COMPARATIVE STUDY BETWEEN THE EFFECT OF GAMMA IRRADIATION AND OZONE GAS ON THE INCIDENCE OF E. COLI O157:H7 IN BEEF BURGER SOLD IN ASSIUT GOVERNORATE

COMPARATIVE STUDY BETWEEN THE EFFECT OF GAMMA IRRADIATION AND OZONE GAS ON THE INCIDENCE OF E. COLI O₁₅₇:H₇ IN BEEF BURGER SOLD IN ASSIUT GOVERNORATE

Lubna M. EBRAHEEM; Ghada M. Mohamed and MAHMOUD A.M. Ammar

Animal Health Research Institute, Assiut Laboratory

Email: mahmoud2014eg@yahoo.com

INTRODUCTION

Beef burger is a raw food of animal origin which forms a significant portion of the diet in many countries. Consumers expect meat products to be safe for consumption when handled and cooked properly. Beef burger occasionally poses a high risk to the consumer due to their potential for carrying disease causing bacteria. Escherichia coli O₁₅₇:H₇ is one of the bacteria that have been identified as the cause of several food borne outbreaks (food and Nutrition 1999). E. coli bacteria are members of the family Enterobactreriaceae, facultative, anaerobic, Gram-negative short-rods and considered a common inhabitant of the gut of worm-blooded animals, including man. Most strains of E. coli are harmless, however, some strains, such as E. coli O₁₅₇:H₇, can cause severe food-borne disease (WHO, 1996).

E. coli O₁₅₇:H₇ attracted attention not only because food-borne transmission is more common, but also because it can cause life-threatening conditions such as hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (Buchanan and Doyle, 1997). The pathogenicity of E. coli O₁₅₇:H₇ is attributed to the production of Shiga toxins1and 2, previously known as Verocytotoxins because of their toxicity on Vero cells (OÕBrien, 1992). Approximately half the HUS patients require kidney dialysis and their illness may last from several days to many months or years with a mortality rate of 3-5% (Food and Nutrition, 1999). People of all ages can get E. coli O₁₅₇:H₇ gastroenteritis, however, young children and the elderly tend to develop severe symptoms such as HUS (Koutkia et al., 1997 and Stewart et al., 1997).

The reservoir of E. coli O₁₅₇:H₇ appeared to be mainly cattle, which is present in the intestines in approximately 1 percent of healthy cattle. Meat from cattle may become contaminated with this strain of bacteria during the slaughtering (McEvoy et al., 2003) and grinding process of the beef (Le Saux       et al., 1993).

The majority of outbreaks have resulted from the transmission of the organism through the consumption of beef, most commonly, under-cooked contaminated ground beef (especially hamburger) thus, the term “Hamburger Disease” (Le Saux et al., 1993 and Dolye et al., 1997).

Public attention has continued to focus on the detection of pathogenic E. coli O₁₅₇:H₇ in beef burger. Since its introduction in the mid-1980, polymerase chain reaction (PCR) technology has proved to be a valuable method for detection of pathogens in food. In the last few years, several papers have been published reporting the development of PCR for the detection of pathogenic E. coli O₁₅₇:H₇ and to identify the H serogroup and the type of shiga toxin produced by this bacterium (Fratamico et al., 2000).

Due to the increased resistance of bacterial pathogens to commercially used antibiotics, there has been an increasing interest in the development of new types of effective and non-toxic antimicrobial compounds such as food preservation methods.

Among these methods, food irradiation which considered the most versatile treatment, nowadays. Microorganisms can be inactivated by impairment of important molecules or organelles, such as DNA and the cytoplasmic membrane (Diehl, 1995).

Hence, gamma radiation became an important tool to be used by the food industry not only as a method of preserving food but also to improve food safety (Mulder, 1988). Irradiation by gamma rays with specific doses reduces or eliminates pathogenic and spoilage microorganisms as E. coli O₁₅₇:H₇ in food. The process has little effect on the nutritional value and organoleptic qualities of food where it remains almost unchanged, but Irradiation can minimally affect some very sensitive vitamins, such as thiamin. Beef burger can be treated with a two-sided approach, penetrating 1.5 inches (Elaine, 1998).

Consumers cannot recognize irradiated food by sight, smell, taste, or feel. Irradiated foods can be recognized by the presence of the international symbol for irradiation on the packaging along with the words "Treated with Radiation," or "Treated by Irradiation". The overall retained nutritional quality of irradiated food depends on a number of factors, including irradiation dose, temperature, food composition and the presence or absence of oxygen (Pmiipf, 2000).

On the other hand ozone stands out for its antimicrobial properties and its high oxidative compound (Tiwari et al., 2008 and 2010). The biocidal effect of ozone is caused by a combination of its high oxidation potential, and its ability to diffuse through biological cell membranes (Kim et al., 1999). It is able to inactivate bacterial vegetative cells and spores, yeast, molds, and viruses with no waste/toxic products   (Tiwari et al., 2010). Thus it is Generally Recognized As Safe (GRAS) for food applications (Dhillon, 2009 and Tiwari et al., 2010). An additional advantage is the lower cost of ozone equipment (Guzel-Seydim et al., 2004). Ozone has proven to be a very effective in many food processing applications (Joel and George, 2011), including red meat and beef applications against several microorganisms (Akbas and Ozdemir 2006) specifically E. coli O₁₅₇:H₇ (Kim and Yousef 2000).

However, ozone is traditionally used in the aqueous phase for surface sanitation and general disinfection. Ozone gas may be an alternative to aqueous ozone in ground beef processing as an antimicrobial intervention (Rice et al, 1982). Before ozone can be applied successfully in food processing, patterns of microbial inactivation by ozone should be elucidated (Kim and Yousef 2000). Therefore, several factors affect the ozone efficacy including the strain of the microorganism, age of the culture, density of the treated population, and presence of ozone-demanding medium components and method of applying ozone (Khadre, et al., 2001).

As there is little information about the occurrence of E. coli O₁₅₇:H₇ in meat products of ground beef specially beef burger in Egypt, therefore, the purpose of the present investigation was designed to evaluate the following: prevalence of E. coli O₁₅₇:H₇ in beef burger, confirmation of the isolated strains by PCR assay and, determine the gamma radiation dose which reduce the level of this microorganism. Also, evaluate if there is a possibility that ozone gas may be an effective on this microorganism intervention in beef burger as well on the sensory attributes.                                                                    

MATERIALS and METHODS

1. Isolation of E. coli O₁₅₇:H₇ from beef burger:

1.1. Collection of samples:

A total of 125 random samples of beef burger were collected from different supermarkets, and groceries in Assiut Governorate. The samples were obtained in their casing as sold to the consumers and collected in presterilized polyethylene containers. The collected samples were transferred directly to the laboratory in an ice box with a minimum of delay, where they prepared for bacteriological examination.

1.2. Preparation of samples:

At the laboratory, frozen samples were thawed by overnight refrigeration. Each sample was aseptically and carefully freed from its casing.

1.3. Selective enrichment broth: (Tarr et al.,1999):

Twenty-five grams of beef burger was aseptically weighed and placed in sterile stomacher bag containing 225 ml of modified vancomycin-trypticase soy broth (m-VTSB) supplemented with vancomycin (40mg/litre), and stomached at low speed for 2 min. The stomached material was transferred to sterile flask and incubated aerobically at 37°C for overnight.

1.4. Isolation on selective plating media: (De Boer and Heuvelink, 2000)

One hundred µL of incubated broth was pipeted onto dried surface of MacConkey sorbitol agar (MSA) plates and incubated at 37°C for 24h. Colorless (pale) colonies (sorbitol-negative colonies) were picked up and purified for further confirmation.

1.5. Identification of presumptive colonies:

The presumptive colonies were confirmed to be        E. coli following the protocol  described by (Varnam and Evans,1991) using the microscopical examination and the biochemical identification: indole production test, methyl red test, voges-proskauer test, simmon’s citrate test, urease test and triple sugar iron reaction.

1.6. Serological identification of E. coli by latex agglutination test: This was done according to the technique adopted by Krishnan et al., 1997.

1.7. Detection of E. coli O₁₅₇:H₇ by PCR:

Total genomic DNA and PCR amplifications for the three strains (E. coli O₁₅₇ confirmed isolates) was done as described by (Toma et al., 2003). For E. coli O₁₅₇:H₇ specific identification, two primers pairs were used. The primer's sequence, the target, the PCR products size and the references, are listed in table (1).

DNA amplification reaction:

The bacterial genomic DNA samples were amplified by PCR in a reaction mixture(25µl) containing  13.25 sterile distilled H2O, 2.5ml 10 x buffer, 0.63ml 10mMNTPs, 1ml 25Mm Mgcl2, 1.25 µl primer F(20pmol/ml), 1.25 µL primer R(20pmol/ml) and fill up to 25 µl PCR grade water. The PCR protocol consisted of the following steps: An initial denaturation (2 min at 95°C) for 30 cycles, primer denaturation (1 min at 95°C) 1 cycle, primer annealing (1 min at 57°C), primer extension (2 min at 72°C) and a final elongation (5 min at 72°C). The PCR products were electrophoresed in 2.5% agarose gel and stained with ethidium bromide.

Table 1: Oligonucleotide sequences used for identification of E. coli O₁₅₇:H₇ by PCR:

2. Effect of gamma radiation or ozone gas on E. coli O₁₅₇:H₇inoculated in beef burger and their effect on sensory quality of beef burger:

Preparation of bacterial inoculum:

The confirmed isolate of E. coli O₁₅₇:H₇(a beef burger isolate), was used. The strain was maintained on tryptic soya agar (Biolife). Tryptic soya broth (Difico Laboratories) was inoculated from purified separate colony of the strain and incubated at 37 ºC for 24h. After this period, a 100 µ L of incubated broth was pipeted onto dried surfaces of MacConkey Sorbitol Agar (MSA) plates and surface spread using sterile glass rod then incubated at 37°C for 24h. Colorless (pale) colonies (sorbitol-negative colonies) were counted and the CFU/mL was calculated. Serial dilutions of the known population broth was prepared by diluting 1mL of the suspension with 9mL of sterile peptone water to yield a final inoculum approximately 10⁶cfu/mL.

Preparation of samples:

Cardboard packages (15 frozen beef burgers each) were acquired at the retail level at Assuit city. Frozen packages were thawed by overnight refrigeration till the temperature reached 2ºC in the center of each beef burger, as measured with a thermometer (HI98509-1, Romania).

Design of the experiment:

Beef burgers were divided into two groups (group A for gamma irradiation) and (group B for ozone gas treatment). Each group was subdivided into seven sub groups (each 7 burgers) where 3 sub groups was subjected individually each for a different dose while another 3 non injected sub groups were treated by the same doses and used for the sensory evaluation of the samples. The seventh (no injected samples) and the eighth (injected samples) sub groups was transported to the place of treatment but not treated (controls).The prepared samples were transported cooled to the place of irradiation or ozone treatment in an insulated ice box.

Inoculation of samples:

Individual beef burgers were aseptically transferred to Styrofoam trays and 1mL of E. coli O₁₅₇:H₇ suspension (10⁶ cfu/g) was evenly distributed inside and on the surface of each beef burger, using sterile syringe (the injection occur in Assiut). After 15 minutes, each tray was wrapped with PVC (polyvinyl chloride) film and transported immediately to the place of treatment under cool and hygienic measures.

Irradiation of beef burgers:

Two subgroups (inoculated and non inoculated subgroup) of beef burgers were submitted to the each irradiation dose at the National Center For Radiation and Technology (NCRRT) at Nasr City, Cairo, Egypt using India Gamma cell C060t. The ƴ- radiation doses were (2,4 and 6 KGY) at dose rate 2.32 KGY/h. Treated inoculated samples and their control were maintained refrigerated until the beginning of microbiological analyses .To study the effect of irradiation on the sensory quality of beef burger, treated non injected subgroups  together with non inoculated non treated (control) were separately cooked immediately after treatment  and subjected to sensory evaluation. The experiment was repeated 3 times.

Treatment with ozone gas: (Bialk and Demirci, 2007)

This study was performed at EL-Azhar University, Faculty of science. Two subgroups (inoculated and non inoculated subgroup) of beef burgers were submitted to a separate dose. The applied doses were 20, 40 and 70 ppm. The samples were allowed to contact the ozone-containing air for 3 minutes in the container. The whole system was installed in a fume cupboard, and the experiment was completed at room temperature (20°C). After the treatment, the ozone gas was passed through a 2% potassium iodide solution to prevent ozone from being released into the environment. The ozone treatment was performed in a fume hood for safety considerations. The microbiological and sensory analyses were the same as in case of irradiation experiment.

Enumeration of survivors: (Bialk and Demirci, 2007)

For enumeration of E. coli O₁₅₇:H₇in treated and control beef burgers, Twenty-five grams of beef burger was aseptically weighed and placed in sterile stomacher bag containing 225 ml peptone water stomached at low speed for 2 min. Ten –fold of the stomached material was prepared using peptone water diluents. One hundred µl of each dilution was pipeted onto dried surface of MacConkey sorbitol agar (MSA) plates and incubated at 37°C for 24h. Colorless (pale) colonies (sorbitol-negative colonies) counted and the cfu/g beef burger was calculated. Repesentive colonies were picked for confirmation as E. coli using proper biochemical tests and the E. coli antiserum O157 assay. Microbiological data were transformed into log10 cfu/g.

Determination of D10-value and reduction %: (Dickson, 2001)

D10value (the dose required to inactivate 90% of a population) were calculated by the formula: D10 value = dose (kGy)/ (log10 count prior to irradiation - log10 count after irradiation).

Reduction % = (log10 count of control - log10 count of treatment) x100/ log10 count of control.

Cooking of beef burger: (Pourkhalili et al., 2013):

The samples were pan fried in sunflower oil for 20min. The internal temperature during frying was determined as 85°C.

Sensory evaluation of cooked beef burger:

In all sensory tests, the panelists consisted of 5 non-expert members from our laboratory, and scores were obtained by rating the quality attributes using the following scale: 9 – excellent, 8 – very good, 7 – good, 6 – below good/above fair, 5 – fair, 4 – below fair/above poor, 3 – poor, 2 – very poor and 1 – extremely poor. Ratings of 5 and above indicated an acceptable sample, while ratings of less than 5 and more than 3 indicated that the samples were of marginal quality and ratings of 3 and below indicated unacceptable samples (Wierbicki, 1985). Juiciness was defined as the degree to which moisture was released from the sample after seven chews between the molars (Rocha-Garaz and Zayas, 1996).

RESULTS

Table 2: Incidence of E. coli and serologically E. coli O₁₅₇ in examined samples of beef burger

Table 3: Detection of E. coli O₁₅₇:H₇ and non H₇ strains by PCR assay

Table 4: Effect of gamma radiation on the sensory quality of cooked beef burger

Mean with the letter N have non-significant differences

Table 5: Effect of ozone gas on the sensory quality of cooked beef burger

Means with letter N have non-significant differences

Fig. 1: Effect of gamma radiation on E. coli O157: H7 inoculated in beef burger

Fig. 2: Effect of ozone gas on E. coli O157: H7 inoculated in beef burger

     M        1         2         3         4         5

Photograph (1): Agarose gel electrophoresis of PCR amplification products using specific primers of O₁₅₇:H₇ (VTcom-u, Vtcom-d).

Lane M: 518 bp ladder as molecular DNA marker.

Lane 1: Control positive for E. coli O₁₅₇:H₇

Lane 2: Control negative for E. coli O₁₅₇:H₇

Lanes 3 and 4 (1 and 17): -ve E. coli O₁₅₇:H₇

Lane 5 (51): +ve E. coli O₁₅₇:H₇

DISCUSSION

Due to the seriousness of E. coli O₁₅₇:H₇ on public health and its continuous detection in meat products, its prevalence in beef burgers at retail level was explored. As shown in table (2 & 3), E. coli was isolated from 24 (19.2%) of 125 samples. Three of positive samples showed negative sorbitol and also presented agglutination with O₁₅₇ antiserum (E. coli of serogroup O₁₅₇), their percentage was 2.4%. The present study gave lower incidence of E. coli in examined beef burger than that recorded by Ouf (2001) (30%) and Fathi (2004) (72.5%). On the other hand Mohamed (2001) and Abd-EL-Malek (2005) reported that (40%) and (13.3%) of beef burger were contaminated by E. coli which is lower than that obtained in our result. Another study by Tutenel et al. (2003) indicated that 0.18% of examined ground beef harbored E. coli O₁₅₇.

Further examination of the threeO₁₅₇ isolates (using polymerase chain reaction (PCR) revealed that only one confirmed to be E. coli O₁₅₇:H₇ with a percentage of 0.8%., while the other two samples confirmed to be E. coli O₁₅₇ non H₇ with a percentage of 1.6%.

Nearly similar results were recorded by (Jamshidi     et al., 2008) who revealed the presence of E. coli O₁₅₇:H₇ in ground beef with a percentage of 1%, also (Ahmed et al., 2013) reported that 1% of beef burger samples were containing E. coli O₁₅₇:H₇. However Cagney et al. (2004), Abdel-Sadek (2012) and Jamshidi et al. (2012) reported a higher prevalence of the same organism in beef burger(2.91%), (10%) and (4%) respectively. On the other hand, chinen et al. (2001) couldn’t isolate this pathogen from hamburger patties.

Direct comparison of results of this study with other studies is difficult due to differences in manufacture practices, variation in enrichment and isolation procedures, also differences in sample size. While there is some evidence that E. coli O₁₅₇:H₇ may be increasingly common in beef production systems (McDowell and Sheridan, 2001).

Because of the effectiveness of gamma irradiation in controlling common food-borne pathogens and in treating packaged food, thereby minimizing the possibility of cross-contamination prior to consumer use, most food safety officials and scientists view irradiation as an effective critical point in Hazard Analysis and Critical Control Points (HACCP) established for meat and poultry processing (Satin, 2002).

In view of control measures to prevent or eliminate the hazards of E. coli O₁₅₇:H₇ in beef burger, the results of the present study clearly showed that D10 value 0.64 KGy, (a dose of 2KGy) of gamma irradiation showed an approximated reduction of 1.1 logarithmic cycle on E. coli O₁₅₇:H₇ population compared with the control, while D10 values 1.32 and 1.64 KGy (a doses of 4 and 6 KGy) reduced approximately 1.5 and 2.3 logarithmic cycles of E. coli O₁₅₇:H₇ count respectively in relation to the control (Fig. 1).

The results show that the population of E. coli O₁₅₇:H₇ decreased gradually with increasing irradiation doses, that irradiation of the inoculated samples at doses 4 and 6 kGy significantly (P < 0.05) decreased the counts of the inoculated pathogen compared with the control, while irradiation of the inoculated samples at dose 2 KGy showed non significant (P > 0.05) effect (Fig. 1). On the counts of the same pathogen (Rodolfo et al., 2002) reported that the D10 values for E. coli O₁₅₇:H₇ in beef burger ranged from 0.17KGy to 0.27KGy, this result did not agree with those reported in our study.

The broad variation in D10 values for beef burgers can be due to differences in composition of the beef burgers belonging to different brands.

According to Monk et al. (1994), some food preservatives also affect the growth or death of microorganisms when food is submitted to irradiation treatment. There for the presence of these compounds could also have influenced the values obtained.

The effect of gamma irradiation on sensory quality of beef burger is of concern due to the formation of free radicals. To evaluate the effect of irradiation on beef burger, non inculcated cooked samples were submitted to sensory evaluation after being exposed to irradiation doses.  In our experiment, the applied doses (2, 4, 6 KGy) did not affect the sensory attributes of the product, (Tab.4). In the same respect (Rodriguez et al., 1993) reported no changes in sensory attributes of beef treated with 2KGy, while (Rodolfo et al., 2002) reported that a dose of 1.2 KGy imparted an unfavorable odor and taste to the beef burger.

Our results indicate that at application doses (2, 4, 6 KGy) of gamma irradiation there is no significant differences between treated samples and control and beef burgerwill not be adversely affected form a sensory standpoint, (Tab. 4). This information can be used to support the use of gamma irradiation in meat to advance food safety practices.

Also, this research investigates the bacterial action of gaseous ozone for the elimination of E. coli O₁₅₇:H₇ from beef burgerby surface exposure technique.

Under identical treatment conditions, (Fig. 2) show that 20 PPM ozone concentration decreased the number of E. coli O₁₅₇:H₇ from 4.7 to 4.4 log10 cfu/g, while 40 PPM ozone concentration reduced the pathogen count to 4.3 log10 cfu/g. At a dose of 70 PPM the greatest reduction in E. coli O₁₅₇:H₇ population was achieved (1 log10 cfu/g).

The results show that all the applied doses of gaseous ozone significantly (P < 0.05) decrease the population of E. coli O₁₅₇:H₇ in beef burger comparing with the control. The sensory analysis showed non-significant difference in the treated cooked beef burger with the three doses of ozone gas compared with the control (Tab 5). Joel and George (2011) treated ground beef with gaseous ozone at various levels, it was determined that approximately 73% of the E. coli was killed using 50 PPM ozone for 3 minutes with no change in color or flavor at ozone levels while approximately 95.8% of the E. coli in the ground beef was killed when the ozone concentration approached 200 PPM. The flavor change (a slight off flavor) occurred in the 200 PPM treated samples while no noticeable color change was present.

Due to the lack of conclusive evidence that ozone gas will be effective on beef burger, and limitation of using only ozone gas as an antimicrobial intervention on beef burger, further research is necessary.

CONCLUSIONS

The application of gamma irradiation at dose 6KGy or ozone gas at concentration of 70 PPM can improve the safety beef burgers through significant reduction of E. coli O₁₅₇:H₇ without any defect in the sensory quality of the product. Gamma irradiation at dose 6KGy is more effective in reduction of E. coli O₁₅₇:H₇ population (a reduction % of 56.o8) than ozone gas at concentration of 70 PPM (a reduction % of 21.14)

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