greater reduction on FCV than MNV titers. This has been shown for other natural antimicrobials, such as oregano, zataria and clove essential oils (Elizaquível et al., 2013) or grape seed extract (GSE) (Su and D'Souza, 2011). However, in this latter publication, MNV was more resistant than HAV to grape seed treatment, unlike our results. Interestingly, the reduction of norovirus surrogates by carvacrol at 4 °C was found to be greater than at 24 °C, although less than at 37 °C. Most of the studies have assessed the virucidal activity of natural compounds at 24 or 37 °C (Table 1), and when performed at 4 °C (Elizaquível et al., 2013; Kovac et al., 2012) the antiviral activity was much lower or ineffective. For the two norovirus surrogates, carvacrol exerted the strongest effect at 37 °C, yet was still active at refrigerated temperatures, which may facilitate the final application in the food industry. In the fresh-cut vegetable industry, water immersion of fresh-cut leafy greens in washing tanks during processing, presents a risk of cross-contamination from leafy greens to water or vice versa (EFSA, 2014b). Use of chlorine has been a standard for industry for decades since it is both relatively cheap and easy to use. However in some
European Union countries such as: Germany, Switzerland, the Netherlands, Denmark, and Belgium, the use of chlorine is prohibited since its use may generate the formation of by-products, such as trihalomethanes and other chemical residues, which has led to the search for new alternatives for disinfection (Van Haute et al., 2013). Therefore, in this study, the antiviral activity of carvacrol was further explored by assessing its activity in lettuce wash water. To investigate the interfering effect of COD, as an indication of the organic quality of water, the antiviral activity of 0.5% carvacrol was determined in lettuce wash water at different COD values. While for FCV efficacy of 0.5% carvacrol was unaffected by COD, for MNV the presence of COD ≥ 300 ppm significantly reduced the efficacy of carvacrol, which may be because part of the carvacrol was blocked by the organic matter while the concentration of free carvacrol was insufficient to inactivate MNV. Assessment of natural compound application in food applications is scarce, so far only GSE has been reported as an effective natural sanitizer against NoV contamination in fresh-cut lettuce processing water with ca. 1.5 to 2 log PFU/ml reduction of MNV, while the role of the wash water COD was deemed insignificant. The application of natural compounds, such as carvacrol, in the sanitation of fresh-cut vegetable water still needs to address some issues, such as treatment time and temperature adaptation to more realistic conditions. With regard to application of carvacrol in food sanitation, lettuce was chosen as the model of fresh leafy greens. Consumption of leafy greens has been associated with foodborne outbreaks, many of which Table 4 Reduction of murine norovirus (MNV) and feline calicivirus (FCV) titers on lettuce at high (ca. 6 log10 TCID50/ml) and low (ca. 4 log TCID50/ml) virus titers after 30 min treatment with carvacrol at room temperature. MNV
FCV
Log10 TCID50/ml Table 3 Reduction of murine norovirus (MNV) and feline calicivirus (FCV) titers after treatment with carvacrol at 0.5% in lettuce wash watera. Lettuce wash water (COD concentration, ppm)
MNV
FCV
0 200 300 400
N4.50A 4.49 ± 0.07A 0.24 ± 0.19B 0.41 ± 0.12B
N3.50A 3.08 ± 0.25A 2.99 ± 0.26A 2.64 ± 0.16A
Within each column for each virus, different letters denote significant differences between treatments (P b 0.05). a Each treatment was done in triplicate.
High virus titer
Low virus titer
Log10 TCID50/ml
Washing treatmenta
Recovered titer
Reduction
Recovered titer
Reduction
Water 0.5% C
6.24 ± 0.19A 6.03 ± 0.31A
– 0.21
6.45 ± 0.21A 6.24 ± 0.38A
– 0.21
1% C Water 0.5% C
5.49 ± 0.14B 3.82 ± 0.19A 3.79 ± 0.12A
0.92 – 0.03
5.45 ± 0.17B 3.91 ± 0.38A 3.66 ± 0.58A
1.00
1% C
2.03 ± 0.38B
1.79
b1.32B
N2.59
0.25
Within each column for each virus, different letters denote significant differences between treatments (P b 0.05). a Each treatment was done in triplicate.
76
C. Sánchez et al. / International Journal of Food Microbiology 192 (2015) 72–76
were due to enteric viruses (Doyle and Erickson, 2008). Moreover, for human NoV, most contamination levels found on leafy greens range from 0 to 5 log genomic copies/g leafy greens (Baert et al., 2011; Kokkinos et al., 2012; Stals et al., 2011). So far, only a few reports are available on the application of natural compounds as a produce wash for foodborne viral reduction. Our results on carvacrol reveal that it exerts a strong antiviral activity against FCV at 1% in lettuce whereas log reduction was achieved on MNV. Similarly, FCV titers were reduced to undetectable levels by GSE at 0.25 mg/ml, but MNV could only be reduced by ca. 1 log PFU on lettuce (Su and D' Souza, 2013). Considering that MNV is a better norovirus surrogate than FCV, and that carvacrol slightly affects HAV infectivity, application of carvacrol alone for food sanitation may not be suitable for complete reduction of foodborne viral contamination. Thus, carvacrol should be considered for use as part of hurdle approaches combining carvacrol with other natural compounds, e.g. GSE, or with other treatments for foodborne virus reduction on leafy greens, e.g. active packaging (Martínez-Abad et al., 2013) or high powered ultrasound (Su et al., 2010). Overall, our findings highlight the potential of carvacrol as an inexpensive natural alternative to reduce viral contamination and, therefore, improve the safety of fresh cut products. Acknowledgments G. Sánchez was supported by the “Ramón y Cajal” Young Investigator program of the Spanish Ministry of Economy and Competitiveness. This study was supported by grant AGL2009-08603 from the Spanish Ministry of Science and Innovation, and ACOMP/2010/279 and ACOMP/2012/199 from the Generalitat Valenciana. References Anonymous, 2013. Surveillance for foodborne disease outbreaks—United States, 2009–2010. Morb. Mortal. Wkly Rep.MMWR 62, 41–47. Azizkhani, M., Elizaquível, P., Sánchez, G., Selma, M.V., Aznar, R., 2013. Comparative efficacy of Zataria multiflora Boiss., Origanum compactum and Eugenia caryophyllus essential oils against E. coli O157:H7, feline calicivirus and endogenous microbiota in commercial baby-leaf salads. Int. J. Food Microbiol. 166, 249–255. Baert, L., Mattison, K., Loisy-Hamon, F., Harlow, J., Martyres, A., Lebeau, B., Stals, A., Van Coillie, E., Herman, L., Uyttendaele, M., 2011. Review: norovirus prevalence in Belgian, Canadian and French fresh produce: a threat to human health. Int. J. Food Microbiol. 15, 261–269. Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in foods—a review. Int. J. Food Microbiol. 94, 223–253. Doyle, M.P., Erickson, M.C., 2008. Summer meeting 2007—the problems with fresh produce: an overview. J. Appl. Microbiol. 105, 317–330. EFSA, 2014a. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2012. EFSA J. 12 (2) (3547, 312 pp.). EFSA, 2014b. Scientific opinion on the risk posed by pathogens in food of non-animal origin. Part 2 (Salmonella and Norovirus in leafy greens eaten raw as salads. EFSA J. 12( (3), 3600.
Elizaquível, P., Azizkhani, M., Aznar, R., Sanchez, G., 2013. The effect of essential oils on norovirus surrogates. Food Control 32, 275–278. Garozzo, A., Timpanaro, R., Bisignano, B., Furneri, P.M., Bisignano, G., Castro, A., 2009. In vitro antiviral activity of Melaleuca alternifolia essential oil. Lett. Appl. Microbiol. 49, 806–808. Gilling, D.H., Kitajima, M., Torrey, J.R., Bright, K.R., 2014a. Antiviral efficacy and mechanisms of action of oregano essential oil and its primary component carvacrol against murine norovirus. J. Appl. Microbiol. 116, 1149–1163. Gilling, D.H., Kitajima, M., Torrey, J.R., Bright, K.R., 2014b. Mechanisms of antiviral action of plant antimicrobials against murine norovirus. Appl. Environ. Microbiol. 80, 4898–4910. Guarda, A., Rubilar, J.F., Miltz, J., Galotto, M.J., 2011. The antimicrobial activity of microencapsulated thymol and carvacrol. Int. J. Food Microbiol. 146, 144–150. Kokkinos, P., Kozyra, I., Lazic, S., Bouwknegt, M., Rutjes, S., Willems, K., Moloney, R., de Roda Husman, A., Kaupke, A., Legaki, E., D'Agostino, M., Cook, N., Rzeżutka, A., Petrovic, T., Vantarakis, A., 2012. Harmonised investigation of the occurrence of human enteric viruses in the leafy green vegetable supply chain in three European countries. Food Environ. Virol. 4 (4), 179–191. Kovac, K., Diez-Valcarce, M., Raspor, P., Hernández, M., Rodríguez-Lázaro, D., 2012. Natural plant essential oils do not inactivate non-enveloped enteric viruses. Food Environ. Virol. 4, 209–212. Li, D., Baert, L., Zhang, D., Xia, M., Zhong, W., Van Coillie, E., Jiang, X., Uyttendaele, M., 2012. Effect of grape seed extract on human norovirus GII.4 and murine norovirus 1 in viral suspensions, on stainless steel discs, and in lettuce wash water. Appl. Environ. Microbiol. 78 (21), 7572–7578. Li, D., Baert, L., Uyttendaele, M., 2013. Inactivation of food-borne viruses using natural biochemical substances. Food Microbiol. 35 (1), 1–9. López-Gálvez, F., Posada-Izquierdo, G.D., Selma, M.V., Pérez-Rodríguez, F., Gobet, J., Gil, M.I., Allende, A., 2011. Electrochemical disinfection: an efficient treatment to inactivate Escherichia coli O157:H7 in process wash water containing organic matter. Food Microbiol. 30, 146–156. Lu, Y., Wu, C., 2010. Reduction of Salmonella enterica contamination on grape tomatoes by washing with thyme oil, thymol, and carvacrol as compared with chlorine treatment. J. Food Prot. 73 (12), 2270–2275. Martínez-Abad, A., Ocio, M.J., Lagarón, J.M., Sánchez, G., 2013. Evaluation of silver-infused polylactide films for inactivation of Salmonella and feline calicivirus in vitro and on fresh-cut vegetables. Int. J. Food Microbiol. 162, 89–94. Nostro, A., Papalia, T., 2012. Antimicrobial activity of carvacrol: current progress and future prospectives. Recent Patents on Anti-Infect. Drug Discov. 7, 28–35. Obaidat, M.M., Frank, J.F., 2009. Inactivation of Escherichia coli O157:H7 on the intact and damaged portions of lettuce and spinach leaves by using allyl isothiocyanate, carvacrol, and cinnamaldehyde in vapor phase. J. Food Prot. 72 (10), 2046–2055. Pilau, M.R., Alves, S.H., Weiblen, R., Arenhart, S., Cueto, A.P., Lovato, L.T., 2011. Antiviral activity of the Lippia graveolens (Mexican oregano) essential oil and its main compound carvacrol against human and animal viruses. Braz. J. Microbiol. 42 (4), 1616–1624. Stals, A., Baert, L., Jasson, V., Van Coillie, E., Uyttendaele, M., 2011. Screening of fruit products for norovirus and the difficulty of interpreting positive PCR results. J. Food Prot. 74 (3), 425–431. Su, X., D' Souza, D.H., 2013. Grape seed extract for foodborne virus reduction on produce. Food Microbiol. 34, 1–6. Su, X., D'Souza, D.H., 2011. Grape seed extract for control of human enteric viruses. Appl. Environ. Microbiol. 77, 3982–3987. Su, X.W., Zivanovic, S., D'Souza, D.H., 2010. Inactivation of human enteric virus surrogates by high-intensity ultrasound. Foodborne Pathog Dis 7, 1055–1061. Van Haute, S., Sampers, I., Holvoet, K., Uyttendaele, M., 2013. Physicochemical quality and chemical safety of chlorine as a reconditioning agent and wash water disinfectant for fresh-cut lettuce washing. Appl. Environ. Microbiol. 79 (9), 2850–2861.
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
The effect of carvacrol on enteric viruses C. Sánchez a, R. Aznar a,b, G. Sánchez a,b,⁎ a b
Departament of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, Burjassot, 46100 Valencia, Spain Departament of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), Av. Agustín Escardino, 7, Paterna, 46980 Valencia, Spain
a r t i c l e
i n f o
Article history: Received 16 July 2014 Received in revised form 18 September 2014 Accepted 27 September 2014 Available online xxxx Keywords: Norovirus Hepatitis A virus Carvacrol Natural compounds Fresh-cut vegetables
a b s t r a c t Carvacrol, a monoterpenic phenol, is said to have extensive antimicrobial activity in a wide range of food spoilage or pathogenic fungi, yeast and bacteria. The aim of this study was to assess its antiviral activity on norovirus surrogates, feline calicivirus (FCV), murine norovirus (MNV), and hepatitis A virus (HAV), as well as its potential in food applications. Initially, different concentrations of carvacrol (0.25, 0.5, 1%) were individually mixed with each virus at titers of ca. 6–7 log TCID50/ml and incubated 2 h at 37 °C. Carvacrol at 0.5% completely inactivated the two norovirus surrogates, whereas 1% concentration was required to achieve ca. 1 log reduction of HAV. In lettuce wash water, carvacrol efficacy on MNV was dependent on the chemical oxygen demand (COD), with no effect over 300 ppm. A 4 log reduction in FCV infectivity was observed when 0.5% carvacrol was used to sanitize lettuce wash water, regardless of COD. Carvacrol was also evaluated as a natural disinfectant of produce, showing 1% carvacrol reduced inoculated NoV surrogates titers in lettuce by 1 log after 30 min contact. These results represent a step forward in improving food safety by using carvacrol as an alternative natural additive to reduce viral contamination in the fresh vegetable industry. © 2014 Elsevier B.V. All rights reserved.
1. Introduction There is increasing awareness of the importance of foodborne diseases caused by enteric viruses; furthermore, several international organizations have detected an upward trend in their incidence. Epidemiological evidence indicates that enteric viruses, in particular human norovirus (NoV) and hepatitis A virus (HAV), are the leading causes of foodborne illnesses in developed countries mainly associated with the consumption of shellfish, soft fruits and leafy greens (Anonymous, 2013; EFSA, 2014a). In the EU, foodborne viruses were identified as the most frequently detected causative agent of foodborne outbreaks in vegetables in 2012, accounting for 25.6% of the reported cases (EFSA, 2014a). The transmission of enteric viruses associated with the consumption of leafy greens is regularly reported after contamination by virusinfected food handlers during harvesting, packaging, or food preparation or by polluted irrigation water. Therefore, it is important to find natural antiviral biopreservatives which are safe, environment friendly, and preferably inexpensive for use in the food industry. The growing demand for the use of natural additives has produced a substantial increase in the number of studies based on natural extracts such as essential oils or their main compounds in the last decade. They are categorized as Generally Recognised as Safe (GRAS), and are
therefore potential alternatives to chemical additives. Furthermore, the antibacterial, antifungal, insecticidal, antitoxigenic and antiparasitic activities of these compounds have been extensively reported (Burt, 2004). In contrast, reports on the antiviral effects of essential oils or their compounds are somewhat limited (Table 1). Carvacrol, a monoterpenic phenol, has emerged due to its wide spectrum activity extended to food spoilage or pathogenic fungi, yeast and bacteria (Nostro and Papalia, 2012). Carvacrol is the primary component of oregano essential oil and has been identified as a natural economical food preservative (Lu and Wu, 2010; Obaidat and Frank, 2009) with potential for incorporation in food packaging (Guarda et al., 2011). It has recently been reported that carvacrol could effectively reduce the infectivity of murine norovirus (MNV) (Gilling et al., 2014a), a human norovirus surrogate, and rotavirus (Pilau et al., 2011). However, the effectiveness of carvacrol against other foodborne viruses, as well as its efficacy in food applications has yet to be explored. In this study, the effect of carvacrol on the infectivity of HAV and two norovirus surrogates, MNV and feline calicivirus (FCV) has been assessed. We have also studied the efficacy of carvacrol in reducing viral loads in fresh-cut lettuce wash water and on a produce model. 2. Material and methods 2.1. Virus strains and cell lines
⁎ Corresponding author at: Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Avda. Agustín Escardino, 7, Paterna, Valencia, Spain. Tel.: +34 96 3900022; fax: +34 96 3939301. E-mail address: [email protected] (G. Sánchez).
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.028 0168-1605/© 2014 Elsevier B.V. All rights reserved.
The cytopathogenic MNV-1 strain (kindly provided by Prof. H. W. Virgin, Washington University School of Medicine, USA), the F9 strain of FCV (ATCC VR-782) and the HM-175 strain of HAV (ATCC VR-1402)
C. Sánchez et al. / International Journal of Food Microbiology 192 (2015) 72–76
73
Table 1 Efficacy of essential oils and their compounds on different enteric viruses. Tested virus
Experimental setup (time, temperature)
Inactivation (log reduction)
References
Essential oils Allspice (4%) Clove (1%) Hyssop (0.2%)
MNV FCV, MNV AdV-2, MNV
3.41 3.75; 0.67 No effect
Gilling et al. (2014b) Elizaquível et al. (2013) Kovac et al. (2012)
Lemongrass (4%) Marjoram (0.2%)
MNV AdV-2, MNV
24 h, 24 °C 2 h, 37 °C 2 and 24 h 4, 25 and 37 °C 24 h, 24 °C 2 and 24 h 4, 25 and 37 °C
2.74 No effect
Gilling et al. (2014b) Kovac et al. (2012)
Mexican oregano Oregano (2%) Oregano (4%) Tea tree (0.025%) Zataria (0.1%)
RV FCV, MNV MNV PV 1, Echo 9, Coxsackie B1, AdV-2 FCV, MNV
No effect 3.75; 1.62 1.10 No effect 4.51; 0.25
Pilau et al. (2011) Elizaquível et al. (2013) Gilling et al. (2014a) Garozzo et al. (2009) Elizaquível et al. (2013)
Main compounds Carvacrol (0.5%) Citral (4%)
MNV MNV
3.87 3.00
Gilling et al. (2014a) Gilling et al. (2014b)
2 h, 37 °C 6 h, 24 °C 1 h, 37 °C 2 h, 37 °C
3 h, 24 °C 24 h, 24 °C
were propagated and assayed in RAW 264.7 (kindly provided by Prof. H. W. Virgin), CRFK (ATCC CCL-94) and FRhK-4 cells (kindly provided by Prof. Albert Bosch, University of Barcelona), respectively. Semi-purified stocks were subsequently produced from the same cells by centrifugation of infected cell lysates at 660 ×g for 30 min. Infectious viruses were enumerated by determining the 50% tissue culture infectious dose (TCID50) with eight wells per dilution and 20 μl of inoculum per well.
FCV at ca. 106 TCID50/ml, and carvacrol was added to the inoculated water to obtain concentrations of 0.5%. Triplicate samples were incubated at 37 °C during 2 h with gentle agitation (150 rpm) in a water bath. Treated and non-treated samples were 10-fold serial diluted, and cell culture assays were performed the same day.
2.2. Cytotoxicity determination of carvacrol on cell lines
Determination of the virucidal activity of carvacrol wash was performed by adapting the procedure described by Su and D' Souza, 2013. Briefly, locally purchased fresh lettuce was cut in pieces of 3 × 3 cm and sterilized with UV light in a safety cabinet under laminar flow for 15 min prior to inoculation of the test viruses. A suspension of FCV or MNV diluted in PBS buffer (at 106 or 104 TCID50for both viruses) was seeded by distributing 30 μl over 3 spots onto the lettuce surface. Inoculated samples were air dried in a laminar flow hood for 45 min. Thereafter, 0.15 ml of water or a carvacrol solution at 0.5 or 1% was added for 30 min to inoculated lettuce samples. After treatments, viruses were eluted from the lettuce surface by pipetting with 0.85 ml DMEM containing 2% FCS, and samples were 10-fold serial diluted, and cell culture assays were performed the same day. Each treatment was done in triplicate.
Carvacrol (≥ 98% purity; Sigma Aldrich) at various concentrations were added to individual wells of confluent RAW 264.7, CRFK and FRhK-4 cells in 96-well plates and incubated 2 h under 5% CO2. Thereafter cells were added with 150 μl of supplemented with 2% of fecal calf serum (FCS) and incubated further for 2 to 15 days. Cytotoxicity effects were determined by visual inspection under the optical microscope. 2.3. Antiviral effects of carvacrol Carvacrol diluted in 50% ethanol was added to virus suspensions in DMEM with 2% FCS (ca. 6–7 log TCID50/ml) and further incubated at 37 °C (unless indicated) in a shaker 2 h. Then, infectious viruses were enumerated by cell culture assays as described above. Each treatment was done in triplicate. Positive controls were virus suspensions added with ethanol in amounts corresponding to the highest quantity present. For exposure time experiments, carvacrol at 0.5% was added to MNV and FCV suspensions at ca. 6–7 log TCID50/ml. Samples were immediately removed (t = 0) and at different time intervals (30, 60 and 120 min), and ten-fold diluted in DMEM with 2% FCS to neutralize the carvacrol action. Then, infectious viruses were enumerated by cell culture assays as described above. Experiments were performed in triplicates. Antiviral activity of carvacrol was estimated by comparing the number of infectious viruses on suspensions without carvacrol and on the carvacrol-treated virus suspensions. 2.4. Antiviral activity of carvacrol in fresh-cut lettuce wash water The process wash water with high chemical oxygen demand (COD) was prepared as described by López-Gálvez et al., 2011. Fresh-cut lettuce wash water was diluted with tap water to obtain a batch with 500 mg/l COD. The process wash water were further diluted with tap water to obtain COD values of ca. 200, 300, and 400 mg/l. Lettuce wash water with different COD values were inoculated with MNV and
2.5. Produce wash test using lettuce
2.6. Statistical analysis The significance of differences among the mean numbers of viruses determined after the various treatments was determined by Student's t test with a significance level of P b 0.05 (Microsoft Office Excel; Microsoft, Redmond, WA, USA). 3. Results 3.1. Determination of cytotoxicity of carvacrol on cell lines Carvacrol was found to be cytotoxic at concentrations that exceeded 1%, for the three cell lines. Thus, this value was the maximum concentration of carvacrol added to determine the antiviral effects of carvacrol against MNV, FCV and HAV. 3.2. Effect of carvacrol on the infectivity of norovirus surrogates and HAV Incubation of MNV and FCV with carvacrol at concentrations of 0.25, 0.5 and 1% for 2 h at 37 °C decreased the titer of the two norovirus surrogates (Table 2). Carvacrol at 0.5 and 1% reduced FCV and MNV titers to undetectable levels, while carvacrol at 0.25% reduced FCV by 3.4 log
74
C. Sánchez et al. / International Journal of Food Microbiology 192 (2015) 72–76
Table 2 Effect of carvacrol against feline calicivirus (FCV), murine norovirus (MNV) and Hepatitis A virus (HAV) after 2 h of incubation at 37 °C. FCV
MNV
Log10 TCID50/ml
Log10 TCID50/ml
HAV Log10 TCID50/ml
Treatmenta
Recovered titer
Reduction
Recovered titer
Reduction
Recovered titer
Reduction
Control 0.25% C 0.5% C 1% C
5.82 2.41 b1.32 b1.32
3.41 N4.53 N4.53
5.70 3.84 b2.13 b2.13
1.86 N3.57 N3.57
6.12 5.96 5.98 5.15
0.16 0.14 0.97
± ± ± ±
0.32A 0.07B 0.00C 0.00C
± ± ± ±
0.00A 1.29B 0.00C 0.00C
± ± ± ±
0.35A 0.12A 0.19A 0.14B
Within each column for each virus, different letters denote significant differences between treatments (P b 0.05). a Each treatment was done in triplicate.
TCID50/ml. On the other hand, the hardier surrogate virus, MNV, was reduced by 1.8 log TCID50/ml after treatment with carvacrol at 0.25%. For HAV, no differences in titers reduction were observed between HAV suspensions treated or non-treated with carvacrol at 0.25 and 0.5%, whereas ca. 1 log reduction was observed when treated with carvacrol at 1% (Table 2). Carvacrol treatments were also performed at low HAV titers (ca. 4 log TCID50/ml) but this viral load rendered the same results (data not shown). Since HAV titers were slightly affected by carvacrol treatment, further experiments focused only on the two norovirus surrogates. Fig. 1 shows the infectivity of MNV and FCV after treatment with carvacrol at 0.5% detected by cell culture at different exposure times. Significant reduction 3.1 log (P b 0.05) in MNV-1 infectivity was observed after 30 min treatment with carvacrol at 0.5%. Complete inactivation of MNV was achieved after 1 hour treatment whereas 30 min treatment was enough to fully inactivate FCV, the other NoV surrogate tested. The virus titer reduction was also found to be dependent on the temperature. As shown in Fig. 2, incubation of norovirus surrogates with 0.5% carvacrol showed titer reductions to undetectable levels of both MNV and FCV at 37 °C, whereas titer reductions of 0.75, and 2.19 log TCID50/ml in MNV and 2.31, and 3.19 log TCID50/ml in FCV were found at 24 and 4 °C respectively. Statistical analyses showed that, overall, 0.5% carvacrol treatment at 37 °C for 2 h caused greater reduction on MNV and FCV than room and refrigerated temperatures (P b 0.05). Moreover, higher inactivation rates were reported for 4 °C than 24 °C for both norovirus surrogates, but yet less than 37 °C. 3.3. Effect of carvacrol in water treatment using model lettuce wash water Reductions in the infectious titers of MNV and FCV inoculated in lettuce wash water with different COD values (200, 300 and 400 mg/l),
A
either with or without 0.5% carvacrol added as a sanitizer, are shown in Table 3. The antiviral effect of carvacrol at 0.5% was very much dependent of COD values for MNV, where no virus reduction was observed in lettuce wash water with COD values of 300 ppm or higher. For FCV, the inactivating effect of carvacrol was slightly decreased as the COD values increased. 3.4. Efficacy of carvacrol on produce The infectivity of NoV surrogates with or without treatment with carvacrol on lettuce samples is shown in Table 4. Sensorial parameters of lettuce were not affected (data not shown). When inoculated at high titers, no significant reduction (P b 0.05) of MNV and FCV infectivity was observed after washing of lettuce with 0.5% carvacrol, whereas ca. 1 log reduction was observed for both surrogates at 1% carvacrol. MNV at low titers was reduced by ca. 1.8 log after washing lettuce with 1% carvacrol, whereas for FCV, virus titers had fallen to below detectable limits when inoculated at low titers (Table 4). 4. Discussion Although our knowledge of the enteric virus inactivation by natural compounds is far from complete, a dramatic increase in publications evaluating the antiviral properties of different natural compounds (reviewed by Li et al., 2013) has been seen in recent years, including some potential applications in the food industry (Azizkhani et al., 2013; Li et al., 2012; Su and D' Souza, 2013). This may be partly due to increased awareness of the prevalence of enteric viruses in food products, likewise the increasing consumer for effective economic, safe, healthy natural products. This study clearly shows that carvacrol was effective in reducing the titers of norovirus surrogates in a dose-dependent manner, where
B
Fig. 1. Infectivity of murine norovirus (MNV) (A) and feline calicivirus (FCV) (B) detected by cell-culture either without (black bars) or with 0.5% carvacrol treatment (gray bars) at different interval times. Each column represents the average of triplicates. Dashed line depicts the detection limit. Star indicates the time that virus reached total inactivation.
C. Sánchez et al. / International Journal of Food Microbiology 192 (2015) 72–76
B
A 8
8
6
6
Log( TCID50/mL)
Log (TCID50/mL)
75
4
2
4
2
0
0 37
24
4
37
Temperature(°C)
24
4
Temperature(°C)
Fig. 2. Infectivity of murine norovirus (MNV) (A) and feline calicivirus (FCV) (B) detected by cell-culture either without (black bars) or with 0.5% carvacrol treatment (gray bars) at different temperatures. Each column represents the average of triplicates. Dashed line depicts the detection limit.
increasing concentrations of carvacrol showed increased reduction in viral titers. The efficacy of carvacrol has also recently been assessed on the MNV S7-PP3 strain. Gilling et al. (2014a) reported 1.77 log TCID50/ ml reduction after 30 min treatment with 0.5% carvacrol at 24 °C whereas in this study MNV-1 infectivity was reduced by 3.01 log at 37 °C for 30 min or 0.79 log at room temperature for 2 h. The differences reported may be due to strain variability and dissolvent used to prepare the carvacrol solution. Furthermore carvacrol treatment resulted in slight reductions on HAV infectivity with a maximum reduction of less than 1 log TCID50/ml at the maximum concentration tested. Overall, this study showed that carvacrol treatment caused greater reduction on FCV than MNV titers. This has been shown for other natural antimicrobials, such as oregano, zataria and clove essential oils (Elizaquível et al., 2013) or grape seed extract (GSE) (Su and D'Souza, 2011). However, in this latter publication, MNV was more resistant than HAV to grape seed treatment, unlike our results. Interestingly, the reduction of norovirus surrogates by carvacrol at 4 °C was found to be greater than at 24 °C, although less than at 37 °C. Most of the studies have assessed the virucidal activity of natural compounds at 24 or 37 °C (Table 1), and when performed at 4 °C (Elizaquível et al., 2013; Kovac et al., 2012) the antiviral activity was much lower or ineffective. For the two norovirus surrogates, carvacrol exerted the strongest effect at 37 °C, yet was still active at refrigerated temperatures, which may facilitate the final application in the food industry. In the fresh-cut vegetable industry, water immersion of fresh-cut leafy greens in washing tanks during processing, presents a risk of cross-contamination from leafy greens to water or vice versa (EFSA, 2014b). Use of chlorine has been a standard for industry for decades since it is both relatively cheap and easy to use. However in some
European Union countries such as: Germany, Switzerland, the Netherlands, Denmark, and Belgium, the use of chlorine is prohibited since its use may generate the formation of by-products, such as trihalomethanes and other chemical residues, which has led to the search for new alternatives for disinfection (Van Haute et al., 2013). Therefore, in this study, the antiviral activity of carvacrol was further explored by assessing its activity in lettuce wash water. To investigate the interfering effect of COD, as an indication of the organic quality of water, the antiviral activity of 0.5% carvacrol was determined in lettuce wash water at different COD values. While for FCV efficacy of 0.5% carvacrol was unaffected by COD, for MNV the presence of COD ≥ 300 ppm significantly reduced the efficacy of carvacrol, which may be because part of the carvacrol was blocked by the organic matter while the concentration of free carvacrol was insufficient to inactivate MNV. Assessment of natural compound application in food applications is scarce, so far only GSE has been reported as an effective natural sanitizer against NoV contamination in fresh-cut lettuce processing water with ca. 1.5 to 2 log PFU/ml reduction of MNV, while the role of the wash water COD was deemed insignificant. The application of natural compounds, such as carvacrol, in the sanitation of fresh-cut vegetable water still needs to address some issues, such as treatment time and temperature adaptation to more realistic conditions. With regard to application of carvacrol in food sanitation, lettuce was chosen as the model of fresh leafy greens. Consumption of leafy greens has been associated with foodborne outbreaks, many of which Table 4 Reduction of murine norovirus (MNV) and feline calicivirus (FCV) titers on lettuce at high (ca. 6 log10 TCID50/ml) and low (ca. 4 log TCID50/ml) virus titers after 30 min treatment with carvacrol at room temperature. MNV
FCV
Log10 TCID50/ml Table 3 Reduction of murine norovirus (MNV) and feline calicivirus (FCV) titers after treatment with carvacrol at 0.5% in lettuce wash watera. Lettuce wash water (COD concentration, ppm)
MNV
FCV
0 200 300 400
N4.50A 4.49 ± 0.07A 0.24 ± 0.19B 0.41 ± 0.12B
N3.50A 3.08 ± 0.25A 2.99 ± 0.26A 2.64 ± 0.16A
Within each column for each virus, different letters denote significant differences between treatments (P b 0.05). a Each treatment was done in triplicate.
High virus titer
Low virus titer
Log10 TCID50/ml
Washing treatmenta
Recovered titer
Reduction
Recovered titer
Reduction
Water 0.5% C
6.24 ± 0.19A 6.03 ± 0.31A
– 0.21
6.45 ± 0.21A 6.24 ± 0.38A
– 0.21
1% C Water 0.5% C
5.49 ± 0.14B 3.82 ± 0.19A 3.79 ± 0.12A
0.92 – 0.03
5.45 ± 0.17B 3.91 ± 0.38A 3.66 ± 0.58A
1.00
1% C
2.03 ± 0.38B
1.79
b1.32B
N2.59
0.25
Within each column for each virus, different letters denote significant differences between treatments (P b 0.05). a Each treatment was done in triplicate.
76
C. Sánchez et al. / International Journal of Food Microbiology 192 (2015) 72–76
were due to enteric viruses (Doyle and Erickson, 2008). Moreover, for human NoV, most contamination levels found on leafy greens range from 0 to 5 log genomic copies/g leafy greens (Baert et al., 2011; Kokkinos et al., 2012; Stals et al., 2011). So far, only a few reports are available on the application of natural compounds as a produce wash for foodborne viral reduction. Our results on carvacrol reveal that it exerts a strong antiviral activity against FCV at 1% in lettuce whereas log reduction was achieved on MNV. Similarly, FCV titers were reduced to undetectable levels by GSE at 0.25 mg/ml, but MNV could only be reduced by ca. 1 log PFU on lettuce (Su and D' Souza, 2013). Considering that MNV is a better norovirus surrogate than FCV, and that carvacrol slightly affects HAV infectivity, application of carvacrol alone for food sanitation may not be suitable for complete reduction of foodborne viral contamination. Thus, carvacrol should be considered for use as part of hurdle approaches combining carvacrol with other natural compounds, e.g. GSE, or with other treatments for foodborne virus reduction on leafy greens, e.g. active packaging (Martínez-Abad et al., 2013) or high powered ultrasound (Su et al., 2010). Overall, our findings highlight the potential of carvacrol as an inexpensive natural alternative to reduce viral contamination and, therefore, improve the safety of fresh cut products. Acknowledgments G. Sánchez was supported by the “Ramón y Cajal” Young Investigator program of the Spanish Ministry of Economy and Competitiveness. This study was supported by grant AGL2009-08603 from the Spanish Ministry of Science and Innovation, and ACOMP/2010/279 and ACOMP/2012/199 from the Generalitat Valenciana. References Anonymous, 2013. Surveillance for foodborne disease outbreaks—United States, 2009–2010. Morb. Mortal. Wkly Rep.MMWR 62, 41–47. Azizkhani, M., Elizaquível, P., Sánchez, G., Selma, M.V., Aznar, R., 2013. Comparative efficacy of Zataria multiflora Boiss., Origanum compactum and Eugenia caryophyllus essential oils against E. coli O157:H7, feline calicivirus and endogenous microbiota in commercial baby-leaf salads. Int. J. Food Microbiol. 166, 249–255. Baert, L., Mattison, K., Loisy-Hamon, F., Harlow, J., Martyres, A., Lebeau, B., Stals, A., Van Coillie, E., Herman, L., Uyttendaele, M., 2011. Review: norovirus prevalence in Belgian, Canadian and French fresh produce: a threat to human health. Int. J. Food Microbiol. 15, 261–269. Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in foods—a review. Int. J. Food Microbiol. 94, 223–253. Doyle, M.P., Erickson, M.C., 2008. Summer meeting 2007—the problems with fresh produce: an overview. J. Appl. Microbiol. 105, 317–330. EFSA, 2014a. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2012. EFSA J. 12 (2) (3547, 312 pp.). EFSA, 2014b. Scientific opinion on the risk posed by pathogens in food of non-animal origin. Part 2 (Salmonella and Norovirus in leafy greens eaten raw as salads. EFSA J. 12( (3), 3600.
Elizaquível, P., Azizkhani, M., Aznar, R., Sanchez, G., 2013. The effect of essential oils on norovirus surrogates. Food Control 32, 275–278. Garozzo, A., Timpanaro, R., Bisignano, B., Furneri, P.M., Bisignano, G., Castro, A., 2009. In vitro antiviral activity of Melaleuca alternifolia essential oil. Lett. Appl. Microbiol. 49, 806–808. Gilling, D.H., Kitajima, M., Torrey, J.R., Bright, K.R., 2014a. Antiviral efficacy and mechanisms of action of oregano essential oil and its primary component carvacrol against murine norovirus. J. Appl. Microbiol. 116, 1149–1163. Gilling, D.H., Kitajima, M., Torrey, J.R., Bright, K.R., 2014b. Mechanisms of antiviral action of plant antimicrobials against murine norovirus. Appl. Environ. Microbiol. 80, 4898–4910. Guarda, A., Rubilar, J.F., Miltz, J., Galotto, M.J., 2011. The antimicrobial activity of microencapsulated thymol and carvacrol. Int. J. Food Microbiol. 146, 144–150. Kokkinos, P., Kozyra, I., Lazic, S., Bouwknegt, M., Rutjes, S., Willems, K., Moloney, R., de Roda Husman, A., Kaupke, A., Legaki, E., D'Agostino, M., Cook, N., Rzeżutka, A., Petrovic, T., Vantarakis, A., 2012. Harmonised investigation of the occurrence of human enteric viruses in the leafy green vegetable supply chain in three European countries. Food Environ. Virol. 4 (4), 179–191. Kovac, K., Diez-Valcarce, M., Raspor, P., Hernández, M., Rodríguez-Lázaro, D., 2012. Natural plant essential oils do not inactivate non-enveloped enteric viruses. Food Environ. Virol. 4, 209–212. Li, D., Baert, L., Zhang, D., Xia, M., Zhong, W., Van Coillie, E., Jiang, X., Uyttendaele, M., 2012. Effect of grape seed extract on human norovirus GII.4 and murine norovirus 1 in viral suspensions, on stainless steel discs, and in lettuce wash water. Appl. Environ. Microbiol. 78 (21), 7572–7578. Li, D., Baert, L., Uyttendaele, M., 2013. Inactivation of food-borne viruses using natural biochemical substances. Food Microbiol. 35 (1), 1–9. López-Gálvez, F., Posada-Izquierdo, G.D., Selma, M.V., Pérez-Rodríguez, F., Gobet, J., Gil, M.I., Allende, A., 2011. Electrochemical disinfection: an efficient treatment to inactivate Escherichia coli O157:H7 in process wash water containing organic matter. Food Microbiol. 30, 146–156. Lu, Y., Wu, C., 2010. Reduction of Salmonella enterica contamination on grape tomatoes by washing with thyme oil, thymol, and carvacrol as compared with chlorine treatment. J. Food Prot. 73 (12), 2270–2275. Martínez-Abad, A., Ocio, M.J., Lagarón, J.M., Sánchez, G., 2013. Evaluation of silver-infused polylactide films for inactivation of Salmonella and feline calicivirus in vitro and on fresh-cut vegetables. Int. J. Food Microbiol. 162, 89–94. Nostro, A., Papalia, T., 2012. Antimicrobial activity of carvacrol: current progress and future prospectives. Recent Patents on Anti-Infect. Drug Discov. 7, 28–35. Obaidat, M.M., Frank, J.F., 2009. Inactivation of Escherichia coli O157:H7 on the intact and damaged portions of lettuce and spinach leaves by using allyl isothiocyanate, carvacrol, and cinnamaldehyde in vapor phase. J. Food Prot. 72 (10), 2046–2055. Pilau, M.R., Alves, S.H., Weiblen, R., Arenhart, S., Cueto, A.P., Lovato, L.T., 2011. Antiviral activity of the Lippia graveolens (Mexican oregano) essential oil and its main compound carvacrol against human and animal viruses. Braz. J. Microbiol. 42 (4), 1616–1624. Stals, A., Baert, L., Jasson, V., Van Coillie, E., Uyttendaele, M., 2011. Screening of fruit products for norovirus and the difficulty of interpreting positive PCR results. J. Food Prot. 74 (3), 425–431. Su, X., D' Souza, D.H., 2013. Grape seed extract for foodborne virus reduction on produce. Food Microbiol. 34, 1–6. Su, X., D'Souza, D.H., 2011. Grape seed extract for control of human enteric viruses. Appl. Environ. Microbiol. 77, 3982–3987. Su, X.W., Zivanovic, S., D'Souza, D.H., 2010. Inactivation of human enteric virus surrogates by high-intensity ultrasound. Foodborne Pathog Dis 7, 1055–1061. Van Haute, S., Sampers, I., Holvoet, K., Uyttendaele, M., 2013. Physicochemical quality and chemical safety of chlorine as a reconditioning agent and wash water disinfectant for fresh-cut lettuce washing. Appl. Environ. Microbiol. 79 (9), 2850–2861.
