Antimicrobial Effects of Essential Oils, Nisin, and Irradiation Treatments against Listeria monocytogenes on Ready-to-Eat Carrots Aude Ndoti-Nembe, Khanh Dang Vu, Nicolas Doucet, and Monique Lacroix
The study aimed at using essential oil (EO) alone or combined EO with nisin and low dose γ -irradiation to evaluate their antibacterial effect against Listeria monocytogenes during storage of carrots at 4 °C. Minicarrots were inoculated with L. monocytogenes at a final concentration of approximately 7 log CFU/g. Inoculated samples were coated by nisin at final concentration of 103 International Unit (IU)/mL or individual mountain savory EO or carvacrol at final concentration of 0.35%, w/w) or nisin plus EO. The samples were then irradiated at 0, 0.5, and 1.0 kGy. The treated samples were kept at 4 °C and microbial analysis of samples were conducted at days 1, 3, 6, and 9. The results showed that coating carrots by carvacrol plus nisin or mountain savory plus nisin and then irradiating coated carrots at 1 kGy could reduce L. monocytogenes by more than 3 log at day 1 and reduced it to undetectable level from day 6. Thus, the combined treatments using nisin plus carvacrol or nisin plus mountain savory and irradiation at 1.0 kGy could be used as an effective method for controlling L. monocytogenes in minicarrots. Abstract:
Introduction In the last decade, food contamination by pathogenic bacteria has been one of the most important considerations of governments and international organizations, such as WHO (Kaferstein and Abdussalam 1999). Bacteria such as Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli O157:H7 have been implicated in different foodborne outbreaks (Farber and Peterkin, 1991; Allerberger and Wagner 2010; Krtini´c and others 2010; Mu˜noz and others 2012). Different kinds of foods, especially ready-to-eat food, are known to harbor pathogenic bacteria. For example, salad vegetables such as cabbage, celery, lettuce, cucumber, radish, and tomatoes are often contaminated with L. monocytogenes, and some of these products have been implicated in listeriosis outbreaks (Francis and others 1999, Kornacki 2005). Gamma irradiation is a process that has been used to eliminate bacteria from food. This technique is also used for extending shelf life and delaying the germination of seeds (Farkas and Moh´acsi-Farkas 2011). However, using radiation to eliminate food pathogens such as L. monocytogenes is limited because the dose required may cause adverse effects on the sensorial quality of food (Lacroix and others 1991; Hsu and others, 2010). Therefore, the combinatorial application of ionizing radiation with other treatments such as essential oils or modified atmosphere packaging to reduce the required doses to kill bacteria in food products has been increasing over the past few years (Turgis and others 2008). Further, combined treatments also may have additive or synergistic effects against target microorganism and reduce the resistance to target microorganism to individual antimicrobial MS 20141665 Submitted 10/8/2014, Accepted 1/28/2015. Authors NdotiNembe, Dang Vu, and Lacroix are with Research Laboratories in Sciences Applied to Food, Canadian Irradiation Center (CIC), INRS-Institut Armand-Frappier, Univ. of Quebec, 531 Boulevard des Prairies, Laval, Quebec, H7V 1B7, Canada. Author Doucet is with INRS-Institut Armand-Frappier, Univ. of Quebec, 531 Boulevard des Prairies, Laval, Quebec, H7V 1B7, Canada. Direct inquiries to author Lacroix (E-mail: [email protected]).
R C 2015 Institute of Food Technologists
doi: 10.1111/1750-3841.12832 Further reproduction without permission is prohibited
agents. Studies have found that the addition of natural or synthetic antimicrobial compounds into food products before applying gamma radiation could lead to an increase in the sensitivity of foodborne pathogens to irradiation (Borsa and others 2004, Mohamed and others 2011; Ndoti-Nembe and others, 2013). Essential oils (EOs) are aromatic oils extracted from plants that are known to have antimicrobial effects (Burt and Reinders 2003; Burt, 2004; Tongnuanchan and Benjakul, 2014). Some EOs are widely used to control food spoilage and foodborne pathogenic bacteria. For example, it has been demonstrated that thyme and oregano EOs inhibit the growth of E. coli O157:H7 effectively (Burt and Reinders 2003). In a study by Oussalah and others, (2007), different EOs were used to evaluate their antimicrobial activity against L. monocytogenes, Salmonella Typhimurium, Staphylococcus aureus, and E. coli O157:H7. The authors found that the most active EOs against 4 pathogenic bacteria were Corydothymus capitatus, Cinnamomum cassia, Cinnamomum verum from bark, Satureja montana and the Origanum heracleoticum. Bacteriocins are small peptides produced by lactic acid bacteria that are known to have antimicrobial effects (Mohamed and others 2011; Abdollahzadeh and others, 2014; Bi and others, 2014). Nisin is one of the most common bacteriocins used in food industries and it is the only bacteriocin that is legally recognized as a food preservative (Thomas and Delves-Broughton 2005). Nisin is produced by Lactococcus lactis, and it has been demonstrated that it is effective against L. monocytogenes, B. cereus, and other Grampositive bacteria (Tu and Mustapha, 2002; Mohamed and others 2011; Abdollahzadeh and others, 2014; Bi and others, 2014). Another important aspect critical to the food industry is the development of methods that help enhance the shelf life and the microbial safety of food products. Different factors can influence the shelf life of a food product, ranging from production processes, the chemical composition of products, or the manipulation of products by consumers (Corbo and others 2009). The most important factor, however, is food contamination by food pathogens. Therefore, controlling the growth of food pathogens during storage can increase the shelf life and microbial safety of food products. Vol. 80, Nr. 4, 2015 r Journal of Food Science M795
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Keywords: carvacrol, Gamma irradiation, Listeria monocytogenes, minicarrots, mountain savory essential oil
Antilisterial activity of treatments . . . In case of ready-to-eat carrot, it has been found that an irradiation dose of 2 kGy could completely control fungal and bacterial growth during storage at 5 °C and the irradiated samples were also acceptable organoleptically (Chaudry and others, 2004). However, the total flora and fungi concentration in these samples of this study were low (approximately 2 log CFU/g). It is not known if the combined treatments consisting of nisin, Eos, and gamma irradiation at low doses could eliminate a high load of pathogenic bacteria in minicarrots or not. Therefore, the objectives of this study were to evaluate the antimicrobial effects of combined treatments using 2 essential oils (mountain savory and carvacrol), nisin, and low doses of gamma irradiation (0.5 and 1.0 kGy) against L. monocytogenes inoculated in carrots at high load (approximately 7 log CFU/g). The antilisterial effects were evaluated during storage of samples at 4 °C for 9 d.
Materials and Methods
M: Food Microbiology & Safety
Essential oils and Bacteriocin preparation Carvacrol (a component of many EOs) and mountain savory (Satureja montana) EO were used in this study. Carvacrol was purchased from Sigma-Aldrich Co. (St. Louis, Mo., U.S.A.) and mountain savory was obtained from Robert et Fils (Montreal, Quebec, Canada). A commercial nisin powder containing 2.5% nisin (balance sodium chloride and denatured milk) was purchased from SigmaAldrich Co. The antibacterial potency of this nisin power was 106 IU/g. A stock nisin solution containing 104 IU/mL was prepared from this powder. The stock solution was prepared by dissolving the nisin powder in acetate buffer at 100 mM pH 5, and the solution was sterilized using a 0.2-μm filter. Bacterial preparation L. monocytogenes HPB 2812 serovar 1/2a was used in this study. It was isolated from homemade salami by the Health Products and Foods Branch of Health Canada (Ottawa, Ontario, Canada). The pathogen was maintained at −80 °C in tryptic soy broth (TSB; Difco, BD, Sparks, Md., U.S.A.) containing glycerol (10%, v/v). Before each experiment, the stock culture of L. monocytogenes was propagated by 2 successive 24 h growth cycles in TSB at 37 °C. Bacterial cells were washed twice with saline solution (0.85% NaCl, w/v), and the cell concentration in suspension was adjusted to approximately 109 CFU/mL (Turgis and others 2008; Takala and others 2011). Minicarrot preparation Peeled minicarrots (ready-to-eat) were purchased from a local store (Maxi & Cie, Laval, Quebec, Canada). Carrots were put in Winpak bags (Winpak Division Ltd., Montreal, Quebec, Canada) and were sterilized by gamma irradiation at a dose of 10 kGy at the Canadian Irradiation Center using a UC-15 A (SS canister) under water calibrator (Nordion Inc., Kanata, Ontario, Canada) equipped with a 60 Co source. Packages of sterilized carrots were kept at −80° C until they were used (Ndoti-Nembe and others, 2013). Inoculation of pathogenic bacteria into carrots An inoculation bath of 2 L of sterile saline water 0.85% NaCl was prepared. The bath was mixed with L. monocytogenes to reach a bacterial concentration of approximately 108 CFU/mL. The bags containing carrots were defrosted and opened under sterile conditions. Each carrot was dipped into the inoculation bath and M796 Journal of Food Science r Vol. 80, Nr. 4, 2015
mixed for 5 min to obtain a final concentration of approximately 107 CFU/g of carrots. Carrots were left to dry for 30 min before coating with antimicrobial compounds (Ndoti-Nembe and others, 2013).
Antimicrobial effect of combined treatments against L. monocytogenes and S. Typhimurium during storage of carrots The emulsion of EO (carvacrol or mountain savory) was prepared by mixing 0.35% EO (w/w), 6.0% DMSO (w/w), 0.5% Tween 80 (w/w), and 93.1% (w/w) sterile water for 1 h at room temperature. The emulsion was used as coating solution for carrots. For the nisin coating of carrots, the final nisin solution concentration was 103 IU/mL. For combined treatments using EO and nisin as coating solution, the final concentration of nisin and EO was 103 IU/mL and 0.35% (w/w), respectively. The inoculated carrots were dipped into beakers containing different coating solutions for 1 min under stirring. In case of control, sterile water was used as immersion solution. It should be mentioned that DMSO 6% were already used to evaluate its capacity to kill the bacteria and it was found that DMSO 6% did not have any antibacterial effect against L. monocytogenes and therefore, DMSO 6% was not tested as a control to reduce the sample quantity. Samples were allowed to dry for 30 min on sterile aluminum sheets in a biological cabinet. The samples were packaged into sterile bags which were then sealed and kept at 4 °C overnight prior to irradiation treatment. The samples were then submitted to an irradiation treatment with 3 different doses of 0, 0.5, and 1 kGy. The irradiation treatment was conducted at the Canadian Irradiation Centre, in a 60 Co underwater calibrator with a dose rate of 14.5 kGy/h (UC-15A, Nordion, Laval, Quebec, Canada). Sample temperature was kept at 4 °C during treatment. After irradiation, samples were kept at 4 °C for 9 d. Microbiological analyses were conducted at days 1, 3, 6, and 9. Microbiological alnalyses Each carrot sample was weighed (ca. 10 ± 0.3 g) and homogenized for 2 min in 90 mL of sterile peptone water (0.1% w/v) using a Lab-blender 400 stomacher (Laboratory Equipment, London, UK). From this mixture, serial dilutions were prepared and plated onto tryptic soy agar (Difco, Becton Dickinson). The plates were then incubated at 37 °C for 24 h for the enumeration of L. monocytogenes. Cell concentrations were expressed as log CFU/g. Statistical analysis All experiments were conducted in 3 replicates. For each replicate and for each dose of irradiation, 3 samples were analyzed. The data were analyzed using IBM SPSS Statistics, and the means comparison among treatments was based on Duncan’s test (P ࣘ 0.05).
Results The antimicrobial effects of combined treatments using essential oils, nisin, and gamma irradiation against L. monocytogenes during storage of carrots is presented in Table 1. In the control (uncoated carrots without irradiation treatment), there was no significant reduction in bacterial concentration at day 1 and 3, then, insignificant increase in bacterial concentration was observed at day 6 as compared to day 1 of storage. At day 9 of storage, there was 0.4 log reduction in the count of L. monocytogenes as compared to day 1 for the control samples. In the uncoated carrots treated with 0.5 kGy irradiation, a reduction of 1.6 log at day 1 and a reduction of
Antilisterial activity of treatments . . . Table 1–Effect of combined essential oils, nisin, and irradiation treatments against populations of Listeria monocytogenes HPB 2812 serovar 1/2a on peeled minicarrots.
Uncoated carrots Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy Nisin coated Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy Carvacrol coated Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy Mountain-savory coated Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy Cavacrol-Nisin coated Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy Mountain savory–Nisin coated Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy
Day 1
Day 3
Day 6
Day 9
7.7 ± 0.1 aAB 6.1 ± 0.1 cdE 5.7 ± 0.1 cdeF
7.6 ± 0.1 aB 4.9 ± 0.1 efG 3.2 ± 0.3 gH
7.9 ± 0.2 aA 6.9 ± 0.1 bD 5.8 ± 0.1 cF
7.3 ± 0.2 aC 6.3 ± 0.6 cE 5.1 ± 0.1 eG
7.7 ± 0.1 aA 6.1 ± 0.3 cdB 4.1 ± 0.3 gD
6.5 ± 0.7 bB 4.6 ± 0.4 fD 2.9 ± 0.1 gF
5.2 ± 0.4 dC 2.5 ± 0.3 hE ND
3.1 ± 0.1 gF ND ND
7.7 ± 0.1 aA 6.2 ± 0.1 cdD 5.4 ± 0.1 de E
7.6 ± 0.2 aA 6.4 ± 0.4 bcCD 5.5 ± 0.5 deE
7.8 ± 0.2 aA 6.9 ± 0.08 bB 5.5 ± 0.14 cdE
6.9 ± 0.8 bB 6.8 ± 0.2 bBC 5.4 ± 0.2 dE
7.3 ± 0.2 abB 6.6 ± 0.4 bcD 5.2 ± 0.1 ef G
7.6 ± 0.1 aA 6.3 ± 0.1 bcdE 5.1 ± 0.1 ef G
7.9 ± 0.2 aA 6.8 ± 0.2 bD 5.6 ± 0.1 cF
7.0 ± 0.1 bC 6.9 ± 0.1 bC 5.2 ± 0.1 de G
6.5 ± 0.1 cA 4.6 ± 0.2 fgBC 3.2 ± 0.2 hD
5.6 ± 0.2 cdeAB 4.7 ± 1.1 efBC 2.6 ± 1.2 gD
3.9 ± 0.1 fD 2.6 ± 0.2 hD ND
2.7 ± 0.1 hD ND ND
6.4 ± 0.2 cA 5.0 ± 0.2 efBC 3.2 ± 0.1 hF
5.3 ± 0.2 efB 4.8 ± 0.5 efC 2.3 ± 0.3 gG
4.3 ± 0.4 eD 3.5 ± 0.1 gEF ND
3.7 ± 0.8 fE ND ND
∗ Values are means ± standard deviations. Means with different uppercase letters within the same group of coated at 3 different irradiation doses (0, 0.5, and 1 kGy) are significantly different from other means as determined by Duncan’s test (P ࣘ 0.05). Means with different lowercase letters within each column are significantly different from other means as determined by Duncan’s test (P ࣘ 0.05). ∗∗ ND, nondetectable (detection limit of 1.5 log CFU/g).
2.7 logs at day 3 as compared to the control (P ࣘ 0.05) at the same day of treatment were observed, respectively; however, in this treatment, there was an increase in bacterial concentration from day 3 to day 6 as compared to day 1 of treatment. In the uncoated carrots treated by irradiation with 1.0 kGy, a reduction of approximately 2 logs at day 1 as compared to the control was observed, and cell concentration was reduced by 2.5 logs at day 3 as compared to day 1 of treatment. Cell concentration was then increased at day 6 and maintained about 5 logs CFU/g until the end of storage. Cell concentration in carrots coated with nisin and without irradiation treatment was reduced by 1.2, 2.7, and 4.6 logs on days 3, 6, 9, respectively, as compared to this treatment at day 1. Cell concentration in carrots coated with nisin and with irradiation treatment of 0.5 kGy was reduced by less than 1 log as compared to the control at day 1. This treatment also caused a reduction of 3.6 logs at day 6 and a reduction to below the detection level (1.5 log/g) at day 9 as compared to day 1 of storage. The combined treatment with nisin and irradiation at 1 kGy was efficient in reducing L. monocytogenes since only 4.1 and 2.9 logs were detected at day 1 and day 3, respectively. Moreover, L. monocytogenes was undetectable from day 6 of storage. There was no significant difference (P > 0.05) in cell concentration between control carrot samples and those coated with carvacrol without irradiation treatment at day 1. Additionally, cell concentration did not significantly chang at day 3 and day 6 of storage (P > 0.05); however, at day 9, there was 0.8-log reduction (P ࣘ 0.05) as compared to day 1. It can be observed that the count of L. monocytogenes in carrots coated with carvacrol and irradiated at 0.5 kGy reduced by about 1.5 logs as compared to the same treatment without irradiation at day 1. Cell concentration in this treatment was increased by less than 1 log during storage. Cell concentration of this treatment was increased less than 1 log during storage. In the case of carrots coated by carvacrol and
irradiated at 1 kGy, there was a reduction of 2 logs in bacterial count at day 1 and the bacterial concentration was unchanged (P > 0.05) during storage. Also, there was no significant difference (P > 0.05) in cell concentration at day 1 between control and samples coated by mountain savory and without irradiation treatment. Moreover, this treatment did not reduce bacterial count during storage. A reduction of 0.7 and 2.1 logs of bacterial count at day 1 in the samples coated by mountain savory and irradiated at 0.5 and 1.0 kGy was observed, respectively. These combined treatments (mountain savory plus irradiation at 0.5 kGy or mountain savory plus irradiation at 1.0 kGy) did not cause any further bacterial reduction during storage. It can be observed that at day 1, cell concentration in carrots coated by carvacrol plus nisin and without irradiation (0 kGy) was reduced by more than one log as compared to the control. This treatment also caused further decrease in cell concentration at day 3 until the end of storage. Cell concentration in carrots coated by carvacrol plus nisin and irradiated at 0.5 kGy was reduced by 1.9 log as compared to this coating formulation without irradiation. In this treatment, cell concentration was further reduced by 2 logs at day 6 and to an undetectable limit at day 9. Interestingly, this coating formulation in combination with irradiation at 1 kGy decreased the count of L. monocytogenes by more than 3 logs at day 1 as compared to this formulation coating without irradiation. In this treatment, the count of the organism showed further reduction at day 3 and was below the detection limit at days 6 and 9 of storage. In carrots coated by mountain savory plus nisin and without irradiation (0 kGy), cell concentration was reduced by more than 1 log as compared to the control at day 1. Cell concentration in this treatment was further decreased during storage to 3.7 logs at day 9. Application of carrot coating by mountain savory plus nisin in combination with irradiation treatment at 0.5 kGy caused a
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Treatments
Antilisterial activity of treatments . . . decrease of more than 1 log in L. monocytogenes concentration at day 1 compared to carrots coated by mountain savory plus nisin without irradiation. The count of the organism in this treatment was further decreased at day 3 and day 6 and was below the detection limit (1.5 log/g) at day 9. Meanwhile coating of carrots by mountain savory plus nisin in combination with irradiation at 1 kGy decreased the count of L. monocytogenes by more than 3 logs at day 1 as compared to this coating formulation without irradiation. Also the count of the organism was further decreased at day 3 and day 6 and was below the detection limit (define the detection limit of the experiment) at day 9 of storage.
Discussion
M: Food Microbiology & Safety
It can be found that irradiation treatment alone at 1.0 kGy was effective at reducing significantly L. monocytogenes inoculated in carrots by approximately 2 log as compared to the control during storage. Comparing all treatments against L. monocytogenes inoculated in carrots, it can be find that coating of carrots by carvacrol plus nisin or by mountain savory plus nisin with irradiation at 1 kGy could reduce L. monocytogenes by more than 3 logs at day 1. The bacterial concentrations in these treatments were also further decreased to undetectable levels at day 6. Thus, we can conclude that nisin contributed to the antimicrobial effects against L. monocytogenes in combination with essential oils (either carvacrol or mountain savory) and irradiation treatment at low dose (1 kGy). Mohamed and others (2011) found that a combined treatment of meat inoculated with 4 × 103 CFU/g of L. monocytogenes with nisin (103 IU/g) coupled to irradiation (1.5 kGy) could effectively eliminate L. monocytogenes, with a number of bacteria lower than 0.03 most probable number (MPN)/g at the end of the treatment. Nisin was also found to increase the antimicrobial effects against L. monocytogenes when used in combination with garlic EO (Rohani and others 2011). In our previous study, it was found that the radiation sensitivity (RS) of L. monocytogenes cell suspension (in vitro) to irradiation was increased by 4.19 and 6.31 times when treated by carvacrol plus nisin and mountain savory plus nisin, respectively, as compared to the control. In case of in situ study, it was found that coated carrots by nisin plus carvacrol increased the RS of L. monocytogenes to irradiation by 2.74 times as compared to the control (NdotiNembe and others, 2013). Thus, it is reasonable that combination of carvacrol or mountain savory EO plus nisin and irradiation can significantly increase the sensitivity of L. monocytogenes during storage. Turgis and others (2012) have evaluated the combined effects of bacteriocins (nisin, bacteriocin MT104, bacteriocin MT162) and irradiation against L. monocytogenes inoculated in sausage meat. The authors found a synergistic antimicrobial effect by using nisin or bacterocin MT 104 in combination with irradiation. The effect was expressed through the increase in relative sensitivity of L. monocytogenes to irradiation treatment. Our results are in agreement with these previous studies. Combined treatments using EO and irradiation against L. monocytogens or L. innocua in carrots have demonstrated that the EO can increase the sensitivity of L. monocytogenes to irradiation treatment, especially under modified atmosphere packaging (Caillet and others 2006b). EOs combined to radiation at low doses can also control the growth of L. innocua during storage of carrots (Caillet and others 2006a). For example, Caillet and others (2006a) evaluated the combined antimicrobial effects of antimicrobial coating (0.025% final concentration of trans-cinnamaldehyde), modified atmosphere packaging (60% O2 , 30% CO2 , and 10% N2 ) and M798 Journal of Food Science r Vol. 80, Nr. 4, 2015
gamma-irradiation (0.25 and 0.50 kGy) against Listeria innocua (103 CFU/g carrots). The authors found that the combined treatment was very effective at inhibiting the growth of L. innocua in carrots. For example, 2.23 logs and 2.26 logs CFU/g were found in the nonirradiated and uncoated samples, as well as in samples coated with the inactive coating and without irradiation treatment. However, after 7 d of storage, no L. innocua was detected in samples treated at 0.5 kGy under normal pressure or in samples treated at 0.25 kGy under modified atmosphere. The authors also found a complete inhibition of L. innocua during the storage period in uncoated and coated samples treated at 0.5 kGy under modified atmosphere. These results indicate that the combination of irradiation and modified atmosphere play an important role in controlling the growth of L. innocua. Similarly, Caillet and others (2006b) found that EOs consisting of trans-cinnamaldehyde, Spanish oregano, winter savory, and Chinese cinnamon could increase the sensitivity of L. monocytogenes inoculated in mini-carrots to irradiation treatment, especially when the samples were packaged in modified atmosphere (60% O2 , 30% CO2 , and 10% N2 ). Thus, it is reasonable to assume that the combined treatments by nisin, Eos, and irradiation could control or reduce the growth of L. monocytogenes inoculated into carrots during storage, even in air-packaged conditions.
Conclusion The present study demonstrates that combinations of nisin and mountain savory essential oil or nisin and carvacrol with low dose irradiation (1 kGy) have eliminated L. monocytogenes from readyto-eat carrots effectively. Coating of carrots by carvacrol plus nisin or by mountain savory plus nisin, then treating coated carrots at 1 kGy could reduce L. monocytogenes by more than 3 logs at day 1. Additionally, the bacterial concentration in these treatments was further decreased to undetectable levels from day 6. The combined nisin plus carvacrol or nisin plus mountain savory and irradiation at 1.0 kGy could be used as an effective method for controlling L. monocytogenes in minicarrots.
Acknowledgments Nordion Inc. is acknowledged for irradiation treatments. The authors sincerely thank the Natural Sciences and Engineering Research Council of Canada (NSERC) through the discovery program and the International Atomic Energy Agency (IAEA) for financial support. This study does not have any conflicts of interest.
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Farber KR, Peterkin PI. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol Rev 55:476–511. Farkas J, Moh´acsi-Farkas C. 2011. History and future of food irradiation. Trends Food Sci Technol 22:121–26. Francis GA, Thomas C, O’Beirne D. 1999. The microbiological safety of minimally processed vegetables. Intl J Food Sci Technol 34:1–22. Hsu WY, Simonne A, Jitareerat P, Marshall JMR. 2010. Low-dose irradiation improves microbial quality and shelf life of fresh mint (Mentha piperita L.) without compromising visual quality. J Food Sci 75:M22–30. Kaferstein F, Abdussalam M. 1999. Food safety in the 21st century. Bull World Health Organ 77:347–51. Kornacki JL. 2005. Controlling Listeria in the food processing environment. Food Technol 59:36–38. Krtini´c G, Đuri´c P, Ili´c S. 2010. Salmonellae in food stuffs of plant origin and their implications on human health. Eur J Clin Microbiol Infec Dis 29:1321–25. Lacroix M, Jobin M, Hamel S, Stahl V, Gagnon M, De Couvercelle C. 1991. Effects of 3 kGy and 7 kGy gamma radiation doses on odour and flavour of fresh chicken breast. Microbiologie Aliments Nutrition 9:375–79. Mohamed HMH, Elnawawi FA, Yousef AE. 2011. Nisin treatment to enhance the efficacy of gamma radiation against Listeria monocytogenes on meat. J Food Prot 74:193–99. Mu˜noz P, Rojas L, Bunsow E, Saez E, S´anchez-Cambronero L, Alcal´a L, Rodr´ıguez-Creixems M, Bouza E. 2012. Listeriosis: an emerging public health problem especially among the elderly. J Infect 64:19–33. Ndoti-Nembe A, Vu KD, Doucet N, Lacroix M. 2013. Effect of combination of essential oils and bacteriocins on the effi cacy of gamma radiation against Salmonella Typhimurium and Listeria m onocytogenes. Intl J Radiat Biol 89:794–800.
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The study aimed at using essential oil (EO) alone or combined EO with nisin and low dose γ -irradiation to evaluate their antibacterial effect against Listeria monocytogenes during storage of carrots at 4 °C. Minicarrots were inoculated with L. monocytogenes at a final concentration of approximately 7 log CFU/g. Inoculated samples were coated by nisin at final concentration of 103 International Unit (IU)/mL or individual mountain savory EO or carvacrol at final concentration of 0.35%, w/w) or nisin plus EO. The samples were then irradiated at 0, 0.5, and 1.0 kGy. The treated samples were kept at 4 °C and microbial analysis of samples were conducted at days 1, 3, 6, and 9. The results showed that coating carrots by carvacrol plus nisin or mountain savory plus nisin and then irradiating coated carrots at 1 kGy could reduce L. monocytogenes by more than 3 log at day 1 and reduced it to undetectable level from day 6. Thus, the combined treatments using nisin plus carvacrol or nisin plus mountain savory and irradiation at 1.0 kGy could be used as an effective method for controlling L. monocytogenes in minicarrots. Abstract:
Introduction In the last decade, food contamination by pathogenic bacteria has been one of the most important considerations of governments and international organizations, such as WHO (Kaferstein and Abdussalam 1999). Bacteria such as Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli O157:H7 have been implicated in different foodborne outbreaks (Farber and Peterkin, 1991; Allerberger and Wagner 2010; Krtini´c and others 2010; Mu˜noz and others 2012). Different kinds of foods, especially ready-to-eat food, are known to harbor pathogenic bacteria. For example, salad vegetables such as cabbage, celery, lettuce, cucumber, radish, and tomatoes are often contaminated with L. monocytogenes, and some of these products have been implicated in listeriosis outbreaks (Francis and others 1999, Kornacki 2005). Gamma irradiation is a process that has been used to eliminate bacteria from food. This technique is also used for extending shelf life and delaying the germination of seeds (Farkas and Moh´acsi-Farkas 2011). However, using radiation to eliminate food pathogens such as L. monocytogenes is limited because the dose required may cause adverse effects on the sensorial quality of food (Lacroix and others 1991; Hsu and others, 2010). Therefore, the combinatorial application of ionizing radiation with other treatments such as essential oils or modified atmosphere packaging to reduce the required doses to kill bacteria in food products has been increasing over the past few years (Turgis and others 2008). Further, combined treatments also may have additive or synergistic effects against target microorganism and reduce the resistance to target microorganism to individual antimicrobial MS 20141665 Submitted 10/8/2014, Accepted 1/28/2015. Authors NdotiNembe, Dang Vu, and Lacroix are with Research Laboratories in Sciences Applied to Food, Canadian Irradiation Center (CIC), INRS-Institut Armand-Frappier, Univ. of Quebec, 531 Boulevard des Prairies, Laval, Quebec, H7V 1B7, Canada. Author Doucet is with INRS-Institut Armand-Frappier, Univ. of Quebec, 531 Boulevard des Prairies, Laval, Quebec, H7V 1B7, Canada. Direct inquiries to author Lacroix (E-mail: [email protected]).
R C 2015 Institute of Food Technologists
doi: 10.1111/1750-3841.12832 Further reproduction without permission is prohibited
agents. Studies have found that the addition of natural or synthetic antimicrobial compounds into food products before applying gamma radiation could lead to an increase in the sensitivity of foodborne pathogens to irradiation (Borsa and others 2004, Mohamed and others 2011; Ndoti-Nembe and others, 2013). Essential oils (EOs) are aromatic oils extracted from plants that are known to have antimicrobial effects (Burt and Reinders 2003; Burt, 2004; Tongnuanchan and Benjakul, 2014). Some EOs are widely used to control food spoilage and foodborne pathogenic bacteria. For example, it has been demonstrated that thyme and oregano EOs inhibit the growth of E. coli O157:H7 effectively (Burt and Reinders 2003). In a study by Oussalah and others, (2007), different EOs were used to evaluate their antimicrobial activity against L. monocytogenes, Salmonella Typhimurium, Staphylococcus aureus, and E. coli O157:H7. The authors found that the most active EOs against 4 pathogenic bacteria were Corydothymus capitatus, Cinnamomum cassia, Cinnamomum verum from bark, Satureja montana and the Origanum heracleoticum. Bacteriocins are small peptides produced by lactic acid bacteria that are known to have antimicrobial effects (Mohamed and others 2011; Abdollahzadeh and others, 2014; Bi and others, 2014). Nisin is one of the most common bacteriocins used in food industries and it is the only bacteriocin that is legally recognized as a food preservative (Thomas and Delves-Broughton 2005). Nisin is produced by Lactococcus lactis, and it has been demonstrated that it is effective against L. monocytogenes, B. cereus, and other Grampositive bacteria (Tu and Mustapha, 2002; Mohamed and others 2011; Abdollahzadeh and others, 2014; Bi and others, 2014). Another important aspect critical to the food industry is the development of methods that help enhance the shelf life and the microbial safety of food products. Different factors can influence the shelf life of a food product, ranging from production processes, the chemical composition of products, or the manipulation of products by consumers (Corbo and others 2009). The most important factor, however, is food contamination by food pathogens. Therefore, controlling the growth of food pathogens during storage can increase the shelf life and microbial safety of food products. Vol. 80, Nr. 4, 2015 r Journal of Food Science M795
M: Food Microbiology & Safety
Keywords: carvacrol, Gamma irradiation, Listeria monocytogenes, minicarrots, mountain savory essential oil
Antilisterial activity of treatments . . . In case of ready-to-eat carrot, it has been found that an irradiation dose of 2 kGy could completely control fungal and bacterial growth during storage at 5 °C and the irradiated samples were also acceptable organoleptically (Chaudry and others, 2004). However, the total flora and fungi concentration in these samples of this study were low (approximately 2 log CFU/g). It is not known if the combined treatments consisting of nisin, Eos, and gamma irradiation at low doses could eliminate a high load of pathogenic bacteria in minicarrots or not. Therefore, the objectives of this study were to evaluate the antimicrobial effects of combined treatments using 2 essential oils (mountain savory and carvacrol), nisin, and low doses of gamma irradiation (0.5 and 1.0 kGy) against L. monocytogenes inoculated in carrots at high load (approximately 7 log CFU/g). The antilisterial effects were evaluated during storage of samples at 4 °C for 9 d.
Materials and Methods
M: Food Microbiology & Safety
Essential oils and Bacteriocin preparation Carvacrol (a component of many EOs) and mountain savory (Satureja montana) EO were used in this study. Carvacrol was purchased from Sigma-Aldrich Co. (St. Louis, Mo., U.S.A.) and mountain savory was obtained from Robert et Fils (Montreal, Quebec, Canada). A commercial nisin powder containing 2.5% nisin (balance sodium chloride and denatured milk) was purchased from SigmaAldrich Co. The antibacterial potency of this nisin power was 106 IU/g. A stock nisin solution containing 104 IU/mL was prepared from this powder. The stock solution was prepared by dissolving the nisin powder in acetate buffer at 100 mM pH 5, and the solution was sterilized using a 0.2-μm filter. Bacterial preparation L. monocytogenes HPB 2812 serovar 1/2a was used in this study. It was isolated from homemade salami by the Health Products and Foods Branch of Health Canada (Ottawa, Ontario, Canada). The pathogen was maintained at −80 °C in tryptic soy broth (TSB; Difco, BD, Sparks, Md., U.S.A.) containing glycerol (10%, v/v). Before each experiment, the stock culture of L. monocytogenes was propagated by 2 successive 24 h growth cycles in TSB at 37 °C. Bacterial cells were washed twice with saline solution (0.85% NaCl, w/v), and the cell concentration in suspension was adjusted to approximately 109 CFU/mL (Turgis and others 2008; Takala and others 2011). Minicarrot preparation Peeled minicarrots (ready-to-eat) were purchased from a local store (Maxi & Cie, Laval, Quebec, Canada). Carrots were put in Winpak bags (Winpak Division Ltd., Montreal, Quebec, Canada) and were sterilized by gamma irradiation at a dose of 10 kGy at the Canadian Irradiation Center using a UC-15 A (SS canister) under water calibrator (Nordion Inc., Kanata, Ontario, Canada) equipped with a 60 Co source. Packages of sterilized carrots were kept at −80° C until they were used (Ndoti-Nembe and others, 2013). Inoculation of pathogenic bacteria into carrots An inoculation bath of 2 L of sterile saline water 0.85% NaCl was prepared. The bath was mixed with L. monocytogenes to reach a bacterial concentration of approximately 108 CFU/mL. The bags containing carrots were defrosted and opened under sterile conditions. Each carrot was dipped into the inoculation bath and M796 Journal of Food Science r Vol. 80, Nr. 4, 2015
mixed for 5 min to obtain a final concentration of approximately 107 CFU/g of carrots. Carrots were left to dry for 30 min before coating with antimicrobial compounds (Ndoti-Nembe and others, 2013).
Antimicrobial effect of combined treatments against L. monocytogenes and S. Typhimurium during storage of carrots The emulsion of EO (carvacrol or mountain savory) was prepared by mixing 0.35% EO (w/w), 6.0% DMSO (w/w), 0.5% Tween 80 (w/w), and 93.1% (w/w) sterile water for 1 h at room temperature. The emulsion was used as coating solution for carrots. For the nisin coating of carrots, the final nisin solution concentration was 103 IU/mL. For combined treatments using EO and nisin as coating solution, the final concentration of nisin and EO was 103 IU/mL and 0.35% (w/w), respectively. The inoculated carrots were dipped into beakers containing different coating solutions for 1 min under stirring. In case of control, sterile water was used as immersion solution. It should be mentioned that DMSO 6% were already used to evaluate its capacity to kill the bacteria and it was found that DMSO 6% did not have any antibacterial effect against L. monocytogenes and therefore, DMSO 6% was not tested as a control to reduce the sample quantity. Samples were allowed to dry for 30 min on sterile aluminum sheets in a biological cabinet. The samples were packaged into sterile bags which were then sealed and kept at 4 °C overnight prior to irradiation treatment. The samples were then submitted to an irradiation treatment with 3 different doses of 0, 0.5, and 1 kGy. The irradiation treatment was conducted at the Canadian Irradiation Centre, in a 60 Co underwater calibrator with a dose rate of 14.5 kGy/h (UC-15A, Nordion, Laval, Quebec, Canada). Sample temperature was kept at 4 °C during treatment. After irradiation, samples were kept at 4 °C for 9 d. Microbiological analyses were conducted at days 1, 3, 6, and 9. Microbiological alnalyses Each carrot sample was weighed (ca. 10 ± 0.3 g) and homogenized for 2 min in 90 mL of sterile peptone water (0.1% w/v) using a Lab-blender 400 stomacher (Laboratory Equipment, London, UK). From this mixture, serial dilutions were prepared and plated onto tryptic soy agar (Difco, Becton Dickinson). The plates were then incubated at 37 °C for 24 h for the enumeration of L. monocytogenes. Cell concentrations were expressed as log CFU/g. Statistical analysis All experiments were conducted in 3 replicates. For each replicate and for each dose of irradiation, 3 samples were analyzed. The data were analyzed using IBM SPSS Statistics, and the means comparison among treatments was based on Duncan’s test (P ࣘ 0.05).
Results The antimicrobial effects of combined treatments using essential oils, nisin, and gamma irradiation against L. monocytogenes during storage of carrots is presented in Table 1. In the control (uncoated carrots without irradiation treatment), there was no significant reduction in bacterial concentration at day 1 and 3, then, insignificant increase in bacterial concentration was observed at day 6 as compared to day 1 of storage. At day 9 of storage, there was 0.4 log reduction in the count of L. monocytogenes as compared to day 1 for the control samples. In the uncoated carrots treated with 0.5 kGy irradiation, a reduction of 1.6 log at day 1 and a reduction of
Antilisterial activity of treatments . . . Table 1–Effect of combined essential oils, nisin, and irradiation treatments against populations of Listeria monocytogenes HPB 2812 serovar 1/2a on peeled minicarrots.
Uncoated carrots Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy Nisin coated Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy Carvacrol coated Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy Mountain-savory coated Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy Cavacrol-Nisin coated Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy Mountain savory–Nisin coated Irradiated 0 kGy Irradiated 0.5 kGy Irradiated 1 kGy
Day 1
Day 3
Day 6
Day 9
7.7 ± 0.1 aAB 6.1 ± 0.1 cdE 5.7 ± 0.1 cdeF
7.6 ± 0.1 aB 4.9 ± 0.1 efG 3.2 ± 0.3 gH
7.9 ± 0.2 aA 6.9 ± 0.1 bD 5.8 ± 0.1 cF
7.3 ± 0.2 aC 6.3 ± 0.6 cE 5.1 ± 0.1 eG
7.7 ± 0.1 aA 6.1 ± 0.3 cdB 4.1 ± 0.3 gD
6.5 ± 0.7 bB 4.6 ± 0.4 fD 2.9 ± 0.1 gF
5.2 ± 0.4 dC 2.5 ± 0.3 hE ND
3.1 ± 0.1 gF ND ND
7.7 ± 0.1 aA 6.2 ± 0.1 cdD 5.4 ± 0.1 de E
7.6 ± 0.2 aA 6.4 ± 0.4 bcCD 5.5 ± 0.5 deE
7.8 ± 0.2 aA 6.9 ± 0.08 bB 5.5 ± 0.14 cdE
6.9 ± 0.8 bB 6.8 ± 0.2 bBC 5.4 ± 0.2 dE
7.3 ± 0.2 abB 6.6 ± 0.4 bcD 5.2 ± 0.1 ef G
7.6 ± 0.1 aA 6.3 ± 0.1 bcdE 5.1 ± 0.1 ef G
7.9 ± 0.2 aA 6.8 ± 0.2 bD 5.6 ± 0.1 cF
7.0 ± 0.1 bC 6.9 ± 0.1 bC 5.2 ± 0.1 de G
6.5 ± 0.1 cA 4.6 ± 0.2 fgBC 3.2 ± 0.2 hD
5.6 ± 0.2 cdeAB 4.7 ± 1.1 efBC 2.6 ± 1.2 gD
3.9 ± 0.1 fD 2.6 ± 0.2 hD ND
2.7 ± 0.1 hD ND ND
6.4 ± 0.2 cA 5.0 ± 0.2 efBC 3.2 ± 0.1 hF
5.3 ± 0.2 efB 4.8 ± 0.5 efC 2.3 ± 0.3 gG
4.3 ± 0.4 eD 3.5 ± 0.1 gEF ND
3.7 ± 0.8 fE ND ND
∗ Values are means ± standard deviations. Means with different uppercase letters within the same group of coated at 3 different irradiation doses (0, 0.5, and 1 kGy) are significantly different from other means as determined by Duncan’s test (P ࣘ 0.05). Means with different lowercase letters within each column are significantly different from other means as determined by Duncan’s test (P ࣘ 0.05). ∗∗ ND, nondetectable (detection limit of 1.5 log CFU/g).
2.7 logs at day 3 as compared to the control (P ࣘ 0.05) at the same day of treatment were observed, respectively; however, in this treatment, there was an increase in bacterial concentration from day 3 to day 6 as compared to day 1 of treatment. In the uncoated carrots treated by irradiation with 1.0 kGy, a reduction of approximately 2 logs at day 1 as compared to the control was observed, and cell concentration was reduced by 2.5 logs at day 3 as compared to day 1 of treatment. Cell concentration was then increased at day 6 and maintained about 5 logs CFU/g until the end of storage. Cell concentration in carrots coated with nisin and without irradiation treatment was reduced by 1.2, 2.7, and 4.6 logs on days 3, 6, 9, respectively, as compared to this treatment at day 1. Cell concentration in carrots coated with nisin and with irradiation treatment of 0.5 kGy was reduced by less than 1 log as compared to the control at day 1. This treatment also caused a reduction of 3.6 logs at day 6 and a reduction to below the detection level (1.5 log/g) at day 9 as compared to day 1 of storage. The combined treatment with nisin and irradiation at 1 kGy was efficient in reducing L. monocytogenes since only 4.1 and 2.9 logs were detected at day 1 and day 3, respectively. Moreover, L. monocytogenes was undetectable from day 6 of storage. There was no significant difference (P > 0.05) in cell concentration between control carrot samples and those coated with carvacrol without irradiation treatment at day 1. Additionally, cell concentration did not significantly chang at day 3 and day 6 of storage (P > 0.05); however, at day 9, there was 0.8-log reduction (P ࣘ 0.05) as compared to day 1. It can be observed that the count of L. monocytogenes in carrots coated with carvacrol and irradiated at 0.5 kGy reduced by about 1.5 logs as compared to the same treatment without irradiation at day 1. Cell concentration in this treatment was increased by less than 1 log during storage. Cell concentration of this treatment was increased less than 1 log during storage. In the case of carrots coated by carvacrol and
irradiated at 1 kGy, there was a reduction of 2 logs in bacterial count at day 1 and the bacterial concentration was unchanged (P > 0.05) during storage. Also, there was no significant difference (P > 0.05) in cell concentration at day 1 between control and samples coated by mountain savory and without irradiation treatment. Moreover, this treatment did not reduce bacterial count during storage. A reduction of 0.7 and 2.1 logs of bacterial count at day 1 in the samples coated by mountain savory and irradiated at 0.5 and 1.0 kGy was observed, respectively. These combined treatments (mountain savory plus irradiation at 0.5 kGy or mountain savory plus irradiation at 1.0 kGy) did not cause any further bacterial reduction during storage. It can be observed that at day 1, cell concentration in carrots coated by carvacrol plus nisin and without irradiation (0 kGy) was reduced by more than one log as compared to the control. This treatment also caused further decrease in cell concentration at day 3 until the end of storage. Cell concentration in carrots coated by carvacrol plus nisin and irradiated at 0.5 kGy was reduced by 1.9 log as compared to this coating formulation without irradiation. In this treatment, cell concentration was further reduced by 2 logs at day 6 and to an undetectable limit at day 9. Interestingly, this coating formulation in combination with irradiation at 1 kGy decreased the count of L. monocytogenes by more than 3 logs at day 1 as compared to this formulation coating without irradiation. In this treatment, the count of the organism showed further reduction at day 3 and was below the detection limit at days 6 and 9 of storage. In carrots coated by mountain savory plus nisin and without irradiation (0 kGy), cell concentration was reduced by more than 1 log as compared to the control at day 1. Cell concentration in this treatment was further decreased during storage to 3.7 logs at day 9. Application of carrot coating by mountain savory plus nisin in combination with irradiation treatment at 0.5 kGy caused a
Vol. 80, Nr. 4, 2015 r Journal of Food Science M797
M: Food Microbiology & Safety
Treatments
Antilisterial activity of treatments . . . decrease of more than 1 log in L. monocytogenes concentration at day 1 compared to carrots coated by mountain savory plus nisin without irradiation. The count of the organism in this treatment was further decreased at day 3 and day 6 and was below the detection limit (1.5 log/g) at day 9. Meanwhile coating of carrots by mountain savory plus nisin in combination with irradiation at 1 kGy decreased the count of L. monocytogenes by more than 3 logs at day 1 as compared to this coating formulation without irradiation. Also the count of the organism was further decreased at day 3 and day 6 and was below the detection limit (define the detection limit of the experiment) at day 9 of storage.
Discussion
M: Food Microbiology & Safety
It can be found that irradiation treatment alone at 1.0 kGy was effective at reducing significantly L. monocytogenes inoculated in carrots by approximately 2 log as compared to the control during storage. Comparing all treatments against L. monocytogenes inoculated in carrots, it can be find that coating of carrots by carvacrol plus nisin or by mountain savory plus nisin with irradiation at 1 kGy could reduce L. monocytogenes by more than 3 logs at day 1. The bacterial concentrations in these treatments were also further decreased to undetectable levels at day 6. Thus, we can conclude that nisin contributed to the antimicrobial effects against L. monocytogenes in combination with essential oils (either carvacrol or mountain savory) and irradiation treatment at low dose (1 kGy). Mohamed and others (2011) found that a combined treatment of meat inoculated with 4 × 103 CFU/g of L. monocytogenes with nisin (103 IU/g) coupled to irradiation (1.5 kGy) could effectively eliminate L. monocytogenes, with a number of bacteria lower than 0.03 most probable number (MPN)/g at the end of the treatment. Nisin was also found to increase the antimicrobial effects against L. monocytogenes when used in combination with garlic EO (Rohani and others 2011). In our previous study, it was found that the radiation sensitivity (RS) of L. monocytogenes cell suspension (in vitro) to irradiation was increased by 4.19 and 6.31 times when treated by carvacrol plus nisin and mountain savory plus nisin, respectively, as compared to the control. In case of in situ study, it was found that coated carrots by nisin plus carvacrol increased the RS of L. monocytogenes to irradiation by 2.74 times as compared to the control (NdotiNembe and others, 2013). Thus, it is reasonable that combination of carvacrol or mountain savory EO plus nisin and irradiation can significantly increase the sensitivity of L. monocytogenes during storage. Turgis and others (2012) have evaluated the combined effects of bacteriocins (nisin, bacteriocin MT104, bacteriocin MT162) and irradiation against L. monocytogenes inoculated in sausage meat. The authors found a synergistic antimicrobial effect by using nisin or bacterocin MT 104 in combination with irradiation. The effect was expressed through the increase in relative sensitivity of L. monocytogenes to irradiation treatment. Our results are in agreement with these previous studies. Combined treatments using EO and irradiation against L. monocytogens or L. innocua in carrots have demonstrated that the EO can increase the sensitivity of L. monocytogenes to irradiation treatment, especially under modified atmosphere packaging (Caillet and others 2006b). EOs combined to radiation at low doses can also control the growth of L. innocua during storage of carrots (Caillet and others 2006a). For example, Caillet and others (2006a) evaluated the combined antimicrobial effects of antimicrobial coating (0.025% final concentration of trans-cinnamaldehyde), modified atmosphere packaging (60% O2 , 30% CO2 , and 10% N2 ) and M798 Journal of Food Science r Vol. 80, Nr. 4, 2015
gamma-irradiation (0.25 and 0.50 kGy) against Listeria innocua (103 CFU/g carrots). The authors found that the combined treatment was very effective at inhibiting the growth of L. innocua in carrots. For example, 2.23 logs and 2.26 logs CFU/g were found in the nonirradiated and uncoated samples, as well as in samples coated with the inactive coating and without irradiation treatment. However, after 7 d of storage, no L. innocua was detected in samples treated at 0.5 kGy under normal pressure or in samples treated at 0.25 kGy under modified atmosphere. The authors also found a complete inhibition of L. innocua during the storage period in uncoated and coated samples treated at 0.5 kGy under modified atmosphere. These results indicate that the combination of irradiation and modified atmosphere play an important role in controlling the growth of L. innocua. Similarly, Caillet and others (2006b) found that EOs consisting of trans-cinnamaldehyde, Spanish oregano, winter savory, and Chinese cinnamon could increase the sensitivity of L. monocytogenes inoculated in mini-carrots to irradiation treatment, especially when the samples were packaged in modified atmosphere (60% O2 , 30% CO2 , and 10% N2 ). Thus, it is reasonable to assume that the combined treatments by nisin, Eos, and irradiation could control or reduce the growth of L. monocytogenes inoculated into carrots during storage, even in air-packaged conditions.
Conclusion The present study demonstrates that combinations of nisin and mountain savory essential oil or nisin and carvacrol with low dose irradiation (1 kGy) have eliminated L. monocytogenes from readyto-eat carrots effectively. Coating of carrots by carvacrol plus nisin or by mountain savory plus nisin, then treating coated carrots at 1 kGy could reduce L. monocytogenes by more than 3 logs at day 1. Additionally, the bacterial concentration in these treatments was further decreased to undetectable levels from day 6. The combined nisin plus carvacrol or nisin plus mountain savory and irradiation at 1.0 kGy could be used as an effective method for controlling L. monocytogenes in minicarrots.
Acknowledgments Nordion Inc. is acknowledged for irradiation treatments. The authors sincerely thank the Natural Sciences and Engineering Research Council of Canada (NSERC) through the discovery program and the International Atomic Energy Agency (IAEA) for financial support. This study does not have any conflicts of interest.
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Vol. 80, Nr. 4, 2015 r Journal of Food Science M799
