Effect of short-time microwave heating on survival of Escherichia coli O 157 in beef hamburgers

Survival of three Escherichia coli strains O 157: B 032, LMG 8223, IVK 805 from the collection of the Department of Veterinary Protection of Public Health, Faculty of Veterinary Medicine, University of Gent, Belgium, in breaded and unbreaded beef hamburgers (n = 144), subjected to short microwave heating was determined. 50 g hamburger samples were contaminated by bacterial suspension injections (the initial contamination level was 106 cfu/g of product). Microwave heating for 30, 60, 90, 120 and 150 s was done using a microwave cooker (Dialog cook, Moulinex, at 480 and 760 W). 30 s heating at 480 W caused test bacteria cells partial reduction that was more pronounced in breaded hamburgers. In the case of E. coli O 157: B 032, the reduction was lower than for the other two strains. At 480 W, none of the E. coli O 157 test strains survived 60 s of heating that corresponded to the hamburgers’ temperature ranging from 75.2 to 89.1°C. Increasing the power to 760 W, decreased the bacteria inactivation time to 30 s while the hamburgers’ temperature ranged from 71.9 to 78.8°C. Indifferent of the cooker power for each of the heating times, breaded hamburgers achieved higher temperatures than the unbreaded ones.


INTRODUCTION
Escherichia coli O 157 (S TE C) that produced S higa-toxin was detected for the first time in 1982 as the etiologic factor of haemorrhagic colitis (HC) in human patients (CDC, 1982).In 1983, Karmali et al. (1983) demonstrat ed that E. coli O 157:H7 and ot her S TE C serologic groups were responsible for the occurrence of t he haemolytic uremic syndrome (HUS).
Epidemiological studies carried out to investigate the above and similar cases of infection with S higa -toxin producing strains of E. coli showed that the food products of animal origin (especially beef and milk), raw fruits and vegetables contaminated with animal excrements, fruit juices and wat er were the main sources of infection (Armstrong et al., 1996;Paton and Paton, 1998).Also, direct animal-human and human-human contacts play an *Corresponding author.E-mail: jan.uradzinski@uwm.edu.pl.Tel: 0048 (89) 523-3995.Fax: 0048 (89) 523-3331.important role in this epidemiological chain.Over 100 STE C serotypes, isolated from patients with HC and/ or HUS symptoms, have been described so far.E. coli O 157: H7 seem to be the most pat hogenic among them, as it causes both sporadic and epidemiological infections, leading even to lethal outcomes (Johnson et al., 1996).
The best-known cases of infections with E. coli O 157 in humans are the epidemic in Japan in 1996 (8576 infected children, 106 HUS cases, three lethal outcomes), and the epidemic in Scotland, also in 1996 (501 cases of infection, 20 lethal outcomes) (Wat anabe et al., 1996).Vegetables (Japan) and meat (Scotland) were t he sources of infection in those epidemics.Those and other similar cases indicate that E. coli O 157 is one of the most dangerous infectious agents in food poisonings in humans.
Cons equently, E. coli O 157 presence in products of animal origin (particularly beef products) resulting from their contamination may pose a serious consumer health hazard.For thos e reasons the knowledge on resistance of thos e bacteria to the environment al and technical factors and considering the wide range of products of animal origin that might be contaminated with E. coli O 157, plays an import ant role.50 g samples of breaded and unbreaded beef hambur gers w ere contaminated w ith the test strains.The contamination w as conducted immediately prior to commencement of heating the hamburgers in the microw ave cooker by injecting them w ith freshly prepared suspension of the specific bacter ial strain (18 to 24 h culturing in a TSB -Tryptone Soya Broth by Oxoid), prepared according to the Mc Farland scale so that the initial contamination w ith the E.coli O 157 bacter ia w as at the level of 10 6 cfu/g of the product heated.The hamburgers temperature after contamination and before heating w as at the level of ca.6°C.

MATERIALS AND M ET HODS
Hamburgers prepared in that w ay w ere microw ave heated in the household microw ave cooker ( Dialog cook, Moulinex) apply ing tw o different pow er settings (480 and 760 W) and different heating times (30, 60, 90, 120 and 150 s).At the end of the heating process, the hamburgers w ere left in the closed cooker for another 30 s to equalise the temperature.Next, they w ere taken out recording at the same time their temperatur e (average temperature value form six different measur ement points).During prepar ation of the hamburgers for quantitative microbiological tests, they w ere homogenised in a stomacher (Lab-blender 400, England) for 60 s.After preparation of consecutive dec imal dilutions the culturing on the Eosin Methylene Blue Agar ( Modified) ( mEMB by Oxoid) w as conducted.Follow ing 48 h of incubation at 37°C, the E. coli O 157 colonies w ere counted on tw o parallel plates and the average values per 1 g of product w ere deter mined.At the same time, the test bacteria counts in the hamburgers that w ere not subjected to heating representing the controls w ere taken.Tw o series of tests were conducted for each bacterial strain apply ing all the specified variants of deter mination (n = 144).Statistical analysis w as conducted by means of the univar iate repeated-measures A NOVA test, t-Student and Mann-Whitney U Test, using the Microsoft Excel 2010 and Statistica 9 PL softw are package.The statistical significance level of α=0,05 w as assumed.

RESULTS
The study results are presented in Figures 1 to 4. Figure 1 presents the survival rates of three E. coli O 157 strains in unbreaded and breaded hamburgers heated for different times in the microwave cooker at 480 W. The figure indicat es that heating bot h types of hamburgers for 30 s caused reduction in the numbers of test bacteria cells that was more pronounced in breaded hamburgers, however the recorded differences were insignificant (univariate repeated-measures ANOVA test, p=0,131).In the case of E. coli O 157: B 032 strain, the reduction level was slightly lower than in the cas e of the other strains.Nevertheless, no statistically significant differences between the s urvival of individual strains of E. coli 0157 in both types of heated hamburgers in the 480 W microwave oven was found (Mann-Whitney U Test, p > 0.05).Extending the heating to 60 s at the same power setting (480 W), caused total destruction of all the t est E. coli O 157 strains.Application of the higher c ooker power (760 W), accelerated the effect of total inactivation of the test bacteria, which was ac hieved after 30 s heating already (Figure 2).That effect was achieved for all three test E. coli strains and for both hamburger types.Statistical analysis of results confirmed the existence of statistically significant differences in t he survival of E. coli 0157 strains tested in the experiment regarding t he power of the oven (480 and 760 W respectively) in both breaded and unbreaded hamburgers (univariate repeated-measures ANOVA test, p=0.000).
The presented reduction level res ults (Figures 1 to 2), represent average values from the two series of tests ; no significant differences bet ween t he results from t he individual test series were found.
To present the complete image of the results obtained, the time and hamburger's temperature after heating was determined (as the average value of measurements taken at six different points at the time of taking the hamburger out from the cooker).Figures 3 and 4 present the correlation bet ween the microwave radiation influence and changes of temperature taking place in breaded and unbreaded hamburgers depending on the heating power applied.Figure 3 indicates that after heating t he hamburgers in the cooker set at 480 W for 30 s, the temperature of the samples increased to 64.1 °C in unbreaded hamburgers and 72.2 °C in breaded hamburgers; with heating time increas ed to 60, 90, 120 and 150 s, the temperatures increased to 75.2 and 89.1, 90.7 °C and 94.1; 91 and 94.5 °C and finally to 93.1 and 96.2 °C respectively.In the case of heating in the cook er set at 760 W, the temperat ure of unbreaded and breaded hamburgers reached the values of 71.9 and 78.8 °C; 82.5 and 92. 3 °C; 90.2 and 94.4 °C; 92.7 and 95.4 °C and 94.6 and 98.1 °C for t he same heating times respectively (Figure 4).Indifferent of the cooker power (480 and 760 W), for every heating time applied, breaded hamburgers reached higher final temperatures than the unbreaded ones.The recorded differences were insignificant statistically (Mann-Whitney U Test, p = 0. 296 and t-Student test p=342).
In the case of unbreaded hamburgers heated at 760 W for 30 s, the temperature of which was similar to t he temperature of breaded hamburgers heated at 480 W also for 30 s (Figures 3 and 4) deserves attention.Despite similar t emperatures (71.9 and 72.2 °C), t he effect of test bacteria reduction in those products was surprisingly different.Almost identical temperature (ca.72 °C), in one case resulted in total inactivation of the test bacteria (Figure 2), while in the other, it resulted in E. coli O 157 survival at the levels ranging from 4.28 t o 5.92 log cfu/g of the product depending on the strain (Figure 1).That situation was recorded in both test series.
This is also the case with E. coli O 157, which is present in slaughter animals (particularly cattle) and from the products of animal origin, can pose a serious threat for consumer health.The studies concerning t he influence of microwave heating on E. coli O 157 survival indicates that the bacteria inactivation effectiveness is influenced by numerous factors.In own studies conducted, it was shown that the destruction of E. coli O 157 strains depends on the cooker power, hamburger type, temperature obtained and product heating time.Similar conclusions were reached by Quesada et al. (2003), who showed clearly during microwave heating of minced beef (for 60, 90 and 120 s) that the E. coli destruction level depends on the temperature and meat heating time.Jamshidi et al. (2010), investigating t he influence of microwave heating on E. coli O 157:H7 survival on the surface of 200 g beef pieces, showed that the sample surface temperature increase was accompanied by gradual reduction in the test bacteria numbers.Total elimination of the pathogens was achieved after 30 s heating and exceeding t he temperature of 70°C.Czechowicz and Zottola (1996), heating beef rolls contaminated with E. coli O 157 in different types of microwave cook ers, proved that the time necessary for inactivation of those bacteria was determined by cook er power and temperature obtained within the rolls.Korean researchers, Kim and Lee (2002), determined that beef immersion in 2% lactic acid solution for 2 or 4 s and next vacuum closing and microwave heating (after 70 s) was the most effective in the reduction of the E. coli O 157 count on the surface of samples tested.Entirely different conclusions concerning the possible reduction in t he bacterial number under the influence of microwaves were reached by Göksoy et al. (2000).Those researchers, investigating the effects of s hort -time exposure to microwaves on selected bacteria det ermined that heating for 30 s and shorter did not caus e changes in the number of bacteria present on the chicken breast surface and t he number of E. coli and Campylobacter jejuni cells may even increase slightly.
Interesting results were obtained by Huang and Sites (2010), who placed beef samples contaminated with E. coli O 157:H7 (5, 0±0,3 log cfu/ g) in the heating chamber at different heights (0.03 m and 0.07 m), subjecting them next to heating in the high power microwave cooker (2, 45 GHz; 1250 W).They showed that heating t he samples positioned higher (applying the same heating parameters) was more effective in elimination of the pathogens investigated.Gomółka-Pawlicka et al. 365 Woo et al. (2000), in their study on damages to bacteria at the cellular level caused under the influence of microwaves, exposed the suspension of bacteria (E.coli and Bacillus subtilis) in 0.9% NaCl to microwave radiation (in the 600 W cooker).Those observations showed that both the type and extent of damages on the surface of the bacteria depended to a large extent on the type of bacteria; t hose damages were much more extensive in the case of E.coli.On the other hand, Tsuji and Yokoigawa (2011), in their studies related to the influence of microwaves on microorganisms investigated t he important issue of the microwaving influence on t he ability of verotoxin production by E. coli O 157:H7.
The results they achieved indic ate that both heating t he suspension of bacterial c ells at 65°C (60 s; 2.45 GHz, 100 W), and the influenc e of microwaves without a clear thermal effect (temperature increase to 37°C; 2.45 GHz, 0.6 W/ml), caused evident inhibition of verotoxin production ability.Consequently, Ts uji and Yokoigawa came to the conclusion that exposure of E. coli O 157: H7 bacterial cells may result not only in the reduction of their number but it can also limit their verotoxin production capacity.The present study showed higher temperature ranges and more expressed reduction of pathogens in breaded hamburgers.Thus, it is likely that coating batterbeing a kind of "thermoisolator" -influenced bot h higher terminal temperature of heating hamburgers, as well as the slower rate of decrease hamburg ers' temperature after heating.This created less favorable t hermal conditions to the survival of bacteria in the product than in hamburger without batter.The special case recorded in our own studies where it was noticed t hat depending on the cooker power applied (480 or 760 W), 30 s of heating the hamburgers and the temperature achieved as a consequence of ca.72°C, was accompanied by the effect of total inactivation of all the E.coli O 157 strains tested while in t he other case, the survival of those bacteria at the level dangerous to human health was observed which deserves attention.The described case provides evidence that achieving the specific range of temperatures in the product heated is not always equivalent to achievement of the desired effect in reduction in the number or total inactivation of microorganisms in the product.The temperature is then an important but only one of many factors influencing t he survival of microorganisms in the products heated in microwave cookers and as a consequence is correlat ed to their safety to health.

Conclusions
Own studies as well as the studies by the referenc ed authors indicate the need for considering the cook er power and duration of microwave influence, the type, size and shape of the product heat ed and its temperature during microwave heating of food products so that the chances of inactivating the possible pat hogens are maximised.Appropriately high temperature of the product heated by microwaving does not always offer t he guaranty of a significant reduction in the number or total inactivation of pathogens possibly contained in t he product.Both the mec hanisms and the scope of changes taking place within t he bacterial cells, on their surface or in their life functions resulting from the ex posure of t he bacteria to microwaving are not fully known yet.That is why undert aking further studies concerning the widely understood influence of microwaves on microorganisms (particularly the bacteria the presence of which in food has direct influence on food safety) seems still fully justified.

Figure 1 .
Figure 1.Survival of Escherichia coli O 157 after microw ave heating (w attage of microw ave oven -480 Watt), according to bacterial strain, type of hamburgers and heating time.

Figure 2 .
Figure 2. Survival of Escherichia coli O 157 after microw ave heating (w attage of microw ave oven -760 Watt), according to bacter ial strain, type of hamburgers and heating time.

Figure 3 .
Figure 3. Temperature of hamburgers after microw ave heating (w attage of microw ave oven -480 Watt) at different times.

Figure 4 .
Figure 4. Temperature of hamburgers after microw ave heating (w attage of microw ave oven -760 Watt) at different times.
Three strains: E. coli O 157: B 032, E. coli O 157: LMG 8223 and E. coli O 157: IVK 805, isolated from food infection cases in Europe were the test organis ms.