Combined Effect of Natural Essential Oils, Modified Atmosphere Packaging, and Gamma Radiation on the Microbial Growth on Ground Beef

1237 Journal of Food Protection, Vol. 71, No. 6, 2008, Pages 1237 1243 Copyright, International Association for Food Protection Research Note Combined Effect of Natural Essential Oils, Modified Atmosphere Packaging, and Gamma Radiation on the Microbial Growth on Ground Beef M. TURGIS, 1 J. HAN, 1 J. BORSA, 2 AND M. LACROIX 1 * 1 Canadian Irradiation Centre, Research Laboratory in Sciences Applied to Food, Institut National de la Recherche Scientifique, Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, Québec, Canada H7V 1B7; and 2 MDS Nordion, 447 March Road, Kanata, Ontario, Canada K2K 2P7 MS 08-019: Received 9 January 2008/Accepted 27 January 2008 ABSTRACT Selected Chinese cinnamon, Spanish oregano, and mustard essential oils (EOs) were used in combination with irradiation to evaluate their ability to eliminate pathogenic bacteria and extend the shelf life of medium-fat-content ground beef (23% fat). Shelf life was defined as the time when the total bacterial count reached 10 7 The shelf life of ground beef was determined for 28 days at 4 C after treatment with EOs. The concentrations of EOs were predetermined such that sensory properties of cooked meat were maintained: 0.025% Spanish oregano, 0.025% Chinese cinnamon, and 0.075% mustard. Ground beef samples containing EOs were then packaged under air or a modified atmosphere and irradiated at 1.5 kgy. Ground beef samples (10 g) were taken during the storage period for enumeration of total mesophilic aerobic bacteria, Escherichia coli, Salmonella, total coliforms, lactic acid bacteria, and Pseudomonas. Mustard EO was the most efficient for reducing the total mesophilic aerobic bacteria and eliminating pathogenic bacteria. Irradiation alone completely inhibited the growth of total mesophilic aerobic and pathogenic bacteria. The combination of irradiation and EOs was better for reducing lactic acid bacteria (mustard and cinnamon EOs) and Pseudomonas (oregano and mustard EOs). The best combined treatment for extending the shelf life of ground beef for up to 28 days was EO plus irradiation (1.5 kgy) and modified atmosphere packaging. Despite technological development during the last decade, worldwide food losses increased. According to Health and Welfare Canada, the annual cost of meat spoilage is greater than $500 million (CAD), and the costs of foodborne illnesses associated with consumption of meat-based products are estimated at $1 billion (CAD) in Canada alone (2). Throughout the world, meat inspection authorities are encouraging or requiring meat packaging plants to implement hazard analysis critical control point systems in their processes, with particular emphasis on the development of such systems for the control of the number of pathogenic bacteria deposited on carcasses during carcass dressing (6). Irradiation is one of the major treatments used to eliminate bacterial contamination. -Radiation is used as a method of food preservation in more than 40 countries, including Canada, France, and the United States (7). Ahmed (1) reported that 37 countries have approved irradiated food products for human consumption, and 25 countries have commercialized the irradiation process. According to Morehouse and Komolprasert (10), increasing interest in use of food irradiation resulted in an approval of irradiation for controlling pathogens in raw red meat and processed meat products by the U.S. Food and Drug Administration in 1997. This process has enormous potential for extending * Author for correspondence. Tel: 450-687-5010, Ext 4489; Fax: 450-686- 5501; E-mail: monique.lacroix@iaf.inrs.ca. product shelf life and eliminating pathogenic bacteria such as Escherichia coli and Salmonella, which are principally responsible for foodborne illnesses associated with ground beef. Low-dose radiation ( 1 kgy) is used to delay sprouting of vegetables and aging of fruits; medium doses (1 to 10 kgy) are used to reduce the level of pathogenic organisms, similar to pasteurization; and high doses ( 1 kgy) are used to achieve sterility of the product. One potential negative aspect of the process is that the doses required to eliminate Salmonella generate off-flavors due to the oxidation of fatty acids and sulfur proteins, and these doses are generally higher than 2.5 kgy (8). One way to limit oxidation reactions is through the use of combined treatments to increase bacterial sensitivity and reduce radiation dose. Essential oils (EOs) have been used in combination with irradiation. EOs can act as flavoring compounds while increasing the radiosensitivity of E. coli and Salmonella. According to Chiasson et al. (3), the addition of 0.5% carvacrol (a compound found in thyme EO) to ground beef could more than double the radiosensitivity of E. coli and Salmonella. When irradiation was combined with modified atmosphere packaging (MAP) (60% O 2, 30% CO 2, and 10% N 2 ), the radiosensitivity of Salmonella increased more than 10 times. However, the presence of carvacrol significantly affected the taste of the meat.

1238 TURGIS ET AL. J. Food Prot., Vol. 71, No. 6 The aim of this study was to evaluate the potential for use of EOs without affecting the taste of ground beef in combination with irradiation under air or a modified atmosphere (i) to extend the shelf life, (ii) to eliminate pathogenic bacteria (E. coli and Salmonella), and (iii) to reduce the total endogenous flora (total mesophilic aerobes, total coliforms, lactic acid bacteria, and Pseudomonas). Based on results of previous studies (12, 13), EOs of Chinese cinnamon, Spanish oregano, and mustard were evaluated for their potential to eliminate spoilage and pathogenic bacteria in medium-fat-content ground beef (23% fat) stored at 4 C for 28 days. MATERIALS AND METHODS Meat handling and irradiation. Ground beef containing 23% fat was purchased at a local supermarket and brought to the Canadian Irradiation Centre (Laval, Québec, Canada) under refrigerated conditions (4 1 C). The ground beef was vacuum packaged and sterilized by irradiation at a dose of 25 kgy under frozen conditions ( 80 C) with an IR-147 underwater calibrator (MDS Nordion, Kanata, Ontario, Canada) equipped with a 60 Co source at a dose rate of 17.698 kgy/h. Preparation of bacterial cultures. The natural bacterial flora was isolated from 200 g of ground beef by homogenizing the sample in 200 ml of sterile peptone water (0.1%, wt/vol) and was maintained at 80 C in tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, Md.) with glycerol (10%, vol/vol). The bacterial culture was used for meat inoculation. Before each experiment, stock cultures were propagated through two consecutive 24-h growth cycles in TSB at 35 C, centrifuged (2,000 g) at4 C for 10 min, and washed twice in saline solution (0.85%, wt/vol) to obtain a working culture containing approximately 10 9 CFU/ml. Antimicrobial compounds. Spanish oregano and Chinese cinnamon EOs were purchased from Robert et Fils (Montréal, Québec, Canada). Mustard EO was from Hilltech (Vankleek Hill, Ontario, Canada). Meat preparation and inoculation procedures. Sixteen samples of medium-fat-content ground beef (450 g) were inoculated with a working bacterial culture of the natural flora from ground beef to obtain a final concentration of 10 7 CFU/g and mixed for 1 min using a sterilized spoon. The EO was added to ground beef at 0.025% (wt/wt) for Spanish oregano and Chinese cinnamon and 0.075% (wt/wt) for mustard (concentrations predetermined such that sensory properties of cooked meat were maintained, and samples were mixed for another 1 min to achieve uniform dispersal of EO throughout the ground beef. Control samples also were prepared with only ground beef (no EO). Meat samples (25 g each) were packaged in 0.5-mil metalized polyester 2-mil ethylene vinyl acetate (EVA) copolymer bags (205 by 355 mm; Winpak, St-Léonard, Québec, Canada) that were sealed under air (78.1% N 2, 20.9% O 2, and trace amounts of other gases) for control or under a modified atmosphere (60% O 2, 30% CO 2, and 10% N 2 ) for MAP and stored at 4 C until irradiation (approximately 15 h). Bacterial radiosensitivity evaluation. Inoculated ground beef samples were irradiated at the Canadian Irradiation Center under refrigerated conditions at 0 and 1.5 kgy. The UC-15A underwater calibrator (MDS Nordion) was equipped with a 60 Co source that provided a dose rate of 17.698 kgy/h. Samples were analyzed immediately after irradiation and at each storage interval (3, 7, 14, 21, and 28 days). Microbiological analysis. Each sample was homogenized for 1 min in sterile peptone water (0.1%, wt/vol) with a Labblender 400 stomacher (Laboratory Equipment, London, UK). From this mixture, serial dilutions were prepared and pour plated on tryptic soy agar (Difco, Becton Dickinson) for the determination of total mesophilic aerobic bacteria (TMA). Chromocult coliform agar (EMD Chemicals Inc., Gibbstown, N.J.) was used to determine total coliforms, E. coli, and Salmonella. Pseudomonas agar (EMD Chemicals) was used for the determination of Pseudomonas, and deman Rogosa Sharpe agar (EMD Chemicals) was used for the determination of lactic acid bacteria (LAB). All media were incubated for 24 h at 35 C. Statistical analysis. All experiments were done in triplicate. For each replicate, three samples were analyzed for each treatment evaluated. Bacterial counts (log CFU per gram) were analyzed with the SPSS program (SPSS, Chicago, Ill.), and comparisons of means between treatments were based on Duncan s multiple range tests (P 0.05) as a function of storage time for each treatment and between treatments on each day of analysis. RESULTS AND DISCUSSION Total mesophilic aerobic bacteria. The TMA populations in ground beef during the storage period are shown in Table 1. Results indicated that the shelf life for the control sample, which contained no EO, packaged under air was 1 day. Also at day 1, the presence of EO did not affect the level of TMA regardless of the concentration of EO used. The effect of irradiation alone reduced the level of TMA by 4.75 log Irradiation plus oregano or mustard EO was the most effective treatment, with 4.24- and 4.72-log reductions, respectively, as compared with the control. MAP alone reduced the level of TMA by 1.07 log CFU/g as compared with the control sample. The combination of MAP and irradiation reduced the level of TMA below the level of detection ( 1.0 log CFU/g) for 28 days. The addition of EO to the ground beef packaged under air did not significantly affect TMA (P 0.05) regardless of the EO used. However, when mustard EO was added and the samples were packaged under a modified atmosphere, this combined treatment extended the shelf life to 3 days as compared with the 1-day shelf life of the control samples packaged under air or a modified atmosphere. When any of the EOs was combined with irradiation treatment and MAP, the shelf life of the ground beef was estimated at more than 28 days. E. coli. The E. coli populations in ground beef during the storage period are presented in Table 2. Mustard EO was the most effective EO for reducing the level of E. coli at day 1, with a reduction of 0.81 log Irradiation treatment alone reduced the level of E. coli by 6 log CFU/g, which was below the detection level ( 1.0 log CFU/g). For irradiated samples with EOs, the level of E. coli was below the level of detection until the end of the experiment regardless of the type of EO added. MAP alone reduced the level of E. coli by 0.67 log When mustard EO was added to the ground beef in MAP, this treatment kept the E. coli population at 3.72 to 3.98 log CFU/g for 14 days.

J. Food Prot., Vol. 71, No. 6 COMBINED TREATMENTS WITH IRRADIATION ON BEEF MICROBIOLOGY 1239 TABLE 1. Total mesophilic aerobic bacteria in ground beef alone (control) or mixed with 0.025% cinnamon EO, 0.025% oregano EO, or 0.075% mustard EO, irradiated or not, and stored under air or a modified atmosphere (MAP) for 28 days a Total mesophilic aerobic bacteria (log CFU/g) on storage day: Control 0 kgy air 7.32 0.09 g A 7.0 e B 7.0 f B 7.0 b B 7.0 b B 7.0 b B Control 1.5 kgy air 2.57 0.12 b A 3.46 0.26 c AB 4.26 0.13 c B 7.0 b C 7.0 b C 7.0 b C Cinnamon EO 0 kgy air 7.51 0.04 h A 7.0 e B 7.0 f B 7.0 b B 7.0 b B 7.0 b B Cinnamon EO 1.5 kgy air 3.41 0.18 d B 2.13 0.18 b A 5.60 0.16 e C 7.0 b D 7.0 b D 7.0 b D Oregano EO 0 kgy air 7.51 0.065 gh B 6.98 0.25 d A 7.0 f C 7.0 b C 7.0 b C 7.0 b C Oregano EO 1.5 kgy air 3.08 0.20 c A 3.18 0.23 c A 4.93 0.10 d B 7.0 b C 7.0 b C 7.0 b C Mustard EO 0 kgy air 7.35 0.12 g A 7.0 e A 7.0 f B 7.0 b B 7.0 b B 7.0 b B Mustard EO 1.5 kgy air 2.60 0.23 b A 2.36 0.21 b A 3.81 0.17 b B 7.0 b C 7.0 b C 7.0 b C Control 0 kgy MAP 6.25 0.11 e A 7.0 e B 7.0 f B 7.0 b B 7.0 b B 7.0 b B Control 1.5 kgy MAP 1.0 a A 1.0 a A 0.59 0.92 a A 1.0 a A 1.0 a A 1.0 a A Cinnamon EO 0 kgy MAP 6.38 0.13 e A 7.0 e B 7.0 f B 7.0 b B 7.0 b B 7.0 b B Oregano EO 0 kgy MAP 6.29 0.09 e A 7.0 e B 7.0 f B 7.0 b B 7.0 b B 7.0 b B Oregano EO 1.5 kgy MAP 2.68 0.39 b A 1.0 a B 1.0 a B 1.0 a B 1.0 a B 1.0 a B Mustard EO 0 kgy MAP 5.63 0.08 e A 7.12 0.13 d B 7.0 f C 7.0 b C 7.0 b C 7.0 b C The combination of MAP and irradiation, with or without EOs, reduced the level of E. coli to below the level of detection during the 28 days of storage. Salmonella. Salmonella was sensitive to the presence of all tested EOs at day 1 (Table 3). Mustard and cinnamon EOs reduced the Salmonella level by 3.26 and 2.93 log CFU/g, respectively. Oregano EO was less effective than other EOs, resulting in a reduction of Salmonella by only 2.11 log Irradiation alone (1.5 kgy) decreased the Salmonella population to below the level of detection ( 1.0 log CFU/g) until the end of the experiment. The MAP reduced the Salmonella level by 1.72 log In combination with MAP, cinnamon, oregano, and mustard EOs reduced the level of Salmonella by 3.09, 2.99, and 3.59 log CFU/g, respectively, at day 1. Mustard EO in combination with MAP reduced the level of Salmonella to below the level of detection for up to 14 days. Regardless of storage atmosphere, irradiation alone reduced the level of Salmonella to below the detection level for up to 28 days. Total coliform bacteria. Total coliform bacteria in ground beef during the storage period are presented in Table 4. Cinnamon and oregano EOs did not affect the total coliform counts at day 1 (P 0.05). Mustard EO reduced the level of total coliforms by 0.8 log CFU/g at day 1. Irradiation without EO reduced the total coliform counts below the level of detection ( 1.0 log CFU/g) with both air and MAP for 28 days. MAP alone reduced the level of total coliforms by 1 log CFU/g compared with the air. The addition of oregano or cinnamon EO had no significant effect (P 0.05) on total coliform counts. However, the addition of mustard EO in combination with MAP prevented an overall increase in number of total coliforms for 14 days. LAB. The LAB populations in ground beef during the storage period are presented in Table 5. LAB growth was not affected by the presence of EOs at day 1. However, irradiation alone (1.5 kgy) reduced the LAB by 5.73 log Irradiation in combination with cinnamon or mustard EO was the most effective treatment, reducing the LAB population to below the detection level ( 1.0 log CFU/g). MAP alone reduced the LAB by 1 log When irradiation was combined with MAP, a reduction of 5.46 log CFU/g was observed. The best combined treatments were irradiation plus any EO and MAP and irradiation plus mustard EO stored in air. These combined treatments reduced the LAB population to below the level of detection during the entire storage period. Pseudomonas. The Pseudomonas populations in ground beef during the storage period are presented in Table 6. Pseudomonas populations were reduced by 1 log CFU/g at day 1 when mustard EO was added to the ground beef. Irradiation alone reduced the level of Pseudomonas by 4.39 log Addition of cinnamon, oregano, or mustard EO in combination with irradiation decreased Pseudomonas populations by 3.53, 4.85, and 6.08 log CFU/g, respectively. MAP alone reduced the level of Pseudomonas by 1 log CFU/g as compared with the conventional air packaging. The combination of MAP and irradiation reduced the Pseudomonas level by 6.54 log The addition of cinnamon or oregano EO to ground beef plus MAP had no significant effect (P 0.05) on Pseudomonas. However, the combination of EO, MAP, and irradiation reduced Pseudomonas populations to below the level of detection for 28 days. In our study, 0.075% mustard EO and 0.025% Chinese cinnamon EO had better antimicrobial activity than did

1240 TURGIS ET AL. J. Food Prot., Vol. 71, No. 6 TABLE 2. Escherichia coli populations in ground beef alone (control) or mixed with 0.025% cinnamon EO, 0.025% oregano EO, or 0.075% mustard EO, irradiated or not, and stored under air or a modified atmosphere (MAP) for 28 days a E. coli (log CFU/g) on storage day: Control 0 kgy air 5.68 0.23 de A 7.0 h B 7.0 e B 7.0 f B 7.0 b B 7.0 b B Control 1.5 kgy air 1.0 a A 2.08 0.08 b B 1.0 a A 1.0 a A 1.0 a A 1.0 a A Cinnamon EO 0 kgy air 5.82 0.08 e A 7.0 h B 7.0 e B 7.0 f B 7.0 b B 7.0 b B Cinnamon EO 1.5 kgy air 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Oregano EO 0 kgy air 6.10 0.20 f A 7.0 h B 7.0 e B 7.0 f B 7.0 b B 7.0 b B Oregano EO 1.5 kgy air 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Mustard EO 0 kgy air 4.87 0.12 c C 4.68 0.15 d BC 4.45 0.27 c AB 4.20 0.20 c A 7.0 b D 7.0 b D Mustard EO 1.5 kgy air 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Control 0 kgy MAP 5.01 0.11 c A 6.10 0.04 f B 7.0 e D 6.81 0.17 e C 7.0 b D 7.0 b D Control 1.5 kgy MAP 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Cinnamon EO 0 kgy MAP 6.17 0.17 f D 5.86 0.08 e C 4.68 0.21 c B 4.28 0.24 c A 7.0 b E 7.0 b E Oregano EO 0 kgy MAP 5.55 0.12 d A 6.43 0.11 g C 6.11 0.27 d B 6.17 0.13 d B 7.0 b D 7.0 b D Oregano EO 1.5 kgy MAP 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Mustard EO 0 kgy MAP 3.98 0.13 b B 3.99 0.21 c B 3.58 0.10 b A 3.72 0.28 b A 7.0 b C 7.0 b C 0.025% Spanish oregano EO. Except for mustard in presence of E. coli and Salmonella or cinnamon in presence of Salmonella, the addition of EOs alone did not have an antimicrobial activity against test bacteria and might have promoted growth during storage. However, when EOs were combined with MAP, an additive effect on bacterial growth was observed. In this study, mustard EO and MAP significantly reduced (P 0.05) the number of TMA, E. coli, total coliforms, LAB, and Pseudomonas and completely reduced Salmonella to below detectable numbers. EOs are known antimicrobial agents that can be used to control food spoilage and foodborne pathogenic bacteria. Lemay et al. (9) studied the effect of mustard EO against a mixed culture of E. coli ATCC 25922, Brochothrix thermosphacta CRDAV452, and a protective culture of Lactobacillus alimentarius BJ33 (FloraCan L-2). When mustard TABLE 3. Salmonella populations in ground beef alone (control) or mixed with 0.025% cinnamon EO, 0.025% oregano EO, or 0.075% mustard EO, irradiated or not, and stored under air or a modified atmosphere (MAP) for 28 days a Salmonella (log CFU/g) on storage day: Control 0 kgy air 3.59 0.09 d C 3.38 0.09 d B 3.92 0.05 f D 3.27 0.10 e A 7.0 b E 7.0 b E Control 1.5 kgy air 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Cinnamon EO 0 kgy air 0.66 0.61 b A 0.63 0.99 ab A 2.27 0.29 cd A 4.08 0.37 f C 7.0 b D 7.0 b D Cinnamon EO 1.5 kgy air 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Oregano EO 0 kgy air 1.48 0.14 c A 3.06 0.03 d C 1.81 0.44 c B 3.19 0.17 e C 7.0 b D 7.0 b D Oregano EO 1.5 kgy air 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Mustard EO 0 kgy air 0.33 0.52 ab A 0.74 0.61 ab A 0.33 0.51 ab A 0.17 0.41 a A 7.0 b B 7.0 b B Mustard EO 1.5 kgy air 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Control 0 kgy MAP 1.87 0.18 c A 3.26 0.15 d C 2.88 0.13 e B 2.80 0.22 d B 7.0 b D 7.0 b D Control 1.5 kgy MAP 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Cinnamon EO 0 kgy MAP 0.50 0.55 ab A 1.94 0.10 c C 0.68 0.75 b AB 1.21 0.24 b B 7.0 b D 7.0 b D Oregano EO 0 kgy MAP 0.60 0.55 b A 2.31 0.03 c B 2.62 0.22 de B 2.39 0.16 c B 7.0 b C 7.0 b C Oregano EO 1.5 kgy MAP 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Mustard EO 0 kgy MAP 1.0 a A 0.38 0.6 ab A 1.0 a A 1.0 a A 7.0 b A 7.0 b A

J. Food Prot., Vol. 71, No. 6 COMBINED TREATMENTS WITH IRRADIATION ON BEEF MICROBIOLOGY 1241 TABLE 4. Total coliform bacteria in ground beef alone (control) or mixed with 0.025% cinnamon EO, 0.025% oregano EO, or 0.075% mustard EO, irradiated or not, and stored under air or a modified atmosphere (MAP) for 28 days a Total coliform bacteria (log CFU/g) on storage day: Control 0 kgy air 6.28 0.14 de A 7.0 f B 7.0 f B 7.0 f B 7.0 b B 7.0 b B Control 1.5 kgy air 1.0 a A 2.00 0.08 b B 1.0 a A 1.0 a A 1.0 a A 1.0 a A Cinnamon EO 0 kgy air 6.34 0.09 e A 7.0 f B 7.0 f B 7.0 f B 7.0 b B 7.0 b B Cinnamon EO 1.5 kgy air 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Oregano EO 0 kgy air 6.63 0.10 f A 7.0 f B 7.0 f B 7.0 f B 7.0 b B 7.0 b B Oregano EO 1.5 kgy air 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Mustard EO 0 kgy air 5.45 0.06 c B 5.94 0.21 c C 5.17 0.04 c B 4.36 0.13 c A 7.0 b C 7.0 b C Mustard EO 1.5 kgy air 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Control 0 kgy MAP 5.28 0.07 c A 7.0 f B 7.0 f B 7.0 f B 7.0 b B 7.0 b B Control 1.5 kgy MAP 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Cinnamon EO 0 kgy MAP 6.07 0.11 d B 7.11 0.11 d C 6.12 0.17 d B 5.44 0.24 d A 7.0 b D 7.0 b D Oregano EO 0 kgy MAP 6.44 0.17 ef A 7.38 0.21 e C 7.31 0.08 e C 6.93 0.11 e B 7.0 b D 7.0 b D Oregano EO 1.5 kgy MAP 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A 1.0 a A Mustard EO 0 kgy MAP 4.42 0.14 b C 6.21 0.28 c D 4.86 0.14 b C 3.70 0.16 b A 7.03 0.07 b E 7.0 b F EO was added to nutrient media containing aerobic mesophilic bacteria or LAB, the level of these bacteria was significantly reduced (P 0.05) compared with the control after 2 days. In our study, mustard EO also was effective for reducing bacterial growth during the storage of mediumfat-content ground beef, and a synergistic or additive effect was obtained when mustard EO was combined with MAP storage. Salmonella and E. coli growth was stopped and total coliforms were reduced to below detectable levels in the presence of mustard EO. De et al. (5) also reported that cinnamon has antimicrobial activity against E. coli ATCC 10536. Penalver et al. (13) found that the EO with the greatest antimicrobial activity against four strains of Salmonella was Origanum vulgar (MIC 1%, vol/vol) followed by Thymus zygis (MIC 2%, vol/vol). Greater inhibitory capacity was observed TABLE 5. Lactic acid bacteria in ground beef alone (control) or mixed with 0.025% cinnamon EO, 0.025% oregano EO, or 0.075% mustard EO, irradiated or not, and stored under air or a modified atmosphere (MAP) for 28 days a Lactic acid bacteria (log CFU/g) on storage day: Control 0 kgy air 7.33 0.11 f A ND b ND ND ND 7.0 b B Control 1.5 kgy air 1.60 1.11 c B ND 2.07 0.44 c B 2.61 0.88 c B ND 1.0 a A Cinnamon EO 0 kgy air 7.58 0.12 f A ND 7.0 e B 7.0 e B ND 7.0 b B Cinnamon EO 1.5 kgy air 1.0 a A ND 0.68 0.75 b B 1.50 1.64 b C ND 1.0 a A Oregano EO 0 kgy air 7.51 0.10 f A ND 7.0 e B 7.0 e B ND 7.0 b B Oregano EO 1.5 kgy air 0.72 1.11 b A ND 1.11 0.58 b A 1.23 0.77 b A ND 1.0 a A Mustard EO 0 kgy air 7.39 0.09 f B ND 6.00 0.35 d A 6.29 0.23 d A ND 7.0 b B Mustard EO 1.5 kgy air 1.0 a A ND 1.0 e A 1.0 a A ND 1.0 a A Control 0 kgy MAP 6.34 0.13 c A ND ND ND ND 7.0 b B Control 1.5 kgy MAP 1.87 0.94 b B ND 1.06 0.17 b B 1.0 a A ND 1.0 a A Cinnamon EO 0 kgy MAP 6.31 0.05 c A ND ND ND ND 7.0 b B Cinnamon EO 1.5 kgy MAP 1.0 a A ND 10 a A 1.0 a A ND 1.0 a A Oregano EO 0 kgy MAP 6.44 0.03 c A ND ND ND ND 7.0 b B Oregano EO 1.5 kgy MAP 1.0 a A ND 1.0 a A 1.0 a A ND 1.0 a A Mustard EO 0 kgy MAP 6.17 0.08 c A ND ND ND ND 7.0 b B Mustard EO 1.5 kgy MAP 1.0 a A ND 1.0 a A 1.0 a A ND 1.0 a A b ND, not determined.

1242 TURGIS ET AL. J. Food Prot., Vol. 71, No. 6 TABLE 6. Pseudomonas populations in ground beef alone (control) or mixed with 0.025% cinnamon EO, 0.025% oregano EO, or 0.075% mustard EO, irradiated or not, and stored under air or a modified atmosphere (MAP) for 28 days a Pseudomonas (log CFU/g) on storage day: Control 0 kgy air 7.31 0.14 f A ND b ND ND ND 7.0 b B Control 1.5 kgy air 2.92 0.34 c C ND 3.13 0.15 c C 2.61 0.08 c B ND 1.0 a A Cinnamon EO 0 kgy air 7.38 0.07 f A ND 7.0 e B 7.0 e B ND 7.0 b B Cinnamon EO 1.5 kgy air 3.78 0.20 d d ND 1.14 0.60 b B 2.50 0.29 c C ND 1.0 a A Oregano EO 0 kgy air 7.42 0.07 f B 6.81 0.26 a A 7.0 e B 7.0 e B ND 7.0 b B Oregano EO 1.5 kgy air 2.46 0.65 c C ND 1.39 0.24 b B 1.81 0.31 b C ND 1.0 a A Mustard EO 0 kgy air 6.12 0.07 e A ND 6.10 0.17 d A 6.47 0.29 d A ND 7.0 b B Mustard EO 1.5 kgy air 1.23 1.25 b B ND 1.28 0.64 b B ND ND 1.0 a A Control 0 kgy MAP 6.38 0.17 e A ND ND ND ND 7.0 b B Control 1.5 kgy MAP 0.77 0.65 a A ND 0.17 0.41 a A 1.0 a A ND 1.0 a A Cinnamon EO 0 kgy MAP 7.31 0.02 f A ND ND ND ND 7.0 b B Cinnamon EO 1.5 kgy MAP 10 a A ND 1.0 a A 1.0 a A ND 1.0 a A Oregano EO 0 kgy MAP 7.19 0.03 f A ND ND ND ND 7.0 b B Oregano EO 1.5 kgy MAP 1.72 0.87 b B ND 1.0 a A 1.0 a A ND 1.0 a A Mustard EO 0 kgy MAP 6.35 0.06 e A ND ND ND ND 7.0 b B Mustard EO 1.5 kgy MAP 1.13 0.78 ab B ND 1.0 a A 1.0 a A ND 1.0 a A b ND, not determined. for EOs rich in phenolic components (e.g., carvacrol and thymol) as compared with those rich in the monoterpenic alcohol linalool. Oussalah et al. (12) studied the use of packaging film containing oregano, cinnamon, and winter savory (0.5%) for controlling the growth of pathogenic bacteria on bologna and ham. In their study, Listeria monocytogenes and Salmonella Typhimurium were initially completely eliminated by the packaging film and the bacteria were not detected during 5 days of storage. Oussalah et al. (11) also found that the incorporation of pimento and oregano EOs (0.5%) into edible film applied onto muscle meat helped reduce the growth of Pseudomonas spp. and E. coli O157:H7. These treatments also protected the meat from lipid oxidation during 7 days of storage. In our study, when oregano or cinnamon EO was added to medium-fat-content ground beef at a concentration that did not affect the sensory properties, the compounds did not have the same level of effectiveness as found in previous studies. Therefore, combined treatments are required to achieve safety and assure quality. The results obtained with MAP are in agreement with those obtained in a previous study by Chiasson et al. (4). We used the same modified atmosphere in the present study. Other authors have observed an increase of the radiosensitivity of E. coli and Salmonella Typhimurium on ground beef stored in presence of high concentrations of O 2. A high concentration of O 2 during irradiation might enhance the lethal effects of radiation because of the formation of oxygen radicals and ozone during treatment (3, 14). In our study, mustard EO was the most effective EO for reducing bacterial growth on ground beef during storage. A synergistic or additive effect was observed when mustard EO was added to ground beef that was stored in MAP. Irradiation completely inhibited the growth of E. coli and Salmonella in medium-fat-content ground beef for up to 28 days. The combination of -radiation (1.5 kgy), mustard EO (0.075%), and MAP effectively eliminated the growth of TMA. The combination of irradiation, MAP, and EOs also inhibited LAB. Therefore, combinations of EOs (at concentrations that do not affect taste), irradiation, and MAP are useful for extending the shelf life and assuring the safety of ground beef during 28 days of storage. ACKNOWLEDGMENTS This research was supported by the Natural Sciences and Engineering Research Council of Canada through the RDC program and by a research contract under a research agreement with MDS Nordion. The authors thank Winpak (Winnipeg, Manitoba, Canada) for providing the meat packaging material. REFERENCES 1. Ahmed, M. 1993. Up-to-date status of food irradiation. Radiat. Phys. Chem. 42:245 251. 2. Anonymous. 1994. Foodborne pathogens, risk and consequences. Task force report no. 122. Council for Agricultural Science and Technology, Ames, Iowa. 3. Chiasson, F., J. Borsa, B. Ouattara, and M. Lacroix. 2004. Radiosensitization of Escherichia coli and Salmonella Typhi in ground beef. J. Food Prot. 67:1157 1162. 4. Chiasson, F., J. Borsa, B. Ouattara, and M. Lacroix. 2005. Combined effect of carvacrol and packaging conditions on radiosensitivity of Escherichia coli and Salmonella Typhi in ground beef. J. Food Prot. 68:2567 2570. 5. De, M., K. De, and A. B. Banerjee 1999. Antimicrobial screening of some Indian spices. Phytother. Res. 13:616 618. 6. Hudson, W. R., G. C. Mead, and M. H. Hinton. 1996. Relevance of abattoir hygiene assessment to microbial contamination of British beef carcasses. Vet. Rec. 139:587 589. 7. International Consultative Group on Food Irradiation. 1999. Facts about food irradiation. A series of fact sheets from the international

J. Food Prot., Vol. 71, No. 6 COMBINED TREATMENTS WITH IRRADIATION ON BEEF MICROBIOLOGY 1243 consultative group on food irradiation. Food and Agriculture Organization and the International Atomic Energy Agency, Vienna. 8. Katta, S. R., D. R. Rao, G. R. Sunki, and C. B. Chawan. 1991. Effect of irradiation of whole chicken carcasses on bacterial loads and fatty acids. J. Food Sci. 56:371 372. 9. Lemay, M. J., J. Choquette, P. J. Delaquis, G. Claude, N. Rodrique, and L. Saucier. 2002. Antimicrobial effect of natural preservatives in cooked and acidified chicken meat model. Int. J. Food Microbiol. 78:217 226. 10. Morehouse, K. M., and V. Komolprasert. 2004. Irradiation of food packaging: an overview, p. 1 11. In V. Komolprasert and K. M. Morehouse (ed.), Irradiation of food and packaging: recent developments. No. 875. American Chemical Society, Washington, D.C. 11. Oussalah, M., S. Caillet, S. Salmieri, L. Saucier, and M. Lacroix. 2004. Antimicrobial and antioxidant effects of milk protein based film containing essential oils for the preservation of whole beef muscle. J. Agric. Food Chem. 52:5598 5605. 12. Oussalah, M., S. Caillet, S. Salmieri, L. Saucier, and M. Lacroix. 2007. Antimicrobial effects of alginate-based films containing essential oils on Listeria monocytogenes and Salmonella Typhimurium present in bologna and ham. J. Food Prot. 70:901 908. 13. Penalver, P., B. Huerta, C. Borge, R. Astorga, R. Romero, and A. Perea. 2005. Antimicrobial activity of five essential oils against origin strains of the Enterobacteriaceae family. APMIS 113:1 6. 14. Thakur, B. R., and R. K. Singh. 1994. Food irradiation chemistry and applications. Food Rev. Int. 10:437 473.