EFFECT OF COOKING PROCESS ON COLIFORMS AND ESCHERICHIA COLI IN SOME SEA FOODS AND THEIR PUBLIC HEALTH SIGNIFICANCE

Materials and methods

1: Samples collection:

 A total of 60 apparently healthy samples of shellfish (30 each of baclawese "Tartufo di mare" and om EL-khloul "Donax trunculus anatinus" were randomly collected from Port Said markets. All specimens were prepared, treated and examined within 4 hours of collection. In case of baclawese the samples were divided into 2 parts; the 1st part directed to counting, isolation and identification of coliforms and Escherichia coli. The 2nd part was cooked before the bacteriological examination. While In case of om EL-khloul the samples were divided into 3 parts; the 1st part directed to counting, isolation and identification of coliforms and Escherichia coli. The 2nd and the 3rd part were cooked and salted before the bacteriological examination.

2: Bacteriological examination:

2-1: Preparation of the samples:

The meat and liquor of each sample was homogenized, and 10 g were drawn off into a sterile dilution cup and blended for 2 min in sterile phosphate buffer dilution water to yield 1:10 dilution of sample. Then serial dilution was prepared using sterile phosphate buffer dilution water according to(FDA, 2002).

2-2: Presumptive test for coliforms: 

Five tubes each containing 10 ml of sterile lauryl tryptose broth (Difco) with Durham's tubes, were inoculated with 1ml from each of the original dilution. Inoculated tubes were thoroughly mixed before being incubated at 35°C for 48 hours. The inspection was done after 24 and 48 hours incubation for positive gas production (FDA, 2002).

2-3: Probable number of total coliforms:

Each positive LT tube was gently agitated then loopful of suspension was transferred to tube of brilliant green bile broth 2% (BGB) containing inverted Durham's tube. All tubes were incubated at 35°C for 48 ± 2 hr and examined for gas production. Enumeration of total coliforms was applied by using table of most probable number (MPN) based on combination of confirmed gassing LT tubes for 5 consecutive dilutions and the results were reported for 100 g of each sample.

2-4: Probable number of fecal coliforms:

Gentle agitation of each gassing LT tube was done and a loopful of the suspension was transferred to tube of EC medium containing inverted Durham's tube. All broth tubes were incubated at 44.5 ± 0.2°C for 48 ± 2 hr. The presence of gas in EC medium was considered to be confirmed evidence of the presence of fecal coliform bacteria in the shellfish sample. Enumeration of fecal coliforms was applied by using table of most probable number (FDA, 2002).

2-5: Enumeration of Escherichia coli:

A loopful of suspension from gassing EC medium tubes was streaked onto Levine's Eosin-Methylene Blue agar plate and iincubates at 35°C for 18-24 hr. Typical colonies with a dark center or a metallic sheen were selected, streaked for purity on nutrient agar, and incubated for 24 h at 35°C. Enumeration of Escherichia coli was applied by using table of MPN based on proportion of EC medium tubes in 5 consecutive dilutions which shown to contain Escherichia coli according to (FDA, 2002).

2-6: Biochemical identification of the isolates:

Pick three typical colonies from each agar plate and transfer to plate count agar (PCA) slants tube and incubate at 35^°C for 18-24 hr. All PCA slants were directed to morphological and biochemical identification according to Holt et al. (1994) and Farmer (1995).

3: Statistical methods:

One-Way ANOVA test was performed on the parameter studied to describe data using Statistical Package for Social Scientists (SPSS) for windows 16.0 (SPSS Inc., Chicago, IL, and USA). Correlations between total coliform, fecal coliform and Escherichia coli counts based on the examined samples of baclawese "Tartufo di mare" and om EL-khloul "Donax trunculus anatinus" in relation to cooking and salting technique. Significant differences in parameters analyzed. P value was considered significant if less than 0.05 and 0.01 at 95% and 99% respectively (SPSS, 2007).

 Discussion

Marine bivalve like baclawese and om EL-khloul are globally important food resources. They are particularly important in developing countries; mostly because they are easily collected in shallow areas and have high nutritional value. These species are sedentary and filter-feeding which favor bioaccumulation of microorganisms, which make them frequently involved in outbreaks of gastroenteritis (Sockett et al., 1985; Potasman and Odeh, 2002).

Members of the coliform groups are referred as general indicator microorganisms to measure the potential presence of enteric pathogens (for example Escherichia coli) in foods, besides the measuring of fecal contamination of the food products and the sanitary condition in the food-processing environment (Greenberg and Hunt, 1985; APHA, 1992; FAO, 1992).

The results in Table 1 revealed that the  incidences of positive fresh baclawese samples for total coliforms, fecal coliforms and Escherichia coli were 100% (30), 100% (30) and 20% (6) respectively. While that of   om EL-khloul were 100% (30), 100% (30) and 10% (3) respectively (Table 2). The incidence of positive samples for coliforms was higher than the results recorded by Betty et al. (2008) but that of Escherichia coli was lower than those recorded by them. The high incidence of positive samples may be due to the water is highly contaminated by shipping and untreated sewage. Also may be attributed to the unsanitary conditions during the collection and transportation of baclawese and om EL-khloul.

The mean values of the total coliforms, fecal coliforms and Escherichia coli counts in fresh baclawese were 2.4 X 10⁶ , 2.3 X 10⁵  and 3.7 X 10² (MPN/100g) while in case of om EL-khloul were 1.8 X 10⁵ ,8 X 10³ and 2 X 10³ (MPN/100g) respectively (Tables 1 and 2). These results agreed with the results recorded by Carlos et al. (2007). On the other hand, our results were higher than that recorded by Hood et al. (1983); Humphey and Gawler (1986) and Colakoglu et al. 2010). Meanwhile the higher counts of fecal coliform and Escherichia coli may be attributed to recent fecal contamination (Caplenas and Kanarek, 1984; Greenberg and Hunt, 1985; APHA, 1992; FDA, 2002).

Regarding to the effect of cooking process on total coliforms, fecal coliforms and Escherichia coli presented in Table 1. It is evident that the incidences of positive cooked baclawese samples for total and fecal coliforms and Escherichia coli were 73.33% (22), 53.30% (16) and 0.00% (0.00) respectively. On the other hand the mean values of the total, fecal coliforms and Escherichia coli counts were 1.8X10⁵, 5.6X10³ and 0.00 for cooked baclawese respectively. While in case of the examined cooked om EL-khloul the incidence of positive samples decreased to 16.7% (5), 13.3% (4) and 0.00% (0.00) respectively (Table 2). The salting effect revealed that the percentage of the examined positive salted om EL-khloul samples were 63.3 % (19), 60 %( 18) and 10 %( 3) for total coliforms, fecal coliforms and Escherichia coli respectively. The mean values of the total, fecal coliforms and Escherichia coli counts were 3.5X10⁴, 9.2X10² and 9.5X10¹ MPN/100g under effect of salting technique respectively results showed that the cooking process either in baclawese or om EL-khloul reduced the total and fecal coliforms and Escherichia coli counts to a different levels. This reduction effect agrees with the result recorded by Desmarchelier and Grau, (1997) and Yilmaz et al. (2005). This reduction was impossible to eliminate all forms of coliforms (Jay, 1978).

Statistically by using one way ANOVA test, a highly significant relationship between the counts of each of total and fecal coliforms and Escherichia coli recovered from fresh baclawese and om EL-khloul samples with that of the cooked samples. On the other hand, the relationship between cooking and salting technique in case of EL-khloul showed a significant relationship. This means that cooking technique was more effective and more efficient than salting technique in reduction effect (Yilmaz et al., 2002).

The obtained results in Table 3 showed that the number of coliforms isolates of fresh baclawese and om EL-khloul were 52, and 47 respectively. In case of cooked samples, the isolates were reduced to 14, and 8 for baclawese and om EL-khl oul repectively.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         The number of isolates in case of salted om EL-khloul was more than the number in the cooked om EL-khloul. This mean cooking technique was more effective and more efficient than salting technique. The coliforms organisms recovered from the examined baclawese samples were identified as Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Citrobacter koseri, Klebsiella pneumonia and Klebsiella Ozaenae. While in om EL-khloul, the isolates were Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Citrobacter koseri and Klebsiella pneumonia. Our results come in agreement with the result recorded by Gulsen et al. (2008).The main and important isolate found in fresh and treated samples was Escherichia coli. On the other hand Citrobacter freundii and Citrobacter koseri found only in total coliform and this may be attributed to that these group of microorganisms cannot tolerate high temperature but fecal coliform as Enterobacter cloacae and Enterobacter aerogenes can tolerate and considered thermotolerant organisms (Caplenas and Kanarek, 1984). The reduction in the number of isolates was more in cooking than in salting technique and this agrees with the result recorded by Zahra et al. (1985) who showed that coliforms were easily killed and decreased markedly after heat treatment but heating process was not enough to ensure the safe eating quality (Bayhan et al., 1990) and impossible to eliminates all forms of coliforms (Jay, 1978). In conclusion, to increase the safety of Shellfish and decrease their coliforms loads and prevention the infections by these microorganisms to the consumers, a groups of measurement should be attempt such as removal the source of pollution as fecal contamination and sewage water and good sanitary condition during collection and transportation. Besides the equipment and utensils used for collection and transportation must be clean. sufficiently cooking of Shellfish and prevent the post cooking cross contamination and prohibiting people who are ill from working in food operations and strictly prevention the consumption of insufficient cooked Shellfish specially in compromised hosts reduce the consumers health problem.

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