A Pilot Study of the Microbiological Quality of Culturally Diverse,Ready-to-Eat Foods from Selected Retail Establishments in Melbourne,Australia

Abstract

In recent years, there has been an increasing number of foodborne outbreaks linked to the consumption of culturally diverse foods. This appears to be because of the increasing quantity of culturally diverse foods available and a preference to store these foods, some of which are considered potentially hazardous, at ambient temperature. This practice may contravene temperature requirements defined by the Food Standards Code. A lack of understanding of the hazardous nature of some culturally prepared foods also poses difficulties in applying the Australian food safety legislation by regulators. This pilot study examined the normal microbiota of four culturally diverse foods: nem chua, che dau trang, kueh talam, and bánh tét nhân mặn, which are traditionally stored and consumed at ambient temperature. Challenge testing was conducted to investigate the ability of these foods to support the growth of foodborne bacterial pathogens. Two of the products (kueh talam and che dau) were found to be microbiologically unsatisfactory because of the high standard plate counts. Challenge testing indicated that kueh talam, che dau, and bánh tét nhân mặn were able to support the growth of Bacillus cereus, Escherichia coli, Staphylococcus aureus, and Salmonella (1–2 log increases over 6 hours at 25°C), suggesting that these foods may require temperature control during storage. However, nem chua was unable to support the growth of test bacteria, probably because of its acidic nature (pH 4.5), suggesting that ambient storage of this food may be safe. This study provided some preliminary evidence to support the need for further sampling and challenge testing of these products.

Introduction

T he incidence of foodborne illness is an increasing problem worldwide, and there is greater concern regarding food safety and new potential hazards (Mishra and Sinija, 2008). Consumer demands for foods that are minimally processed, easily prepared, and ready-to-eat (RTE) present major challenges to food safety and quality (Mishra and Sinija, 2008). Recent studies have indicated that RTE foods and food preparation surfaces may be reservoirs for microbial contamination (Mankee et al., 2005; Ghosh et al., 2007; Abadias et al., 2008; Christison et al., 2008).

There are ∼5 million cases of foodborne illness in Australia each year, resulting in 80 deaths and costing the community in excess of $1 billion (OzFoodNet, 2005). In recent years, several foodborne outbreaks in Australia have been attributed to the consumption of culturally diverse RTE foods. These outbreaks may be because of the increasing availability of these types of foods as well as a lack of understanding surrounding the potentially hazardous nature of the foods. Vietnamese pork rolls have been implicated in two significant outbreaks of Salmonella in 1997 and 2003 involving more than 900 cases and one fatality (OzFoodNet Working Group, 2002). In 2002, more than 270 people were treated for foodborne gastroenteritis caused by consumption of food contaminated with Bacillus cereus and Staphylococcus spp. at a community festival celebrating Islamic New Year (Communicable Diseases Surveillance, 2002). In 2005, more than 170 people became ill after consuming dips from a Turkish restaurant contaminated with Salmonella (OzFoodNet, 2005). Despite these incidents, there has been limited research into the potentially hazardous nature of culturally diverse RTE foods in Australia. The lack of adequate food safety information with respect to these foods has also been recognized internationally (Mauer et al., 2006).

The Australian Food Standards Code requires “potentially hazardous foods,” defined as foods that are able to support the growth of pathogenic microorganisms at certain temperatures, to be maintained at temperatures either below 5°C or above 60°C; or between 5°C and 60°C for no longer than 4 hours, to minimize this growth and prevent the formation of toxins in the food (FSANZ, 2006). However, potentially hazardous food may be displayed at an alternative temperature if the food business is able to demonstrate that such storage conditions do not adversely affect the microbiological quality of the food (FSANZ, 2006). For example, food businesses that sell sushi (nigiri and nori rolls) in Victoria, Australia, are able to store these products for up to 12 hours at temperatures between 5°C and 15°C, provided that the pH of the rice is <4.8 (Victorian Department of Human Services, 2004).

In this pilot study, we investigated the microbiological safety of four traditional Asian RTE foods readily available in retail establishments in Melbourne. These foods were chosen because local food regulators suggested that they are representative of the challenges associated with the regulation of culturally diverse foods in Australia with regard to temperature control practices of potentially hazardous foods. This pilot study aimed to assess the levels of microbial contamination of the samples, and their potential to support the growth of foodborne pathogens and Escherichia coli when stored at room temperature.

Materials and Methods

Description of RTE foods investigated

Nem chua is a Vietnamese-style fermented sausage made from pork meat mixed with salt and garlic and consumed without cooking.

Che dau trang is a Vietnamese pudding-like sweet that is traditionally made from sticky rice, white beans, and coconut milk.

Kueh talam is a Malaysian sweet consisting of two layers. The top white layer is made from rice flour and coconut milk, while the bottom layer is made from green pea flour, pandan leaf, and palm sugar.

Bánh tét nhân mặn is the name for sticky rice products wrapped in banana leaves. The combinations can be sweet, filled with sticky rice, banana, and coconut milk; or savory, filled with sticky rice, pork, onion, and pepper. The bánh tét nhân mặn used in this study was the savory variety and came wrapped in banana leaves that were bound with nylon string.

Each of the products described above was prepackaged in plastic containers or wrapped in plastic wrap and displayed for sale at room temperature (∼20–25°C) at the time of purchase.

Sample collection and preparation

The four products were selected based on the anecdotal evidence from local food regulators, indicating an increasing availability of these foods and a need for greater knowledge and evidence regarding the potential hazardous nature of the products. Duplicate samples of each product were purchased from four independent retail establishments, transported on ice in an insulated container, and delivered to the Microbiology Laboratory, Faculty of Life and Social Sciences, Swinburne University of Technology. Samples were prepared and tested within 30 minutes of purchase. Ten grams of each sample was added to 90 mL of sterile saline in blender bags and homogenized in a Stomacher (Lab-blender 400; FSE, Melbourne, Australia) for 2 minutes. Samples were serially diluted in sterile physiological saline.

Microbiological analysis

All tests were performed in duplicate according to the Australian Food Standards AS5013 Food Microbiology (Standards Australia, 2004).

Standard plate count

One milliliter of the 10^–1 dilution of prepared sample was aseptically dispensed into the base of a sterile Petri dish. Approximately 15 mL molten Plate Count Agar (Oxoid, Basingstoke, UK) was then added to the dish and mixed thoroughly. This process was repeated using the 10^–2 and 10^–3 dilutions of each prepared sample. Once set, the plates were incubated at 37°C overnight.

Coliforms and E. coli

One milliliter of the 10^–1 dilution of prepared sample was aseptically dispensed into the base of a sterile Petri dish. Approximately 15 mL molten Violet Red Bile Agar (Oxoid) was then added to the dish and the contents mixed thoroughly. This process was repeated using each prepared sample. Plates were then incubated at 37°C overnight.

Yeasts and moulds

A volume (0.1 mL) of the 10^–1 dilution of prepared sample was aseptically dispensed onto a Malt Extract Agar (Oxoid) plate and spread evenly over the entire surface using a sterile glass spreader. The plates were then incubated at 25°C for 48 hours.

Coagulase-positive staphylococci

A volume (0.1 mL) of the 10^–1 dilution of prepared sample was aseptically dispensed onto a Baird Parker Agar (Oxoid) plate and spread evenly over the entire surface using a sterile glass spreader. The plates were then incubated at 37°C for 48 hours.

Salmonella and Shigella

Twenty-five grams of each sample was added to 225 mL of tetrathionate broth (Difco, Franklin Lakes, NJ). The suspension was then incubated at 37°C for 2 hours. A loopful of the suspension was then streaked onto a Xylose Lysine Desoxycholate (Oxoid) plate and incubated at 37°C for 48 hours.

Challenge testing to investigate the ability of foods to support growth of foodborne pathogens and E. coli

Ten grams of each food was added to 90 mL of sterile saline in blender bags and homogenized in a Stomacher for 2 minutes. The food suspension was then transferred to McCartney bottles and sterilized by autoclaving at 121°C for 15 minutes. The pH of each sample was measured with pH indicator strips (pH range 0–14; Merck, Darmstadt, Germany). The bacteria used were B. cereus (ATCC 11778), E. coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), and Salmonella enterica serovar Typhimurium (ATCC 13311). Bacteria were cultured on Nutrient Agar (Oxoid) plates at 37°C before being grown in Tryptone Soy Broth (Oxoid) overnight. All overnight cultures were standardized by matching to the McFarland 0.5 Turbidity Standard using sterile saline to produce inocula of approximately 1.5 × 10⁸ cfu/mL. A volume (0.01 mL) of standardized culture was added to 3 mL of sterile sample, mixed by vortexing, and incubated at 25°C. At 1-hour intervals, for a total of 6 hours, a 0.1 mL aliquot was dispensed into 0.9 mL of sterile saline. Serial 10-fold dilutions were performed in sterile saline and 0.1 mL aliquots spread onto the nutrient agar plates. The plates were then incubated at 25°C overnight. A viable count was then performed, and the number of cfu/mL was recorded.

Results and Discussion

The analysis of each product was conducted to determine the microbiological quality of the samples (Table 1). The purpose of the analysis was to provide an indication of the types of viable microbes that were able to contaminate the products under real storage conditions. The tests described below were conducted as part of the microbial survey. The standard plate count (SPC), or viable count, is commonly used to indicate the microbiological quality of food. These tests vary according to the type of food and processing (FSANZ, 2001). For the types of foods tested in this study, the satisfactory level of microorganisms is <10⁶ cfu/g. Enterobacteriaceae are indicator organisms and include bacteria found in the intestinal tract, including pathogens such as Salmonella. Enterobacteriaceae are useful indicators of hygiene and postprocessing contamination of heat-processed foods. Their presence in high numbers (>10⁴/gram) in RTE foods indicates that an unacceptable level of contamination has occurred, or there has been underprocessing (FSANZ, 2001). However, Salmonella should not be detected in 25 g of sample. E. coli is undesirable in RTE foods, and levels exceeding 100 cfu/g are unacceptable because they indicate poor hygiene conditions that have led to the contamination or inadequate heat treatment (FSANZ, 2001). Yeasts and moulds, although not considered pathogens, may affect the quality and taste of food and cause spoilage. Moulds can be indicative of aging and can produce toxins that may affect human health. Coagulase-positive staphylococci are indicative of contamination through poor food handling and/or inadequate temperature control, and occur largely as a result of human contact (FSANZ, 2001). Levels of >10⁴ cfu/g are considered potentially hazardous because consumption of food with this level of contamination may result in foodborne illness. Salmonella and Shigella are pathogenic in small quantities, and therefore RTE foods should be free of these organisms. Their presence indicates poor food preparation and handling practices such as inadequate cooking or cross-contamination (FSANZ, 2001). Food handlers may be asymptomatic carriers of Salmonella.

Table 1.

Microbiological Survey of Ready-to-Eat Foods and Growth of Foodborne Pathogens and Escherichia coli

				Growth of
Sample	pH	SPC (log cfu/g)	Yeasts/moulds (log cfu/g)	Bacillus cereus	Escherichia coli	Staphylococcus aureus	Salmonella
Kueh talam	6.5	>6.48	>3.48	++	++	+	++
Che dau	6.0	>6.48	>3.48	++	++	+	++
Bánh tét nhân mặn	6.0	4.77 + 0.04	1.80 + 0.06	++	+	+	++
Nem chua	4.5	2.00 + 0.12	0	–	–	–	–

Microbial counts expressed as mean (n = 4) log₁₀ cfu/g ± standard deviation. + , 1 log increase in viable bacteria in 6 hours; ++, 2 logs increase in viable bacteria in 6 hours; –, no growth or <1 log growth of bacteria in 6 hours.

SPC, standard plate count.

Although Salmonella, Shigella, coliforms, and E. coli were not detected in kueh talam and che dau, their respective high SPC indicated unsatisfactory microbiological quality, according to the guidelines for the microbiological examination of RTE foods (FSANZ, 2001). There is a possible correlation between the results of the SPC and the presence of yeasts and moulds, such that the yeasts and moulds were also able to grow on the SPC agar. Another possible explanation for the high SPC is the presence of a large number of spoilage organisms.

The survey results of nem chua and bánh tét nhân mặn indicated acceptable microbiological quality; however, testing for the presence of other foodborne pathogens, such as Clostridium perfringens, B. cereus, Campylobacter spp., and Listeria monocytogenes was not conducted. The presence of these or other organisms would reduce the microbiological safety of this product.

The sampling strategy used in this study means that the results are not representative of the microbiological quality of these products in general, but provide an indication of the microbiota that may be commonly found in these foods.

All but one of the foods tested was able to support the growth of foodborne pathogens and E. coli (Table 1). The time frame selected for these studies was 6 hours, because it is outside the “2-hour/4-hour rule” exception to temperature control (FSANZ, 2006). The tests were incubated at 25°C to imitate the ambient temperature of retail outlets where the products are sold. Kueh talam, che dau, and bánh tét nhân mặn were best able to support the growth of pathogens with 1–2 log increases in viable bacteria over the 6-hour test period. Bánh tét nhân mặn may be cooked prior consumption and thus reduce the number of contaminating Salmonella to acceptable levels. However, the heat-stable toxin produced by B. cereus would not be inactivated by cooking. The low pH of nem chua is probably the reason why the food did not support the growth of the challenge bacteria. This may also explain the low level of microbial contaminants detected in this food during the microbiological analysis. A possible limitation of the study is that samples were sterilized and diluted before testing which may have affected the chemistry of the foods. Sterilization would also have removed any competition for nutrients by resident nonpathogenic microbiota, which may have increased the ability of the challenge bacteria to grow and multiply in the food. Therefore, further challenge testing of the foods directly (without dilution) should be performed.

Conclusions

The results of this pilot study indicated that the culturally diverse RTE foods tested may pose a food safety hazard, especially given that the foods are normally stored at room temperature and that two of the four samples did not meet the standards prescribed by the Food Standards Australia New Zealand guidelines with respect to the microbial quality. In this pilot study, two of the four samples tested were deemed microbiologically unacceptable according to the Australian food standards. In addition, three foods were also able to support the growth of foodborne pathogens and E. coli under ambient incubation conditions. In this instance, the correct storage of these RTE foods, including appropriate refrigeration, should be considered. This pilot study has also indicated that the investigation into the microbial quality of culturally diverse RTE foods of this nature is further warranted.

Footnotes

Acknowledgment

We thank Mr. Ngan Nguyen for advice on testing methods.

Disclosure Statement

No competing financial interests exist.

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