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Efficacy of radio frequency cooking in the reduction of Escherichia coli and shelf stability of ground beef
ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 23 (2006) 112–118 www.elsevier.com/locate/fm
Efficacy of radio frequency cooking in the reduction of Escherichia coli and shelf stability of ground beef Q. Guoa, P. Piyasenab, G.S. Mittala,, W. Sib, J. Gongb a
School of Engineering, University of Guelph, Guelph, Ont., Canada N1G 2W1 Food Research Program, Agriculture & Agri-Food Canada, Guelph, Ont., Canada N1G 2W1
b
Received 2 December 2004; received in revised form 21 February 2005; accepted 21 February 2005
Abstract The effectiveness of radio frequency (RF) cooking on the inactivation of Escherichia coli in ground beef and its effect on the shelf stability of ground beef were investigated with a comparison to hot water-bath cooking. E. coli K12 was used as a target bacterium instead of E. coli O157:H7. The ground beef samples inoculated with E. coli K12 (ampr) were heated until the centre temperature of each sample reached 72 1C. These samples were then stored at 4 1C for up to 30 days. The enumeration of E. coli K12, background E. coli and coliform counts in ground beef samples was carried out for shelf-life study. Although both methods significantly reduced E. coli K12 (ampr), E. coli and coliform counts and extended the shelf-life, RF cooking had a shorter cooking time, and more uniform heating. Thus, RF cooking of meat has a high potential as a substitute for the hot water-bath cooking. r 2005 Elsevier Ltd. All rights reserved. Keywords: E. coli; Ground beef; Meat cooking/heating; Radio frequency; Shelf-life
1. Introduction Escherichia coli O157:H7 has been identified as a major foodborne pathogen. It is responsible for haemorrhagic colitis, haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura (Doyle, 1997). The bacterium was first recognized in 1982 during an investigation of two outbreaks of bloody diarrhoea in Oregon and Michigan (Doyle et al., 2001). Since then, it has been implicated in many other outbreaks. The Center for Disease Control and Prevention has estimated that foodborne diseases caused by E. coli O157:H7 account for 62458 cases of illness, 1843 hospitalizations and 52 deaths in the US each year. Most of the cases were caused by consumption of inadequately cooked contaminated ground beef (Doyle, 1991). The most notable outbreak of 1993 was caused by Corresponding author. Tel. +1 519 824 4120x52431; fax: +1 519 836 0227. E-mail address: [email protected] (G.S. Mittal).
0740-0020/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2005.02.004
undercooked hamburger containing E. coli O157:H7 and affected over 500 people and ultimately led to the death of 4 children (Ayres, 1995). Adequate cooking is the most effective means to eliminate E. coli O157:H7 from meat products and to assure their microbiological safety. Many conventional methods of cooking, including hot air or water, steam, frying with fat or oil, and radiant heating, are applied to cook ground beef. However, such food heating methods require that food is heated externally through conduction, convection or radiation. Because of these heat transfer modes, the conventional cooking methods need longer cooking time and decrease heating uniformity to ensure that the centre is cooked while the surface may overcook (Laycock et al., 2003). Different from conventional cooking methods, radio frequency (RF) cooking is an innovative heating technique that can save cooking time, and heat food uniformly (Rowley, 2001). RF cooking is a technique based on electro-technologies, such as ohmic heating, or microwave dielectric heating (Piyasena et al., 2003).
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Unlike other heat transfer modes, RF cooking heats foods directly through energy conversion, from electrical energy to heat (Rowley, 2001). The energy conversion occurs within the food itself, and heat is absorbed directly by the food. Therefore, RF cooking achieves high-energy efficiency and uniform cooking (Orsat et al., 2001). The application of RF cooking in the food industry can be traced back to the 1940s. RF cooking was initially used to cook meat, bake bread, and dehydrate or blanch vegetables. Later, RF cooking was used to thaw frozen products in the 1960s and to dry cookies in the 1980s. Recently, RF cooking has been evaluated for sterilization and pasteurization of meat products (Piyasena et al., 2003). RF cooking can be used to reduce high levels of microbial contamination and improve food quality. The effectiveness of RF cooking on the reduction of microbial contamination of fresh carrots (Orsat et al., 2001) and sausage (Houben et al., 1991) has been investigated. Its effect on food quality has also been studied on ground beef (Laycock et al., 2003). They found that RF cooking led to shorter cooking time, lower juice losses, and acceptable colour and texture. However, little research has been reported in the reduction of microbial contamination of ground beef by RF cooking. Furthermore, there is limited information on the shelf-life of RF cooked products. Thus, the objective of this study was to investigate the effectiveness of RF cooking on the inactivation of E. coli and its effect on the shelf stability of cooked ground beef. The non-pathogenic E. coli K12 strain, possessing the ampicillin resistance, was used as a target bacterium, as RF cooking may not have selectivity against bacterial strains within a species. In addition, the use of E. coli K12 requires less stringent controlled working conditions as compared to the use of E. coli O157:H7 which requires a laboratory licensed for use of human level 2 pathogens.
2. Materials and methods 2.1. Equipment The RF equipment used for this study was similar to the one used by Laycock et al. (2003). A RF heater of 1.5 kW at a frequency of 27.12 MHz (Strayfield Ltd, Reading, UK) was used. The heater included two parts: an RF generator and an applicator circuit. The applicator contained two electrodes, tuning capacitor plates, an inductance coil and an applicator cylinder. The food samples in the cylinder can be dielectrically heated between the two electrodes. The RF power was adjusted by changing the distance between the tuning plates of the capacitor. The RF power increased as the
113
distance decreased. This was accomplished by manually adjusting a gear mechanism outside the RF oven. An active current of 425 mA for ground beef was maintained by using the gear mechanism. The input or incident power was calculated from the actual current and the high voltage of the RF unit as explained by Piyasena and Dussault (2003). The actual current is the difference between the active current and the standing current of 175 mA (Strayfield, 1993). The high voltage (V) across the electrodes for this RF unit has been estimated to be 4600 V (Strayfield, 1993). The incident power for this unit at 425 mA was 1150 W. Power absorbed (Pw) by the product during continuous steady flow could also be calculated as explained by Strayfield (1993). The power loss is the different between incident power and Pw. 2.2. Ground beef The ground beef used for the experiments was obtained from a local meat processor and kept frozen (1870.2 1C) before use. The ground beef samples were homogeneous in the components and appearance to ensure uniform initial quality. Prior to inoculation with the strain of E. coli K12 (ampr), the meat was thawed at 470.5 1C in a refrigerator over a 24-h period. All raw samples were shaped following the container’s dimensions. The cylinder was made of Polytetrafluoroethylene (PFTE), which is RF inert. The cylinder (ID ¼ 0.0952 m, OD ¼ 0.121 m, height from inside ¼ 0.203 m, height from outside ¼ 0.215 m), with the product inside, was set between two electrodes (Laycock et al., 2003). 2.3. Micro-organism A non-pathogenic E. coli K12 strain W3110 (ATCC27325) was obtained from the bacterial collection of Food Research Program, Agriculture & AgricFood Canada. It was stored at 70 1C in Trypticase Soy Broth (TSB) containing 15% (v/v) glycerol. To facilitate detection and enumeration of the target bacterium, the E. coli K12 strain was introduced with the ampicillin resistance (ampr), which was subsequently used throughout the present study. The organism E. coli K12 (ampr) was subcultured periodically to maintain viability. 2.4. Experimental procedure To evaluate the influence of RF cooking on the inactivation of E. coli in ground beef, the experiment was conducted according to the following procedures. 2.5. Transformation of E. coli K12 Plasmid pUC19 (Fermentas Life Science Ltd., Hanover, USA) carrying the gene of ampr was introduced
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into E. coli K12 by high-voltage electroporation. Briefly, 2.5 ml of log-phase E. coli K12 culture with the optical density at 600 nm (OD600) of 0.6 was harvested by centrifugation at 4200 rpm (Centrifuge, model RC5C, Sorvall Instruments, Asheville, USA) for 20 min at 4 1C. The bacterial pellet was washed twice in 500 ml of icecold distilled water. After the final wash, the pellet was resuspended in 2 ml of 10% glycerol, and 40 ml of the cell suspension was then mixed with 2 mg of pUC19 plasmid DNA for electroporation. The sample was subjected to a 2.5 kV, 400 O, 25 mF electric pulse for 3 s in a 0.2 cm cuvette using a Gene Pulser and Pulse Controller apparatus (Bio-Rad, Hercules, USA). One milliliter of SOC medium (2% Bacto tryptone, 0.5% Bacto yeast extract, 0.06% NaCl, 0.05% KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM Glucose) was immediately added and mixed. The cells were transferred to an Eppendorf (culture) tube and incubated for 1 h at 37 1C in a shaking incubator (Orbital Shaker, Model#420, Thermo Forma, Westmont, USA). After incubation, the cells were plated on Luria–Bertani (LB) [1% (w/v) NaCl, 1% (w/v) tryptone, 0.5% (w/v) yeast extract] agar containing 100 mg/ml ampicillin, followed by incubation at 37 1C for 16 h. Colonies grown on the LB plates were the transformants, E. coli K12 (ampr), with the ampicillin resistance. The introduced ampr gene was shown to be stable during subcultures and storage of the transformant. 2.6. Determination of the relationship between the optical density at 600 nm (OD600) and the counts of E. coli K12 E. coli K12 was subcultured in LB broth at 37 1C. An overnight subculture was used to determine the relationship between the OD600 values and the number of colony forming units (cfu) of the bacterium. The OD600 values were measured using a spectrophotometer (model Helios Alpha, Spectronic Instruments, Garforth, UK). To determine the counts of E. coli K12, the cell suspensions were diluted serially in twofold to obtain the OD600 value between 0.1 and 1, and 0.1 ml of the diluted cell suspension was plated on to LB plates containing 100 mg/ml ampicillin. The number of cfu was counted and expressed as cfu/ ml, and was plotted against the OD600 values. These experiments were repeated three times to ensure reproducibility. 2.7. Preparation of test cultures The cultures were prepared by inoculation of E. coli K12 in LB broth containing 100 mg/ml ampicillin and incubation at 37 1C for 16 h. The cultures were subsequently used for inoculation of ground beef.
2.8. Inoculation of ground beef A known amount (around 1 kg) of ground beef was transferred to a sterile bowl and inoculated with 200 ml of E. coli K12 cell suspension to yield approximately 107 cfu/g of sample. The inoculated ground beef was mixed for 2 min by gloved hands to ensure even distribution of the micro-organism. Ground beef inoculated with E. coli K12 but not cooked, was used as a control. Ground beef, neither inoculated with E. coli K12 nor cooked, served as a blank control. Cooked ground beef sample was a negative control. The final concentration of the micro-organism in the samples was later confirmed by enumeration on agar. 2.9. Microbial enumeration To count the number of surviving bacterial cells per gram, sterile 0.1% peptone-water (PW) was added to each meat sample to obtain 1:10 (w/v) slurry. The samples were homogenized for 2 min by a stomacher (Lab system, Model#400, Seward Ltd., Norfolk, UK) at a high speed. Decimal serial dilutions were prepared in 0.1% PW and 0.1 ml of appropriate diluted samples were plated onto LB agar plates containing ampicillin (100 mg/ml) and 1 ml of appropriate diluted samples were plated on to 3 M E. coli/coliform Petrifilm (3 M Microbiology Products, London, Canada). When there was a need, the undiluted sample was also plated. The LB plates were incubated at 37 1C for 16 h before cfu counting. The colonies on the 3 M Petrifilm were determined after 48 h of incubation at 37 1C. For each sampling point, three plates were used to calculate the average cfu/g of ground beef. 2.10. Cooking and temperature measurement The methods of cooking and temperature measurement are similar to those used by Laycock et al. (2003). Three fiber-optic temperature probes (FOT-L, Fiso Technologies, Quebec, Canada) and signal processor (model #UMI-4, Fiso Technologies, Quebec, Canada) were used to measure the temperatures during RF cooking. One probe recorded the centre temperature of the samples (75 mm from the top surface), the second measured the temperature 10–15 mm from the side (75 mm from the top surface), and the third measured the temperature 10–15 mm from the other side (15 mm from the top surface) (Fig. 1). During RF cooking, the temperature was manually recorded every 15 s until the centre temperature of the samples reached 72 1C, at which point the RF generator was deactivated. After cooking, the samples were stored separately at 470.5 1C in a refrigerator for microbial tests. Samples of same mass were also cooked in a water-bath (model#1417, VWR International, Sheldon
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1.5cm
4.76cm
to determine significant differences (Po0:01) among means.
1.5cm
1.5cm
Thermal probes
3. Results and discussion
7.5cm
3.1. Relationship between the OD600 values and E. coli K12 counts 14.96cm
Ground beef
115
Container
The relationship between the OD600 values and E. coli K12 counts were determined using linear regression (Fig. 2). The OD600 values from 0.01 to 0.1 exhibited a linear relationship with the E. coli K12 counts. 3.2. Comparison of cooking methods
Fig. 1. The arrangement of thermal probes in meat samples during RF cooking.
Manufacturing, Cornelius, OR, USA) for comparative purposes. The dimensions of the samples were 9.52 cm in diameter and 14.96 cm in height. To keep their shape, they were packed in a sheet of aluminium foil. The samples were then put in sterilized stomached bags in the water-bath. The water temperature started at 65 1C and was gradually (0.2 1C/min) increased to 90 1C, until the centre temperature of the samples reached 72 1C. Three thermocouples (Omega, Laval, Canada; model HH23, microprocessor thermometer, type T thermocouple) were used to record the temperature at every 2 min. One temperature probe was inserted in the product centre. 2.11. Shelf-life study RF or water-bath cooked ground beef samples (25 g) were aseptically weighed and packed in stomacher bags and stored at 470.5 1C in a refrigerator for up to 4 weeks to determine shelf stability. The number of E. coli K12 (ampr), background E. coli and coliform in the samples were enumerated. The microbial counts were recorded after refrigerated storage for 0 (baseline), 3, 7, 10, 15, 21, 30 days at 4 1C.
4.00E+13 y = 4E+13x+ 1E+12 R2 = 0.9877
3.00E+13 cfu/ml
9.52cm
The average cooking time was from 4.25 to 150.33 min for the central temperature of samples to reach 72 1C, depending on cooking methods (Table 1). There was a significant difference (Po0:01) among cooking methods with respect to the cooking times. The time for RF cooking (4.2570.25 min) was much shorter than that of the water-bath (150.3371.53 min). The results suggested that RF cooking can save cooking time and thus provide high-energy efficiency. When the temperature data at three locations in the ground beef were analysed (Table 1), there was a significant difference (Po0:01) between RF cooking and water-bath cooking. The wider temperature variation within samples was observed for water-bath cooking compared to RF cooking. The difference between the surface and centre of samples was 12.077.8 1C in waterbath cooking, indicating overcooking of outer section of the samples. This might have affected the organoleptic quality of cooked ground beef. In contrast, the temperature variation in RF cooking was much lower. The temperature difference between the surface and centre of samples heated by RF cooking was only 2.271.4 1C. Thus, RF cooking eliminates the risk of
2.00E+13
2.12. Statistical analysis
1.00E+13
For each cooking method, three replications were conducted. The averages of the three-plate counts for three replications were converted to units of log10 cfu/g. The data were analysed by general linear model (GLM) procedure using SAS (SAS, 2003). Tukey-test was used
0.00E+00 0
0.2
0.4
0.6
0.8
1
OD value (600 nm) Fig. 2. A standard curve of E. coli K12 (ampr) versus optical density (OD) values at 600 nm.
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Table 1 Cooking times and temperatures of RF and water-bath cookings of ground beef samples Cooking method
RF Water-bath
Cooking timea (min)
4.2570.25b 150.3371.53a
Highest temperature (1C) in the ground beef Left side
Center
Right side
74.3470.75b 87.1172.57a
72.3970.11a 72.2870.17a
75.3472.36b 86.8872.08a
Means with different letters in a column are significantly different (Po0:01) a Mean7standard deviation.
overheating food portions. It is more effective than the water-bath for uniform cooking of ground beef.
1.5 1
0.5 0 A
For the shelf-life test, E. coli and coliform survivals in ground beef were examined on the 3 M Petriflims, and E. coli K12 survival in ground beef was tested by LB plates containing 100 mg/ml ampicillin. The results are presented in Fig. 5. After RF or water-bath cooking, no colonies were detected on 3 M Petriflim and LB plates containing ampicillin for 30 days. Therefore, all the lines showed as one line as zero (cooking treatments, log (1)). There was no significant difference (Po0:01) between RF and water-bath cooking. These results show that ground beef samples could be stored at 4 1C for at least 30 days after RF cooking.
B
C
Fig. 3. Population of E. coli/coliform in ground beef samples after RF and water-bath cookings: A ¼ without cooking, B ¼ RF cooking, and C ¼ water-bath cooking.
3M Petrifilm LB plates containing ampicillin
8
6
4
2
0 A
3.4. Shelf-life studies
coliform
2 log10 cfu/g
To validate the efficiency of RF cooking on the inactivation of E. coli, the ground beef samples without inoculation were investigated first, which served as a control. The initial count of E. coli/coliform in the samples was low (o103 cfu/g). Scanga et al. (2000) also found that contaminated ground beef usually contained o103 cfu/g E. coli. After cooking with either RF or water-bath heating, no E. coli/coliform colonies were detected on the 3 M Petrifilms. In addition, no colonies were detected on the LB plates with ampicillin (Fig. 3). Therefore, RF cooking is as effective as the water-bath heating in inactivating E. coli and coliform initially present in the ground beef samples. To compare the rates of E. coli reduction in ground beef cooked by RF and water-bath, a high level of inoculation of E. coli K12 (107 cfu/g) was used. No colonies were detected on 3 M Petrifilms and LB plates with ampicillin after RF or water-bath cooking (Fig. 4). The reductions of E. coli K12 by RF cooking and waterbath heating were both 7 log10 cfu/g and there was no significant difference (Po0:01) between the two methods. These results suggest that both RF and water-bath cooking had a similar degree of efficiency in inactivating E. coli K12.
E.coli
2.5
log10 cfu/g
3.3. Microbial inactivation
3
B
C
Fig. 4. Population of E. coli K12 (ampr) in ground beef after RF and water-bath cookings: A ¼ without cooking, B ¼ RF cooking, and C ¼ water-bath cooking.
The population of E. coli decreased during storage at 4 1C in the ground beef with or without inoculation of E. coli K12 (Fig. 6). E. coli counts decreased by 4.0 log10 cfu/g and 1.93 log10 cfu/g of ground beef, respectively, in the samples with or without inoculation after 30 days of storage at 4 1C. The decrease of E. coli in ground beef during storage was also reported by Ansay et al. (1999). This observation was probably due to microbial competition, as the growth and survival
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117
13 11
Log10 cfu/g
9 7 5 3 1 -1 0
3
7
10 Storage (days)
15
21
30
E. coli on 3M Petriflim (inoculation/no treatment)
Coliform on 3M Petriflim (inoculation/no treatment)
Colonies on LB amp plate (inoculation/no treatment)
E. coli on 3M Petriflim (no inoculation/no treatment)
Coliform on 3M Petriflim (no inoculation/no treatment)
Colonies on LB amp plate (no inoculation/no treatment)
Cooking treatments showing log (1) line
Fig. 5. Counts of E. coli and coliform in ground beef samples during storage at 4 1C.
13
9
11 9
5
log10 cfu/g
log10 cfu/g
7
3
7 5 3
1
1 -1 0
3
7
10 15 Storage (days)
21
30
E. coli on 3M Petriflim (inoculation/no treatment) E. coli on 3M Petriflim (no inoculation/no treatment) Fig. 6. Counts of E. coli on 3 M Petrifilm in the ground beef during storage at 41C.
of E. coli in meat products can be affected by other spoilage organisms. Fresh meat products normally contain psychrotrophic, aerobic, and Gram-negative bacteria (Jay et al., 2003) that can compete with E. coli for available nutrients. In this study, there were spoilage bacteria in the ground beef. The uncooked ground beef samples began to spoil after 3 days of storage. The ground beef gave bad smell. This change indicated that the samples began to spoil, because the first indication of spoilage in the fresh meat is the production of off odours (Adams and Moss, 2000). Fresh beef that undergoes aerobic spoilage at refrigerated temperatures is spoiled by psychrotrophic bacteria (Jay et al., 2003). Moreover, in this study, some colonies were detected on LB agar containing ampicillin, that was plated with the ground beef without inoculation with E. coli K12
-1 0
3
7
10 15 Storage (days)
21
30
colonies on LB amp plate (inoculation/no treatment) colonies on LB amp plate (no inoculation/no treatment) Fig. 7. Counts of the colony on LB plates containing100 mg/ml ampicillin during storage in the ground beef at 4 1C.
(Fig. 7). These colonies were not E. coli as indicated by the Enterotube (BBL Eenterotube II, Becton Dickson Ltd, USA) test. They also grew fast on the plates. These observations suggest the development of spoilage bacterium in the samples although further verification is required. Ansay et al. (1999) reported that the number of E. coli decreased by 1.4 log10 cfu/g when the raw ground beef packed in bags was stored for 4 weeks at 2 1C. Our study indicated that the decrease of E. coli was much higher (4.0 log10 cfu/g with inoculation and 1.93 log10 cfu/g without inoculation) in the uncooked ground beef samples. The difference between our study and Ansay et al. (1999) may be due to the difference in storage
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temperature, E. coli strains, the thawing methods, the plating media, or ground beef samples with different background microflora. The temperature is a critical factor for micro-organism growth and survival (Doyle et al., 2001). Barkocy et al. (2002) suggested that the strain differences could affect the decrease in E. coli. In addition, both thawing methods and plating media can have a noticeable effect on the recovery of E. coli from frozen ground beef (Sage and Ingham, 1998; Clavero and Beuchat, 1995). The background microflora is also important for E. coli survival (Vold et al., 2000). In this study, some ampicillin resistant colonies were detected, from ground beef samples which were not inoculated with E. coli K12 (Fig. 7). The Enterotube test was used to identify the bacteria in this study. Preliminary results suggest that these bacteria were Pseudomonas rather than E. coli. Jay et al. (2003) reported that the main spoilage organism in ground beef was Pseudomonas. Upon verification of the bacterial colonies obtained, our results suggest that Pseudomonas strains may have the ampicillin-resistance from natural source or farming practice. This result was in agreement with the study of Normand et al. (2000). They reported that Pseudomonas had the stable and increasing ampicillin-resistance in veterinary community practice in UK. Furthermore, the growth of ampicillin-resistant Pseudomonas may have resulted in an over estimation of the level of E. coli K12 (ampr) in the inoculated beef samples that were not cooked.
4. Conclusions RF cooking showed significant effects on reducing E. coli, thus maintaining shelf-life and saving cooking time. E. coli was almost eliminated by RF cooking and the shelf-life could be maintained to 30 days. Although both RF and water-bath cookings had a similar effect on reducing E. coli counts and maintaining of shelf-life, RF cooking significantly reduced cooking time and temperature variation, that implies more uniform heating. RF cooking has high potential as a substitute for hot ware bath cooking.
Acknowledgments Authors appreciate the support and suggestions from Mr. Mike Cottrill, Ms. Cheryl Deflice, Dr. Hai Yu and Mr. Xianhua Yin of Food Research Program, Agriculture & Agri-Food Canada during the experiments of this study.
References Adams, M.R., Moss, M.O., 2000. Food Microbiology. The Royal Society of Chemistry, Cambridge, UK, pp. 121–160. Ansay, S.E., Darling, K.A., Kaspar, C.W., 1999. Survival of E. coli O157:H7 in ground-beef patties during storage at 2, 2, 15 and then 2 1C, and 20 1C. J. Food Prot. 62, 1243–1247. Ayres, J.C., 1995. Microbiological implications in the handing, slaughtering, and dressing of meat animals. Adv. Food Res. 6, 109–161. Barkocy, Gallagher, G.A., Dong, H.K., Koohmaraie, M., 2002. Fate of field-isolated E. coli O157 in ground beef at different storage temperatures. J. Food Prot. 65, 1106–1109. Clavero, M.R.S., Beuchat, L.R., 1995. Suitability of selective plating media for recovering heat- or freeze-stress E. coli O157:H7 from tryptic soy broth and ground beef. Appl. Environ. Microbiol. 61, 3268–3273. Doyle, M., 1997. E. coli O157:H7. The Nemesis of Cattle Industry, December 3–4, pp. 42–45. Doyle, M.P., 1991. E. coli O157: H7 and its significance in foods. Int. J. Food Microbiol. 12, 289–302. Doyle, M., Zhao, T., Meng, J., Zhao, S., 2001. E. coli O157:H7 in Food. In: Doyle, M.P., Beuchat, L.R., Montville, T.J. (Eds.), Microbiology: Fundamentals and Frontiers. ASM Press, Washington, DC, pp. 171–192. Houben, J., Schoenmakers, L., vanPutten, E., vanRoon, P., Krol, B., 1991. Radio-frequency pasteurization of sausage emulsions as a continuous process. J. Microwave Power Electromagn. Energy 26, 202–205. Jay, J.M., Vilai, J.P., Hughes, M.E., 2003. Profile and activity of the bacterial biota of ground beef held from freshness to spoilage at 5–7 1C. Int. J. Food Microbiol. 81, 105–111. Laycock, L., Piyasena, P., Mittal, G.S., 2003. Radio frequency cooking of ground, comminuted and muscle meat products. Meat Sci. 65, 959–965. Normand, E.H., Gibson, N.R., Reid, S.W.J., Carmichael, S., Taylor, D.J., 2000. Antimicrobial-resistance trends in bacterial isolates from companion-animal community practice in the UK. Prev. Veter. Med. 46, 267–278. Orsat, V., Gariepy, Y., Raghavan, G.S.V., Lyew, D., 2001. Radiofrequency treatment for ready-to-eat fresh carrots. Food Res. Int. 34, 527–536. Piyasena, P., Dussault, C., 2003. Continuous heating a model viscous solution in a 1.5 kW radio-frequency heater: influence of active current, flow rate and salt content on temperature rise. J. Agric. Bioresource Eng. 45 (3), 27–34. Piyasena, P., Dussault, C., Koutchma, T., Ramaswamy, H.S., Awuah, G.B., 2003. Radio frequency heating of foods: principles, applications and related properties—a review. Crit. Rev. Food Sci. Nutr. 43, 587–606. Rowley, A.T., 2001. Radio frequency heating. In: Richardson, P. (Ed.), Thermal Technologies in Food Processing. Woodhead Publishing Ltd., Cambridge, UK, pp. 163–177. Sage, J.R., Ingham, S.C., 1998. Survival of E. coli O157:H7 after freezing and thawing in ground beef patties. J. Food Prot. 61, 1181–1183. SAS, 2003. Statistical Analysis System User Guide, Version 8.1. SAS Institute, Cary, NC. Scanga, J.A., Grona, A.D., Belk, K.E., Sofos, J.N., Bellinger, G.R., Smith, G.C., 2000. Microbiological contamination of raw beef trimmings and ground beef. Meat Sci. 56, 145–152. Strayfield, 1993. User Manual: 1.5 kW Radio Frequency Tube Heater. Strayfield, Reading, UK. Vold, L., Holck, A., Wasteson, Y., Nissen, H., 2000. High levels of background flora inhibits growth of E. coli O157:H7 in ground beef. Int. J. Food Microbiol. 56, 219–225.
Efficacy of radio frequency cooking in the reduction of Escherichia coli and shelf stability of ground beef Q. Guoa, P. Piyasenab, G.S. Mittala,, W. Sib, J. Gongb a
School of Engineering, University of Guelph, Guelph, Ont., Canada N1G 2W1 Food Research Program, Agriculture & Agri-Food Canada, Guelph, Ont., Canada N1G 2W1
b
Received 2 December 2004; received in revised form 21 February 2005; accepted 21 February 2005
Abstract The effectiveness of radio frequency (RF) cooking on the inactivation of Escherichia coli in ground beef and its effect on the shelf stability of ground beef were investigated with a comparison to hot water-bath cooking. E. coli K12 was used as a target bacterium instead of E. coli O157:H7. The ground beef samples inoculated with E. coli K12 (ampr) were heated until the centre temperature of each sample reached 72 1C. These samples were then stored at 4 1C for up to 30 days. The enumeration of E. coli K12, background E. coli and coliform counts in ground beef samples was carried out for shelf-life study. Although both methods significantly reduced E. coli K12 (ampr), E. coli and coliform counts and extended the shelf-life, RF cooking had a shorter cooking time, and more uniform heating. Thus, RF cooking of meat has a high potential as a substitute for the hot water-bath cooking. r 2005 Elsevier Ltd. All rights reserved. Keywords: E. coli; Ground beef; Meat cooking/heating; Radio frequency; Shelf-life
1. Introduction Escherichia coli O157:H7 has been identified as a major foodborne pathogen. It is responsible for haemorrhagic colitis, haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura (Doyle, 1997). The bacterium was first recognized in 1982 during an investigation of two outbreaks of bloody diarrhoea in Oregon and Michigan (Doyle et al., 2001). Since then, it has been implicated in many other outbreaks. The Center for Disease Control and Prevention has estimated that foodborne diseases caused by E. coli O157:H7 account for 62458 cases of illness, 1843 hospitalizations and 52 deaths in the US each year. Most of the cases were caused by consumption of inadequately cooked contaminated ground beef (Doyle, 1991). The most notable outbreak of 1993 was caused by Corresponding author. Tel. +1 519 824 4120x52431; fax: +1 519 836 0227. E-mail address: [email protected] (G.S. Mittal).
0740-0020/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2005.02.004
undercooked hamburger containing E. coli O157:H7 and affected over 500 people and ultimately led to the death of 4 children (Ayres, 1995). Adequate cooking is the most effective means to eliminate E. coli O157:H7 from meat products and to assure their microbiological safety. Many conventional methods of cooking, including hot air or water, steam, frying with fat or oil, and radiant heating, are applied to cook ground beef. However, such food heating methods require that food is heated externally through conduction, convection or radiation. Because of these heat transfer modes, the conventional cooking methods need longer cooking time and decrease heating uniformity to ensure that the centre is cooked while the surface may overcook (Laycock et al., 2003). Different from conventional cooking methods, radio frequency (RF) cooking is an innovative heating technique that can save cooking time, and heat food uniformly (Rowley, 2001). RF cooking is a technique based on electro-technologies, such as ohmic heating, or microwave dielectric heating (Piyasena et al., 2003).
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Unlike other heat transfer modes, RF cooking heats foods directly through energy conversion, from electrical energy to heat (Rowley, 2001). The energy conversion occurs within the food itself, and heat is absorbed directly by the food. Therefore, RF cooking achieves high-energy efficiency and uniform cooking (Orsat et al., 2001). The application of RF cooking in the food industry can be traced back to the 1940s. RF cooking was initially used to cook meat, bake bread, and dehydrate or blanch vegetables. Later, RF cooking was used to thaw frozen products in the 1960s and to dry cookies in the 1980s. Recently, RF cooking has been evaluated for sterilization and pasteurization of meat products (Piyasena et al., 2003). RF cooking can be used to reduce high levels of microbial contamination and improve food quality. The effectiveness of RF cooking on the reduction of microbial contamination of fresh carrots (Orsat et al., 2001) and sausage (Houben et al., 1991) has been investigated. Its effect on food quality has also been studied on ground beef (Laycock et al., 2003). They found that RF cooking led to shorter cooking time, lower juice losses, and acceptable colour and texture. However, little research has been reported in the reduction of microbial contamination of ground beef by RF cooking. Furthermore, there is limited information on the shelf-life of RF cooked products. Thus, the objective of this study was to investigate the effectiveness of RF cooking on the inactivation of E. coli and its effect on the shelf stability of cooked ground beef. The non-pathogenic E. coli K12 strain, possessing the ampicillin resistance, was used as a target bacterium, as RF cooking may not have selectivity against bacterial strains within a species. In addition, the use of E. coli K12 requires less stringent controlled working conditions as compared to the use of E. coli O157:H7 which requires a laboratory licensed for use of human level 2 pathogens.
2. Materials and methods 2.1. Equipment The RF equipment used for this study was similar to the one used by Laycock et al. (2003). A RF heater of 1.5 kW at a frequency of 27.12 MHz (Strayfield Ltd, Reading, UK) was used. The heater included two parts: an RF generator and an applicator circuit. The applicator contained two electrodes, tuning capacitor plates, an inductance coil and an applicator cylinder. The food samples in the cylinder can be dielectrically heated between the two electrodes. The RF power was adjusted by changing the distance between the tuning plates of the capacitor. The RF power increased as the
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distance decreased. This was accomplished by manually adjusting a gear mechanism outside the RF oven. An active current of 425 mA for ground beef was maintained by using the gear mechanism. The input or incident power was calculated from the actual current and the high voltage of the RF unit as explained by Piyasena and Dussault (2003). The actual current is the difference between the active current and the standing current of 175 mA (Strayfield, 1993). The high voltage (V) across the electrodes for this RF unit has been estimated to be 4600 V (Strayfield, 1993). The incident power for this unit at 425 mA was 1150 W. Power absorbed (Pw) by the product during continuous steady flow could also be calculated as explained by Strayfield (1993). The power loss is the different between incident power and Pw. 2.2. Ground beef The ground beef used for the experiments was obtained from a local meat processor and kept frozen (1870.2 1C) before use. The ground beef samples were homogeneous in the components and appearance to ensure uniform initial quality. Prior to inoculation with the strain of E. coli K12 (ampr), the meat was thawed at 470.5 1C in a refrigerator over a 24-h period. All raw samples were shaped following the container’s dimensions. The cylinder was made of Polytetrafluoroethylene (PFTE), which is RF inert. The cylinder (ID ¼ 0.0952 m, OD ¼ 0.121 m, height from inside ¼ 0.203 m, height from outside ¼ 0.215 m), with the product inside, was set between two electrodes (Laycock et al., 2003). 2.3. Micro-organism A non-pathogenic E. coli K12 strain W3110 (ATCC27325) was obtained from the bacterial collection of Food Research Program, Agriculture & AgricFood Canada. It was stored at 70 1C in Trypticase Soy Broth (TSB) containing 15% (v/v) glycerol. To facilitate detection and enumeration of the target bacterium, the E. coli K12 strain was introduced with the ampicillin resistance (ampr), which was subsequently used throughout the present study. The organism E. coli K12 (ampr) was subcultured periodically to maintain viability. 2.4. Experimental procedure To evaluate the influence of RF cooking on the inactivation of E. coli in ground beef, the experiment was conducted according to the following procedures. 2.5. Transformation of E. coli K12 Plasmid pUC19 (Fermentas Life Science Ltd., Hanover, USA) carrying the gene of ampr was introduced
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into E. coli K12 by high-voltage electroporation. Briefly, 2.5 ml of log-phase E. coli K12 culture with the optical density at 600 nm (OD600) of 0.6 was harvested by centrifugation at 4200 rpm (Centrifuge, model RC5C, Sorvall Instruments, Asheville, USA) for 20 min at 4 1C. The bacterial pellet was washed twice in 500 ml of icecold distilled water. After the final wash, the pellet was resuspended in 2 ml of 10% glycerol, and 40 ml of the cell suspension was then mixed with 2 mg of pUC19 plasmid DNA for electroporation. The sample was subjected to a 2.5 kV, 400 O, 25 mF electric pulse for 3 s in a 0.2 cm cuvette using a Gene Pulser and Pulse Controller apparatus (Bio-Rad, Hercules, USA). One milliliter of SOC medium (2% Bacto tryptone, 0.5% Bacto yeast extract, 0.06% NaCl, 0.05% KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM Glucose) was immediately added and mixed. The cells were transferred to an Eppendorf (culture) tube and incubated for 1 h at 37 1C in a shaking incubator (Orbital Shaker, Model#420, Thermo Forma, Westmont, USA). After incubation, the cells were plated on Luria–Bertani (LB) [1% (w/v) NaCl, 1% (w/v) tryptone, 0.5% (w/v) yeast extract] agar containing 100 mg/ml ampicillin, followed by incubation at 37 1C for 16 h. Colonies grown on the LB plates were the transformants, E. coli K12 (ampr), with the ampicillin resistance. The introduced ampr gene was shown to be stable during subcultures and storage of the transformant. 2.6. Determination of the relationship between the optical density at 600 nm (OD600) and the counts of E. coli K12 E. coli K12 was subcultured in LB broth at 37 1C. An overnight subculture was used to determine the relationship between the OD600 values and the number of colony forming units (cfu) of the bacterium. The OD600 values were measured using a spectrophotometer (model Helios Alpha, Spectronic Instruments, Garforth, UK). To determine the counts of E. coli K12, the cell suspensions were diluted serially in twofold to obtain the OD600 value between 0.1 and 1, and 0.1 ml of the diluted cell suspension was plated on to LB plates containing 100 mg/ml ampicillin. The number of cfu was counted and expressed as cfu/ ml, and was plotted against the OD600 values. These experiments were repeated three times to ensure reproducibility. 2.7. Preparation of test cultures The cultures were prepared by inoculation of E. coli K12 in LB broth containing 100 mg/ml ampicillin and incubation at 37 1C for 16 h. The cultures were subsequently used for inoculation of ground beef.
2.8. Inoculation of ground beef A known amount (around 1 kg) of ground beef was transferred to a sterile bowl and inoculated with 200 ml of E. coli K12 cell suspension to yield approximately 107 cfu/g of sample. The inoculated ground beef was mixed for 2 min by gloved hands to ensure even distribution of the micro-organism. Ground beef inoculated with E. coli K12 but not cooked, was used as a control. Ground beef, neither inoculated with E. coli K12 nor cooked, served as a blank control. Cooked ground beef sample was a negative control. The final concentration of the micro-organism in the samples was later confirmed by enumeration on agar. 2.9. Microbial enumeration To count the number of surviving bacterial cells per gram, sterile 0.1% peptone-water (PW) was added to each meat sample to obtain 1:10 (w/v) slurry. The samples were homogenized for 2 min by a stomacher (Lab system, Model#400, Seward Ltd., Norfolk, UK) at a high speed. Decimal serial dilutions were prepared in 0.1% PW and 0.1 ml of appropriate diluted samples were plated onto LB agar plates containing ampicillin (100 mg/ml) and 1 ml of appropriate diluted samples were plated on to 3 M E. coli/coliform Petrifilm (3 M Microbiology Products, London, Canada). When there was a need, the undiluted sample was also plated. The LB plates were incubated at 37 1C for 16 h before cfu counting. The colonies on the 3 M Petrifilm were determined after 48 h of incubation at 37 1C. For each sampling point, three plates were used to calculate the average cfu/g of ground beef. 2.10. Cooking and temperature measurement The methods of cooking and temperature measurement are similar to those used by Laycock et al. (2003). Three fiber-optic temperature probes (FOT-L, Fiso Technologies, Quebec, Canada) and signal processor (model #UMI-4, Fiso Technologies, Quebec, Canada) were used to measure the temperatures during RF cooking. One probe recorded the centre temperature of the samples (75 mm from the top surface), the second measured the temperature 10–15 mm from the side (75 mm from the top surface), and the third measured the temperature 10–15 mm from the other side (15 mm from the top surface) (Fig. 1). During RF cooking, the temperature was manually recorded every 15 s until the centre temperature of the samples reached 72 1C, at which point the RF generator was deactivated. After cooking, the samples were stored separately at 470.5 1C in a refrigerator for microbial tests. Samples of same mass were also cooked in a water-bath (model#1417, VWR International, Sheldon
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1.5cm
4.76cm
to determine significant differences (Po0:01) among means.
1.5cm
1.5cm
Thermal probes
3. Results and discussion
7.5cm
3.1. Relationship between the OD600 values and E. coli K12 counts 14.96cm
Ground beef
115
Container
The relationship between the OD600 values and E. coli K12 counts were determined using linear regression (Fig. 2). The OD600 values from 0.01 to 0.1 exhibited a linear relationship with the E. coli K12 counts. 3.2. Comparison of cooking methods
Fig. 1. The arrangement of thermal probes in meat samples during RF cooking.
Manufacturing, Cornelius, OR, USA) for comparative purposes. The dimensions of the samples were 9.52 cm in diameter and 14.96 cm in height. To keep their shape, they were packed in a sheet of aluminium foil. The samples were then put in sterilized stomached bags in the water-bath. The water temperature started at 65 1C and was gradually (0.2 1C/min) increased to 90 1C, until the centre temperature of the samples reached 72 1C. Three thermocouples (Omega, Laval, Canada; model HH23, microprocessor thermometer, type T thermocouple) were used to record the temperature at every 2 min. One temperature probe was inserted in the product centre. 2.11. Shelf-life study RF or water-bath cooked ground beef samples (25 g) were aseptically weighed and packed in stomacher bags and stored at 470.5 1C in a refrigerator for up to 4 weeks to determine shelf stability. The number of E. coli K12 (ampr), background E. coli and coliform in the samples were enumerated. The microbial counts were recorded after refrigerated storage for 0 (baseline), 3, 7, 10, 15, 21, 30 days at 4 1C.
4.00E+13 y = 4E+13x+ 1E+12 R2 = 0.9877
3.00E+13 cfu/ml
9.52cm
The average cooking time was from 4.25 to 150.33 min for the central temperature of samples to reach 72 1C, depending on cooking methods (Table 1). There was a significant difference (Po0:01) among cooking methods with respect to the cooking times. The time for RF cooking (4.2570.25 min) was much shorter than that of the water-bath (150.3371.53 min). The results suggested that RF cooking can save cooking time and thus provide high-energy efficiency. When the temperature data at three locations in the ground beef were analysed (Table 1), there was a significant difference (Po0:01) between RF cooking and water-bath cooking. The wider temperature variation within samples was observed for water-bath cooking compared to RF cooking. The difference between the surface and centre of samples was 12.077.8 1C in waterbath cooking, indicating overcooking of outer section of the samples. This might have affected the organoleptic quality of cooked ground beef. In contrast, the temperature variation in RF cooking was much lower. The temperature difference between the surface and centre of samples heated by RF cooking was only 2.271.4 1C. Thus, RF cooking eliminates the risk of
2.00E+13
2.12. Statistical analysis
1.00E+13
For each cooking method, three replications were conducted. The averages of the three-plate counts for three replications were converted to units of log10 cfu/g. The data were analysed by general linear model (GLM) procedure using SAS (SAS, 2003). Tukey-test was used
0.00E+00 0
0.2
0.4
0.6
0.8
1
OD value (600 nm) Fig. 2. A standard curve of E. coli K12 (ampr) versus optical density (OD) values at 600 nm.
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Table 1 Cooking times and temperatures of RF and water-bath cookings of ground beef samples Cooking method
RF Water-bath
Cooking timea (min)
4.2570.25b 150.3371.53a
Highest temperature (1C) in the ground beef Left side
Center
Right side
74.3470.75b 87.1172.57a
72.3970.11a 72.2870.17a
75.3472.36b 86.8872.08a
Means with different letters in a column are significantly different (Po0:01) a Mean7standard deviation.
overheating food portions. It is more effective than the water-bath for uniform cooking of ground beef.
1.5 1
0.5 0 A
For the shelf-life test, E. coli and coliform survivals in ground beef were examined on the 3 M Petriflims, and E. coli K12 survival in ground beef was tested by LB plates containing 100 mg/ml ampicillin. The results are presented in Fig. 5. After RF or water-bath cooking, no colonies were detected on 3 M Petriflim and LB plates containing ampicillin for 30 days. Therefore, all the lines showed as one line as zero (cooking treatments, log (1)). There was no significant difference (Po0:01) between RF and water-bath cooking. These results show that ground beef samples could be stored at 4 1C for at least 30 days after RF cooking.
B
C
Fig. 3. Population of E. coli/coliform in ground beef samples after RF and water-bath cookings: A ¼ without cooking, B ¼ RF cooking, and C ¼ water-bath cooking.
3M Petrifilm LB plates containing ampicillin
8
6
4
2
0 A
3.4. Shelf-life studies
coliform
2 log10 cfu/g
To validate the efficiency of RF cooking on the inactivation of E. coli, the ground beef samples without inoculation were investigated first, which served as a control. The initial count of E. coli/coliform in the samples was low (o103 cfu/g). Scanga et al. (2000) also found that contaminated ground beef usually contained o103 cfu/g E. coli. After cooking with either RF or water-bath heating, no E. coli/coliform colonies were detected on the 3 M Petrifilms. In addition, no colonies were detected on the LB plates with ampicillin (Fig. 3). Therefore, RF cooking is as effective as the water-bath heating in inactivating E. coli and coliform initially present in the ground beef samples. To compare the rates of E. coli reduction in ground beef cooked by RF and water-bath, a high level of inoculation of E. coli K12 (107 cfu/g) was used. No colonies were detected on 3 M Petrifilms and LB plates with ampicillin after RF or water-bath cooking (Fig. 4). The reductions of E. coli K12 by RF cooking and waterbath heating were both 7 log10 cfu/g and there was no significant difference (Po0:01) between the two methods. These results suggest that both RF and water-bath cooking had a similar degree of efficiency in inactivating E. coli K12.
E.coli
2.5
log10 cfu/g
3.3. Microbial inactivation
3
B
C
Fig. 4. Population of E. coli K12 (ampr) in ground beef after RF and water-bath cookings: A ¼ without cooking, B ¼ RF cooking, and C ¼ water-bath cooking.
The population of E. coli decreased during storage at 4 1C in the ground beef with or without inoculation of E. coli K12 (Fig. 6). E. coli counts decreased by 4.0 log10 cfu/g and 1.93 log10 cfu/g of ground beef, respectively, in the samples with or without inoculation after 30 days of storage at 4 1C. The decrease of E. coli in ground beef during storage was also reported by Ansay et al. (1999). This observation was probably due to microbial competition, as the growth and survival
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117
13 11
Log10 cfu/g
9 7 5 3 1 -1 0
3
7
10 Storage (days)
15
21
30
E. coli on 3M Petriflim (inoculation/no treatment)
Coliform on 3M Petriflim (inoculation/no treatment)
Colonies on LB amp plate (inoculation/no treatment)
E. coli on 3M Petriflim (no inoculation/no treatment)
Coliform on 3M Petriflim (no inoculation/no treatment)
Colonies on LB amp plate (no inoculation/no treatment)
Cooking treatments showing log (1) line
Fig. 5. Counts of E. coli and coliform in ground beef samples during storage at 4 1C.
13
9
11 9
5
log10 cfu/g
log10 cfu/g
7
3
7 5 3
1
1 -1 0
3
7
10 15 Storage (days)
21
30
E. coli on 3M Petriflim (inoculation/no treatment) E. coli on 3M Petriflim (no inoculation/no treatment) Fig. 6. Counts of E. coli on 3 M Petrifilm in the ground beef during storage at 41C.
of E. coli in meat products can be affected by other spoilage organisms. Fresh meat products normally contain psychrotrophic, aerobic, and Gram-negative bacteria (Jay et al., 2003) that can compete with E. coli for available nutrients. In this study, there were spoilage bacteria in the ground beef. The uncooked ground beef samples began to spoil after 3 days of storage. The ground beef gave bad smell. This change indicated that the samples began to spoil, because the first indication of spoilage in the fresh meat is the production of off odours (Adams and Moss, 2000). Fresh beef that undergoes aerobic spoilage at refrigerated temperatures is spoiled by psychrotrophic bacteria (Jay et al., 2003). Moreover, in this study, some colonies were detected on LB agar containing ampicillin, that was plated with the ground beef without inoculation with E. coli K12
-1 0
3
7
10 15 Storage (days)
21
30
colonies on LB amp plate (inoculation/no treatment) colonies on LB amp plate (no inoculation/no treatment) Fig. 7. Counts of the colony on LB plates containing100 mg/ml ampicillin during storage in the ground beef at 4 1C.
(Fig. 7). These colonies were not E. coli as indicated by the Enterotube (BBL Eenterotube II, Becton Dickson Ltd, USA) test. They also grew fast on the plates. These observations suggest the development of spoilage bacterium in the samples although further verification is required. Ansay et al. (1999) reported that the number of E. coli decreased by 1.4 log10 cfu/g when the raw ground beef packed in bags was stored for 4 weeks at 2 1C. Our study indicated that the decrease of E. coli was much higher (4.0 log10 cfu/g with inoculation and 1.93 log10 cfu/g without inoculation) in the uncooked ground beef samples. The difference between our study and Ansay et al. (1999) may be due to the difference in storage
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temperature, E. coli strains, the thawing methods, the plating media, or ground beef samples with different background microflora. The temperature is a critical factor for micro-organism growth and survival (Doyle et al., 2001). Barkocy et al. (2002) suggested that the strain differences could affect the decrease in E. coli. In addition, both thawing methods and plating media can have a noticeable effect on the recovery of E. coli from frozen ground beef (Sage and Ingham, 1998; Clavero and Beuchat, 1995). The background microflora is also important for E. coli survival (Vold et al., 2000). In this study, some ampicillin resistant colonies were detected, from ground beef samples which were not inoculated with E. coli K12 (Fig. 7). The Enterotube test was used to identify the bacteria in this study. Preliminary results suggest that these bacteria were Pseudomonas rather than E. coli. Jay et al. (2003) reported that the main spoilage organism in ground beef was Pseudomonas. Upon verification of the bacterial colonies obtained, our results suggest that Pseudomonas strains may have the ampicillin-resistance from natural source or farming practice. This result was in agreement with the study of Normand et al. (2000). They reported that Pseudomonas had the stable and increasing ampicillin-resistance in veterinary community practice in UK. Furthermore, the growth of ampicillin-resistant Pseudomonas may have resulted in an over estimation of the level of E. coli K12 (ampr) in the inoculated beef samples that were not cooked.
4. Conclusions RF cooking showed significant effects on reducing E. coli, thus maintaining shelf-life and saving cooking time. E. coli was almost eliminated by RF cooking and the shelf-life could be maintained to 30 days. Although both RF and water-bath cookings had a similar effect on reducing E. coli counts and maintaining of shelf-life, RF cooking significantly reduced cooking time and temperature variation, that implies more uniform heating. RF cooking has high potential as a substitute for hot ware bath cooking.
Acknowledgments Authors appreciate the support and suggestions from Mr. Mike Cottrill, Ms. Cheryl Deflice, Dr. Hai Yu and Mr. Xianhua Yin of Food Research Program, Agriculture & Agri-Food Canada during the experiments of this study.
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