PUBLIC HEALTH HAZARDS OF SOME BACTERIAL PATHOGENS ASSOCIATED WITH MEAT AND STUDYING THE MOST EFFECTIVE METHODS OF COOKING ON THEIR DESTRUCTION

PUBLIC HEALTH HAZARDS OF SOME BACTERIAL PATHOGENS ASSOCIATED WITH MEAT AND STUDYING THE MOST EFFECTIVE METHODS OF COOKING ON THEIR DESTRUCTION

GHADA M. MOHAMED^*; Lubna M. EBRAHEEM and M.A.M. AMMAR

^*Animal Health Research Institute, Assiut Regional Lab. (AHRI), Egypt.

Email: mahmoud2014eg@yahoo.com                                                                   Assiut University web-site: www.aun.edu.eg

Introduction

Meat is a major constituent of the human diet. It is an essential food item, (Rao et al., 2009) and one of the main sources of protein, vitamins, minerals, lipids and savory sensation, (Zweifer et al., 2008). Most meat has high water content corresponding to the water activity approximately 0.99 which is suitable for microbial growth, (Rao et al., 2009). Meat is subjected to changes by its own enzyme, by microbial action and its fat may be oxidized chemically. Microorganisms grow on meat causing visual, textural and organoleptic change when they release metabolites, (Jackson et al., 2001). Meat is a good material for bacterial growth; its quality depends on the initial bacterial contamination. This contamination causes meat deterioration, lowers quality and sometimes illness may be caused by bacterial pathogens or their toxins through meat and meat products.

 In fact, tissue from healthy animal is sterile, (Lawrie, 1984) but the immune system are destroyed during the slaughter process, (Romans et al., 2001). However, contamination of meat occur during slaughtering, preparation of carcasses, (Huffman, 2002) or from feces, soil, and water (Jay,1996), where microorganisms came chiefly from the exterior of the animal and its intestinal tract, and that more added from knives, clothes, air, carts and equipment in general, (Lawrie, 1984). Retail cut could also result in greater microbial load because of the large amount of exposed surface area, (Forest et al., 1985).

Food-borne pathogens of concern in beef carcass decontamination are E. coli O₁₅₇:H₇, Salmonellae and Stapylococcus aureus, (Huffman, 2002). Contaminated raw meat is one of the main sources of food-borne illnesses (Bhandare et al., 2007 and Podpečan et al., 2007) and death in developing countries costing billions of dollars in medical care (Fratamico et al., 2005) and record of 3900 deaths each year, (Buzby et al., 1997). Changes in eating habits, mass catering complex, lengthy food supply procedures with increased international movement and poor hygiene practices are major contributing factors of illness and death, (Hedberg et al., 1992).

The symptoms of food poisoning may vary depending on the type of bacteria causing the illness. Symptoms include nausea, stomach cramps, vomiting, diarrhea, fever and headache. Some food-borne pathogens cause other symptoms FAO (1999), for instance, pathogenic Listeria cause listeriosis In pregnant women and meningitis in Immuno-depress individuals while Salmonellosis is caused by Salmonellae, (Estes, 2003 ). One the other hand, 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 most used meat preservation step is thermal processing which is the application of heat to food in order to destroy pathogenic microorganisms below the concentration of their ability to produce disease, (Richardson, 2002). Heat treatments is influenced by many factors, some of them are due to inherent resistance of microorganisms (Brown, 1994), which include differences among species, strains, spores and vegetative cells of bacteria (Tomlins, 1976), while others are due to environmental influences (Brown, 1994) such as the composition of the heating menstruum (amount of carbohydrates, proteins, lipids, solutes.), water activity, pH, added preservatives and method of heating, (Smelt et al., 1994). The optimum temperature for the multiplication of most food poisoning bacteria is between 5 - 63^oC, whilst, at temperatures over 70^oC most bacteria are killed and below 5^oC most food poisoning bacteria can only multiply slowly or not at all. Most cooking methods if performed properly will heat foods to over 70 ^oC, so applying such a temperature for a carefully calculated time period will prevent many food borne illnesses that would otherwise manifest if the raw food was eaten. The fundamental types of cooking are grilling, frying and boiling. Grilling is cooking of food using a direct dry heat, frying is the cooking of food in oil or fat while boiling is the cooking of foods in a liquid which is the common type of cooking, (EUFIC, 2010).

Although cooking, moreover, improves the hygienic quality of the food by inactivation of pathogenic microorganisms (Bognár, 1998), it also, causes a complex series of physical and chemical changes to occur. These changes vary depending on the type of food being cooked and the method used to cook it. The changes may be advantageous e.g., improving the flavour, texture and colour of the food, or they may be disadvantageous e.g., reducing the nutrient value of the food, or the generation of undesirable compounds (EUFIC, 2010).

So, it is important to select the proper cooking method and the degree of cooking which employed further effects on the number and the types of microorganisms. Since, consumption of healthy food is one of the significant factors affecting the health; such studies are extremely important and will be helpful in supervision and control of quality of meat.

The present investigationaimed to evaluate the prevalence of some microorganisms of public health importance (Escherichia coli, Salmonella, Staphylococcus aureus and Listeria monocytogenes) in fresh beef and studying the growth and survival behavior of these pathogens when subjected different types of cooking.

Materials and Methods

Collection of samples:

A total of 100 random samples (500g each) of fresh beef were collected from butcher shops with different sanitation levels in Assiut city. The samples were transferred separately and aseptically in an ice box without delay to the laboratory where they were examined.

Isolation procedures:

1- Isolation of Staph. aureus: Feingold and Martin (1982).

2- Isolation of Salmonella spp: According to the method recorded by APHA (1992).

3- Isolation of Listeria monocytogenes: Oxoid Manual (1990).

4- Isolation of E. coli: AOAC (1990).

In each case enrichment procedures was applied using 25g sample followed by selective plating as recommended by the corresponding reference. All isolates were identified morphologically using staining reaction (APHA, 1992) and motility test (Baron et al., 1994), as well as, biochemically using catalase, coagulase, triple sugar iron (TSI) agar test (Baron et al., 1994), citrate utilization, indole production, methyl red, urease, voges-Proskauer tests (Koneman et al., 1992), nitrate reduction test (Cowan and Steel, 1974), sugar fermentation reaction (APHA, 1992), and Christine-Alkine- Munch- Peterson (CAMP) test (Herrera, 2001).

E .coli isolates were serologically identified according to Kok et al. (1996) by using rapid diagnostic E.coli antisera sets (DENKA SEIKEN Co., Japan) for diagnosis of the enteropathogenic types. Serological identification of Salmonellae was carried out according to Kauffman – White scheme (Kauffman, 1974).

For further confirmation of L. monocytogenes isolates were inoculated into 10% aqueous stock solution of mannitol, rhammose and xylose, (Collee and Miles, 1989). While Further Identification of Staphylococcus aureus is done by Thermostable nuclease test "D-Nase activity", (Lachia et al., 1971).

Heat- inactivation experiment:

Preparation of cultures:Murphy et al. (2000)

For each trial, a 24h culture was prepared individually for E. coli O₁₅₇:H₇, Salmonella enteritidis, L. monocytogenes and Staph. aureus by culturing in Tryptic Soya Broth  and incubation at 37°C. The count for each /ml was determined by surface plating decimal dilutions on MacConkeys sorbitol gar, MacConkeys agar, Paird-parker agar and PALCAM agar (Oxoid, Basingstoke, UK), respectively. Just prior to the thermal treatment calculated broth portions were mixed to form a mixed cocktail of test strains.

Meat samples:(Patel et al., 2004).

Fresh boneless strip lions (longissimus muscle) were purchased from a local retailer. After removing the fat from the surface, the strip lions were sliced into 25 g portions (3 cm thickness x 2cm x 4cm) using sterile knife. The inoculation was applied by injecting 1.5 ml of the mixed strain cocktail (10⁶ CFU of individual strain /g) in the center of each meat sample. The inoculated samples were placed onto a sterile tray covered with aluminum foil and then stored at 4°C for 30 minutes to permit bacterial attachment to meat and heat equilibrium.

Thermal treatment:

The target cooking internal temperature of the samples was the temperature recommended by (FAO, 2007) for intact beef (65°C, medium-rare cooking) and then for an excess of 6 minutes using three different heating methods. For heating, one single inoculated refrigerated food item was placed in boiling water (100°C) , heated oil (150°C) or heated oven (180°C) and then heated for times ranging from 0 (not heated) to 15°min.

For the boiling-inactivation test, a cooking pan (24°cm diameter) with water (2.5 L) was brought to the boil. The water was constantly heated and as the weight of the added matrices was small (25°g) compared to that of the water (2.5L), the temperature profile of the water was constant at 100°C, (de Jong et al., 2012).In roasting, the samples in a metal baking tray lined with aluminum foil were placed on an oven rack in the center of electric oven at 180°C for the specified time, (Jefferies, 2011). For frying, the samples were fried in a common fryer at 150°C (oil temperature) for the specified time, (Miller et al., 2011). Due to variations in initial temperature between trials, a standardized start time of when samples were 20°C employed to determine cooking time. The temperature of water, oil or oven was monitored to be constant along the heating times using digital thermometer (thermometer ST-131 waterproof digital).

Enumeration of survivors and analysis for quality changes:

Just after removal of the sample from the heating medium, the internal temperature of the sample was determined by inserting the actual sensor in the last two centimeters of the temperature probe (thermometer ST-131 waterproof digital) to the geometric center of the sample. The sample was then placed in sterilized plastic bag and immersed in a mixture of ice-water. Testing of survivors and analysis for quality changes were carried out when samples cooled to room temperature.

Enumeration of survivors:Murphy et al. (2000)

Prior to thermal treatment, 25g sample each of inoculated and non-inoculated samples was combined with 225 mL of sterile peptone solution (0.1%) in a sterile stomacher bag and blended in a stomacher for 2 minutes. Counting of test microorganisms was carried out by spread-plating decimal dilutions on appropriate media. Cultivability of the inoculated bacteria in the meat after heat-treatment was determined by preparing sample suspensions and spread plating appropriate dilutions on appropriate media. The plates were incubated at 37°C for72h for all the test microorganisms except listeria where they were incubated for 144 h. The plates were counted each 24h until the number of colony formation unit no longer increased. Suspected colonies of the test microorganisms were confirmed by biochemical and serological methods. Average values of bacterial counts, from duplicate plate samples, were converted to log for each bacterium. In order to validate complete destruction of tested microorganisms, sample enrichment were performed on samples that contained no growth at the experimental detection limit of 1 log CFU/g. Ten ml of each sample time that produced no growth were diluted with 90 ml of Tryptic Soya Broth. Each enrichment solution was incubated at 37°C for 24 h. After incubation, the solutions were streak plated onto appropriate media and incubated at 37°C for 48h.

Analysis for quality changes:Nikmaram et al. (2011)

Cooking losses were determined by measuring the difference in sample's weight before cooking and then after cooking when samples cooled to room temperature.

Cook loss % =  Weight of raw sample _ Weight of cooked sample                                                                     

                               __________________________________   x 100

                                                  Weight of raw sample

The pH of meat homogenate was measured after microbial analysis using digital pH meter (Gallenhamp No.101284).

Calculation of D-values: Juneja et al. (2001)

D-values (time to inactivate 90% of the population) were calculated from the straight portion of the survival curves by plotting the log of survival counts compared with their corresponding heating times, using SPSS (2007).

RESULTS

 Table 1: Incidence of bacterial pathogens in raw beef samples.

Table 2: Effect of cooking methods on E. coli O₁₅₇: H₇  and Salmonella entertidis.

U D: Under detectable level (less than 1 log 10 CFU/g)

a, b, c: data with different litters are significantly different at P< 0.05

Table 3: Effect of cooking methods on Listeria monocytogenes and Staph. aureus

UD: Under detectable level (less than 1 log 10 CFU/g)

a, b, c: data with different litters are significantly different at P< 0.05

Table 4: Effect of cooking methods on weight loss % and pH values.

a, b, c: data with different litters are significantly different at P< 0.05

Fig. 1: The rate of heating in different cooking methods

Fig. 2: Effect of cooking methods on E. coli O₁₅₇: H₇ 

Fig. 3: Effect of cooking methods on Salmonella enteritidis

Fig. 4: Effect of cooking methods on Staph. Aureus

Fig. 5: Effect of cooking methods on L. monocytogenes

Fig. 6: Effect of cooking methods on weight loss %

Fig. 7: Effect of cooking methods on pH values

DISCUSSION

The present study evaluated the microbial quality of raw meat sold in butcher shops in Assiut City, Egypt. Our findings showed that out of 100 meat samples analyzed for microbial quality 90 (90%) were contaminated with different kinds of microorganisms namely E. coli 48 (48%), Salmonellae spp. 18 (18%), Staphylococcus spp. 16 (16%), and listeria spp. 8 (8%) as showed in Table (1). Our result indicated the predominance of Gram-negative organisms such as E.coli and Salmonella as reported by (zakpaa et al., 2009) and (Iroha et al., 2011).

The distribution of the isolated pathogens were listed in the same table these results show that most of E.coli isolates were E.coli O₁₁₁: H₄ 21(43.75%) followed by E.coli O₁₂₆: H₁₁ 13 (27.08%) then E.coli O₁₂₈: H₁₁ 11(22.92%) and E.coli O₁₅₇: H₇ 3 (6.25%).

In 2013 (Ahmad et al., 2013 and Archana et al., 2013) reported the presence of high percent of E.coli in beef samples 75% and 65% respectively, also in 2014 (Sami et al., 2014) could isolate (60%) of E.coli from beef samples while (zhao et al., 2001 and Iroha et al., 2011) could isolate lower percent (19%) of this organism than that reported in our study.

Ebrahim et al. (2012) reported that 8.2% of beef samples were E.coli O₁₅₇ positive where 1.2% of them was E.coli O₁₅₇: H₇, while (Mboto et al., 2012) could not detect E.coli O₁₅₇: H₇ in any of the fresh meat samples examined.

E.coli O₁₅₇: H₇ is an enteric organism associated with animal and slaughter hygiene, it may be present in the feces and intestines of healthy bovines (Mcevoy et al., 2004). Therefore, meat can be contaminated during slaughter operation. The severity of the illness and the low infective dose (<100) make this organism among the most serious food borne pathogens (Meng et al., 2007).

Three species of the Salmonella genus were isolated from tested samples, the highest prevalence of them was Salmonella typhimurium (44.4%), while Salmonella enteritidis and Salmonella anatum rank as a second (27.8%) for each.

The percent of Salmonella spp. Isolated by (Ahmad et al., 2013, Archana et al., 2013 and Sami et al., 2014) from beef samples was higher than that obtained in our study, their percentages were 35%, 45% and 26.6% respectively, while the number of Salmonella spp. isolated by (zhao et al., 2001 and Iroha et al., 2011) was lower than that obtained in our result, their percentages were 1.9% and 4% respectively. 

Also the same table showed that 16% of tested samples was staphylococcus spp. whereas 12 (75%) of them recorded as Staphylococcus aureus and 3 (18.75%) recorded as Staphylococcus epidermidis, while 1(6.25%) recorded as Staphylcococcus gallinarum, all of them were coagulase positive and this was disagreement with (Goja et al., 2013) who reported that most of Staphylococci isolates from fresh beef samples were coagulase negative.

The isolated spp. of Staphylococci in our study were considered to be well known pathogens to humans and animals, specially Staph aureus, their presence could be due to the insanitary condition of the butcher and absence of the health services in butcheries.

The number of Staph aureus 2 (2%) isolated by (Iroha et al., 2011) was lower than that obtained in our result, while (Ahmad et al., 2013) and (Sami et al., 2014) could isolate higher percent of the same spp. than ours, their percentages were 70% and 46% respectively.

Four (50%) of isolated listeria spp. were characterized as Listeria monocytogens, and 2(25%) of them were characterized as Listeria innocua while the percent of Listeria welshimeri and Listeria invanovii was 1 (12.5%) for each. 

The presence of zoonotic bacteria in meat indicates poor anti- mortem inspection of the animals as well as unhygienic meat processing (Barros et al., 2007).

The inactivation of infectious pathogens using a heat treatment is a critical control point in the safe preparation of meat, the major benefit of thermal processing is the overall improvement of product quality and safety. There are three main thermal processing methods to treat meat (boiling, frying and roasting).

The objectives of this part of the study were to evaluate the thermal inactivation of inculcated E.coli O₁₅₇: H₇, Salmonella enteritidis, Staph aureus and listeria monocytogenes in fresh meat using boiling, frying and roasting treatments and to compare between the thermal lethality kinetics of these pathogens after using the three methods of inactivation.

Heating rate was presented in (Fig.1), it was 2°C /min by boiling, 7.5°C /min by frying, while it was 3.6°C /min at roasting. When the data were fitted to relate log of survivors to time in each of the experiments resulted a linear model. The form of this model was: Y= aX + E

Where, Y: is log CFU (log of colony forming per gram), a: represents the slope of the model for log CFU / g vs. time, X: represents time in minutes and E: represents error. Estimates of a could be used to then estimate D values. The D values for the individual experiments were obtained as the inverse negative of the slope (a) of the linear regression line.

The experimental inactivation of inoculated E.coli O₁₅₇: H₇ in fresh meat and fitted curves are included in (Fig.2). The target internal Temp. (65°C) of all treated samples (with selected organisms) was attained in 3, 2 and 3 min (come up time) of boiling, frying and roasting treatments, respectively. Table (2) showed that at this Temp, the log cycles reductions of E.coli O₁₅₇: H₇ were 1.3, 1.5 and 2.6 at boiling, frying and roasting treatments respectively compared with the control. Statistical analysis revealed that at the third minute of each treatment there was significant difference between boiling and frying on reduction of E.coli O₁₅₇: H₇ count, also between boiling and roasting, while there was no significant difference between frying and roasting on reducing the count of the same organism.

By boiling, the count of the organism reached to undetectable level (inverted columns, Fig. 2) at internal Temp. 80°C.This cooking end point of boiled samples was achieve with holding time 2 min,(time to reach internal Temp. of  65°C and excess 2 min exposure). By frying, an internal Temp. of 83°C (cooking end point) resulted after  holding time of 3 min, while the count reached to undetectable level in roasted samples at internal Temp. 74°C with holding time 5 min.

D-values are used in the food industry to determine the effectiveness of the heat inactivation process, these values were calculated for E.coli O₁₅₇: H₇ from the survival curves (Fig. 2), they were 1.1, 1.1 and 1.2 minutes at boiling, frying and roasting treatments respectively.

Kawang (2014), reported that pathogenic cells like E.coli O₁₅₇: H₇ on the meat surface may be translocate and trapped in sterile internal tissues, protecting themselves from thermal destruction if the meat is undercooked.

The survivor curves of Salmonella enteritidis were constructed by plotting recovered CFU/g of sample versus heating time (Fig. 3). As expected, as heating temperature increased, survival of S. enteritidis decreased.

At the time of internal Temp. 65°C and depending on the method of heat treatment (as shown in Table 2 and Fig.3), S. enteritidis decreased 2 Log cycle for all heat treatments compared with the control, and the statistical analysis revealed that there was no significant difference between these treatments on reducing the count of the organism at the third minute, also this Table showed that, the count of S.enteritidis reached to undetectable level at internal Temp 80°C, 80°C and 71°C at the cooking end point of boiling, frying and roasting treatments respectively, each with a holding time 2 minutes.

D-values were calculated in meat samples inoculated with S. enteritidis and obtained D- values of 1.1, 1.0 and 1.2 minutes at boiling frying and roasting treatments, respectively.

When the different heat treatments were applied on inculcated meat samples with Staph aureus and at internal Temp. 65°C, nearly results were observed, Staph aureus decreased 2.2, 2.3 and 2.3 1og cycles at boiling frying and roasting treatments, respectively (Table 3 and Fig. 4). The statistical analysis at the third minute of these treatments revealed that there was significant difference, between boiling and frying, also between frying and roasting, while no significant difference detected between boiling and roasting, and the count of the organism reached to undetectable level at internal Temp. 80°C, 80°C and 71°C at the cooking and point of boiling, frying and roasting treatments respectively, each with a holding time 2 minutes (Table 3 and Fig.4).

D- Values of Staph aureus in inoculated meat were calculated from survival curves, (Fig.4) they were 1.1, 0.9 and 1.1 minutes at boiling, frying and roasting treatments respectively.

The survival of L. moncytogenes at different cooking methods is given in (Table 3 and Fig. 5), L. monocytogenes suffered 2.2, 2.1 and 1.4 1og cycles reduction when the internal Temp. of inculcated meat samples reached 65°C of boiling, frying and roasting treatments respectively, the results of statistical analysis indicated that there were significant differences between the three treatments at the third minute.

The same table showed that the count of L. monocytogenes reached to undetectable limit at internal Temp. 80°C, 80°C and 78°C at the cooking end point of boiling, frying and roasting treatments receptively with holding time 2 min for each of boiling and frying treatments, while this time was 6 min for roasting treatment.

D- Values of L.monocytogens in inculcated meat samples were detected to be, 1.1, 0.8 and 1.1 minutes at boiling, frying and roasting treatments, respectively (Fig. 5).

The high temperature used in thermal processing destroys microbial cells by destabilizing the structural and functional integrity of the cytoblasmic membrane, (Hoover, 1993).

The results of different studies indicate the existence of considerable variation among the reports on the heat resistance of these inoculated organisms, there are several factors altering the level of heat resistance of these organisms, such as differences among the strains, inoculum level, preparation of the product, substrate specific effects, experimental condition, protocols, recovery media and methods. Thus, direct comparisons between the studies are difficult, although it reasonable to accept that at least some of these factors underlie the observed variations in the heat resistance.

In general the thermal resistance by inoculated organisms is variable, and semi logarithmic survivor curves showed a linear decline in population over heating time.

Some changes occur during thermal processing of meat leading to weight loss and change in pH. Cooking losses were determined by measuring the difference in meat samples weigh before cooking, and then after cooking when samples were cooled to room temperature, this recorded in (Table 4) in which boiling caused weight losses ranging from 8.1% to 17.47%, frying caused losses ranging from 15% to 23.53%, while the range of weight losses in roasting treatment was 4.77% to 23.5%.

Statistical analysis revealed that at the cooking end point of the three treatments there was significant difference between boiling and frying, also between boiling and roasting, while no significant difference appeared between frying and roasting this difference is most likely due to different cooking methods.

As expected, the higher cooked internal temperature resulted higher cooking losses (Table 4 and Fig. 6). Prolonged cooking time causing extra moisture loss via evaporation and the release of excess juice inside the meat samples. There by boiling may be the suitable cooking method for meat due to reducing cooking loss.

Sun (2006) observed an increase in cooking loses with an increasing internal Temp. of meat with greatest increments in cooking loses observed within Temp. range 50°C - 70°C, also they reported that denaturation of proteins during thermal processing can cause loss of up to 20% - 40%, mainly in the form of moisture and fat. Also, Sun (2006) observed that the low-steam cooking condition significantly increased cooking yield and the low-steam cooked samples were significantly different form high-steam samples producing a higher cooking yield.

The pH of control samples was 5.1 (Table 4 and Fig. 7). Boiling treatment caused increase in pH, resulted in pH values ranging from 5.1-7.1, but this correlation not clear in both frying and roasting treatments. The increase in pH for cooked meat is due to the reduction of free acidic groups as meat temperature increased during heating process, (Li, 2014). Doyle and Mazzotta (2000) reported that bacteria are more resistant to heat at pH 7.0 or higher. 

Statistical analysis revealed that there was significant difference between boiling and frying treatments also between boiling and roasting treatments, while no significant difference appeared between frying and roasting treatments. 

Conclusions:

The presence of food borne pathogens such as E.coli, Salmonella, Staphylococcus and Listeria in raw meat indicate poor ante-mortem inspection of the animals as well as unhygienic meat processing, it can be concluded that the cooking process carried out at an internal temperature of 65°C is not sufficient for eliminating the high contamination (10⁶ CFU/g) of E.coli O₁₅₇: H₇, S. enteritides, Staph. aureus and L. monocylogenes, and the survival of these pathogens after this temperature indicate the possibility of a public health hazard. Results of this work emphasis the necessity for cooking meat at an internal temperature of 83ºC, that as cooking time and temperature increase, levels of microbial destruction increase, also higher cooked internal temperature resulted higher cooking losses and changes in pH. 

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