Microbial profile of some ready-to-cook frozen food items sold in Dhaka city, Bangladesh

M. Shamimuzzaman, Raji Kanti Roy, Toma Rani Majumer, Nirmal Chanra Barman,Nazia Nawsha Lina, M. Tarek Hasan, Bipla Kumar Dash,*

a Department of Microbiology, Jashore University of Science and Technology, Jashore 7408, Bangladesh

b Department of Nutrition and Food Technology, Jashore University of Science and Technology, Jashore 7408, Bangladesh

c Department of Genetic Engineering and Biotechnology, Jashore University of Science and Technology, Jashore 7408, Bangladesh

d Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia 7003, Bangladesh

Keywords:

Frozen food

Foodborne pathogens

Dhaka city

Microbiological quality

Aerobic plate count

A B S T R A C T

This study aimed to assess the microbiological quality of 159 animal- and plant-based ready-to-cook (RTC)frozen food items currently popular in Dhaka. Microbiological analyses were carried out to enumerate total aerobic counts of mesophilic bacteria, yeast and mold and to detect some common foodborne pathogens according to standard microbiological criteria. Higher microbial loads in all animal-based food types, other than chicken strips were found. In contrast, plant-based items were relatively safe except paratha, dal-puri,rooti chapati, and aloo-puri. Several common pathogenic bacterial species were screened by analyzing their morphological, cultural, and biochemical characteristics. Escherichia coli was prevalent in most samples,while Salmonella species were least reported. A considerable number of samples exhibited enough microbial loads at infection or intoxication levels.

1. Introduction

Food is one of the five necessities for human beings, and its safety in terms of microbial load and nutritional balance is so preferred. In recent years, especially the young and office working city dwellers in Bangladesh have inclined to consume RTC frozen foods. The key factors for increased intake of these RTC foods are their quicker processing time, organoleptic appeal, and availability throughout the year [1]. The modern sedentary lifestyle and increased number of nuclear families have also encouraged people to depend more on these frozen food items [2]. Although the nutritional values and cost of these frozen items are sometimes compared to the raw and homemade items,the consumers of the former group are increasing notably with time.

After processing and partial cooking, RTC frozen foods are preserved at freezing temperature (–18 °C or below) to halt spoilage microorganisms’ activities and thereby preserve the quality and maintain nutritional values and freshness for a long time. These frozen foods can be processed from animals and plants, including fruits,vegetables, and wheat-based products. Currently, over 40 frozen RTC food items, including paratha, roti chapati, samosa, meatball, chicken nugget, burger, and spring rolls, are available in Dhaka, Bangladesh,whose market value has crossed 95 million USD with more than 20% yearly expansion [3]. This progressing demand has made domestic companies start producing these food items locally.

Although frozen items have an excellent overall safety record,a few previous outbreaks of foodborne illness associated with these foods demand careful monitoring of the critical production and processing steps of raw materials [4]. Most reported foodborne pathogens, includingSalmonellaspp.,Staphylococcusspp.,Clostridiumspp.,Listeriaspp., andBacillus cereus, are nonpsychrophilic, i.e., cannot multiply in freezing conditions [5].Maintaining the standard food processing, storage, and handling practices is mandatory to exclude, whenever possible, these pathogens in products or minimize their presence to the most significant degree before freezing [6]. Food spoilage bacteria deteriorate frozen food products and develop unpleasant odors, sour tastes, and bad smells. They change textures or flavors to make the foods unsuitable for consumption. Several studies reported direct associations of various gastrointestinal disorders, as well as typhoid fever and tuberculosis with the consumption of contaminated foodstuffs [7-9].Foodborne intoxication may also occur due to consuming frozen foods contaminated with higher toxin-producing bacterial and fungal counts, as these toxins cannot be destroyed even by cooking at high temperatures. Interestingly, toxins of different microbial species may not be responsible for altering food texture, odor, or flavor [10].

To date, knowledge about the microbial quality of popular RTC frozen foods in Bangladesh is minimal. Sultana et al. [11]studied bacterial count and antibiotic resistance pattern of bacterial isolates obtained from 20 RTC frozen food samples of animal origin sold in Dhaka. In another study, Shammi [12]reported the presence ofVibriospp.,Salmonellaspp., andShigellaspp. in 8 animal-based frozen food samples by enrichment culture technique. However, these reports are insufficient in terms of types and number of food samples tested and microbiological parameters analyzed. Therefore, the present study aimed to provide a broader insight into the microbiological quality of 16 distinct (159 samples in total) animal- and plant-based RTC frozen foods sold in Dhaka.

2. Materials and methods

2.1 Collection and preparation of samples

Total 159 frozen RTC food samples were purchased randomly from local markets in Dhaka. The samples comprised 2 broad categories: animal- and plant-based frozen foods. Animal-based foods included 10 different frozen items from 3 distinct sources: chicken(nuggets, wings, sausage, cutlet, ball, stripes, samosa, and spring rolls), beef (samosa), and shrimp (spring rolls). Likewise, plantbased foods contained 6 different frozen items: paratha, dal-puri,roti chapati, vegetable spring roll, samosa, and aloo-puri. Following collection, samples were first delivered immediately to the laboratory in the frozen state. Then, measured samples (25 g, in triplicate) were dissolved in 225 mL buffered peptone water (BPW; Oxoid, UK) and homogenized unless otherwise stated. Subsequently, homogenized samples were diluted serially by 10-fold (10-1to 10-6) using BPW to analyze aerobic bacteria and total yeast and mold counts. Diluted samples were also screened forStaphylococcus aureus,Bacillus cereus,Listeriaspp.,Salmonellaspp.,Vibriospp., andEscherichia coli. Standard aseptic conditions were maintained in every step of handling samples.

Standard microbial food safety limits (satisfactory, unsatisfactory,or borderline) given by different regulatory authorities were employed to access the safety concern of the RTC frozen foods.Several standardization institutions like the European Commission,the Philippines Food and Drug Administration, United States Food and Drug Administration, and Australian Standards set up specific standard limits for different microorganisms in various food products.In Bangladesh, such formal rules and regulations for food standards and risk assessment of frozen foods have not been developed yet.Therefore, existing global standards were used to measure the food safety of food samples tested in this study.

2.2 Enumeration of aerobic plate count (APC)

The standard pour plate method was followed to enumerate aerobic plate count for mesophilic bacteria, as described previously [13].Sterile Petri plates containing an aliquot of 1 mL appropriate dilutions were poured with autoclaved melted (at around 45 °C) plate count agar (PCA; Difco BD, USA). After incubation at 37 °C for 24–48 h,plates having well-spaced colonies (30–300) were counted by a colony counter (Stuart Scientific, UK). The final count per gram was obtained by multiplying the average number of colonies per plate by reciprocal dilution and expressed as a logarithmic value of lg(CFU/g)of a sample [14]. Representative colonies were isolated, purified, subcultured on nutrient agar slants, and maintained at 4 °C until further characterization.

2.3 Enumeration of total yeast and mold

The standard spread plate technique was followed to determine total yeast and mold count. An aliquot of 0.1 mL suspension and its decimal dilutions (10-1and 10-2) were spread on Sabouraud dextrose agar (Merck, Germany) and incubated at 25 °C for 5 days. Creamy white or whitish colonies were characterized as yeasts and fluffy or rough colonies as molds [15].

2.4 Detection of S. aureus

An aliquot of 0.2 mL suspension and its 10-1and 10-2dilutions were spread on Baird Parker agar (Merck, Germany) plates and incubated at 37 °C for 48 h. Convex, shiny and black or grey colonies surrounded by a clear zone were typicalS. aureus. Shiny black or grey colonies with or without a narrow white edge and clear halos were considered atypical. A representative number of these colonies were cultured in brain heart infusion (BHI) broth (Oxoid, UK) to confirmS. aureusby tube coagulase and thermostable nuclease tests [16].

2.5 Detection of B. cereus

An aliquot of 0.1 mL suspension (from 10-1and further decimal dilutions, when necessary) was cultured on mannitol egg yolk polymyxin agar (Oxoid, UK) and incubated at 30 °C for 18–40 h. Bright pink colonies with egg yolk precipitation zones were considered typicalB. cereus. A representative number of these colonies were transferred to BHI broth with 0.1% glucose for enterotoxin production studies. According to sections F and H of BAM online, further biochemical tests were performed to confirm typical colonies asB. cereus[17].

2.6 Detection of Listeria spp.

Listeriaspp. were detected following the procedure described by Hitchins et al. [18]. Twenty-five-gram samples blended with 225 mL bufferedListeriaenrichment broth (BLEB, with additives;Oxoid, UK) were incubated at 30 °C for 24–48 h for pre-enrichment.At 24 and 48 h, BLEB suspensions were streaked on both Oxford agar (OXA; Oxoid, UK) and Oxoid chromogenicListeriaagar(OCLA; Oxoid, UK) plates and incubated for up to 48 h. Plates were checked after 24 and 48 h for typical colonies. On OXA, after 24 h incubation at 35 °C, typicalListeriacolonies appeared grey to black surrounded by an approximately 1 mm black halo. Following 48 h incubation, typicalListeriacolonies appeared black with a black halo and approximately 2–3 mm sunken center.Listeriaspecies appeared as 1–3 mm blue or green colonies on OCLA at 37 °C. Additionally,L. monocytogenesandL. ivanoviicolonies appeared opaque white with halos surrounding the colonies. Up to 5 typical colonies from each OXA and OCLA were streaked for purity on tryptone soya yeast extract agar (HiMedia, India) and incubated at 35 °C for 24–48 h.Typical colonies were 1–2 mm entire-edged convex, colorless, and opaque. Several biochemical and Christie–Atkins–Munch-Peterson tests were performed for further confirmation ofListeriaspecies.

2.7 Detection of Salmonella spp.

The procedure described by Andrews et al. [19]was employed to detectSalmonellaspecies. Ten milliliter pre-enriched BPW suspensions (incubated at 37 °C for 18 h) selectively enriched in 100 mL sterilized selenite cystine broth (Oxoid, UK) were incubated at 37 °C for 24–48 h. One loopful enriched inocula was streaked on preincubated bismuth sulfite agar (BSA; Oxoid, UK) and xylose lysine deoxycholate (XLD) agar (Oxoid, UK) plates. TypicalSalmonellaspp. produced pink colonies with or without black centers on XLD agar and brown, grey, or black colonies on BSA agar.Suspected colonies were identified by their cultural, morphological,and biochemical characteristics, namely, growth in triple sugar iron(TSI) agar, urease test, MR-VP test, oxidase test, citrate test, and fermentation of carbohydrates (glucose, sucrose, arabinose, mannose,mannitol, and inositol) and decarboxylase (lysine, arginine, and ornithine) following the taxonomic guide ofBergey’s Manual of Determinative Bacteriology, 8th edition [20]. Cultures with positive biochemical reactions were confirmed by agglutination test withSalmonellapolyvalent (O) somatic antisera.

2.8 Detection of Vibrio spp.

Vibriospp. were detected following the procedure described by Kaysner et al. [21]. Preincubated thiosulfate-citrate-bile salts-sucrose(TCBS) agar (HiMedia, India) and cellobiose polymyxin B colistin(CPC) agar (Merck, Germany) plates streaked with one loopful pre-enriched BPW suspensions were incubated at 37 °C for 24 h.TypicalVibriospp. colonies on TCBS agar were large, yellow, and smooth, whereas, on CPC agar, they were small, smooth, opaque,and green to purple. Suspected colonies were further identified by analyzing cultural, morphological, and biochemical characteristics,including, oxidase test and fermentation of carbohydrates(glucose, sucrose, arabinose, mannose, mannitol, and inositol) and decarboxylase (lysine, arginine, and ornithine) according to the taxonomic guide ofBergey’s Manual of Determinative Bacteriology,8th edition [20]. An agglutination test using the polyvalentV. cholerae(O) antiserum was performed for the final confirmation ofVibriospecies.

2.9 Detection of E. coli

E. coliwere detected following the procedure described by Popovic et al. [22]. Tryptone bile glucuronide agar (Oxoid, UK)plates streaked with one loopful pre-enriched BPW suspensions were incubated at 44 °C for 24 h. TypicalE. colicolonies appeared distinctive green or blue. IMViC and gas production tests in EC broth(Oxoid, UK) at 45 °C confirmed suspected colonies.

2.10 Statistical analysis

Data were analyzed using the statistical package for social science(SPSS version 22.0; SPSS Inc., Chicago). Descriptive statistical analyses like frequency and percentages, means, standard deviation(SD), and range were performed to summarize and represent the information obtained from the 8 microbial parameters used in this study. To correlate the type of studied frozen items with the magnitude of microbial contamination at the bivariate level, Chisquare test, independent samplet-test, and one-way ANOVA followed by Tukey-Kramer HSD post-hoc analyses were employed at 5% level of significance.

3. Results and discussion

The distribution of mean and range of microbial counts (lg(CFU/g))in different chicken-based RTC frozen food samples are presented in Table 1. The types of chicken-based frozen items significantly affected their loads of microbial contamination. While chicken spring roll had the maximum APC of 7.24 (lg(CFU/g)), followed by that of chicken sausage of 7.21 (lg(CFU/g)), the least APC was present in chicken strips (3.65 (lg(CFU/g)). Except strips and wings, most chicken items themselves were significantly different inListeriacontamination.

The distribution of mean and range of microbial counts (lg(CFU/g))in different plant-based RTC frozen food samples are presented in Table 2. Their type significantly influenced the magnitude of microbial contamination present in plant-based products. Among 6 types of plant-derived frozen items, on average, vegetable spring roll and roti chapati had the maximum and minimum average of APC, respectively. While these products contained significantly altered counts of APC, yeast and mold, andS. aureus, they presented exceptions forB. cereus,Listeriaspp.,Vibriospp., andE. colicounts.Only vegetable samosa and spring rolls were significantly distinct forListeriaspp. andVibriospp. counts.

Table 1Distribution of mean and range of microbial counts (lg(CFU/g)) in different chicken-based ready-to-cook frozen food samples.

Table 2Distribution of mean and range of microbial counts (lg(CFU/g)) in different plant-based ready-to-cook frozen food samples.

The distribution of mean and range of microbial counts (lg(CFU/g))in different samosa and roll-based food samples are presented in Table 3. Based on their primary sources mainly 3 types of samosas(chicken, beef, and vegetable) and spring rolls (chicken, shrimp, and vegetable) are available in local markets of Dhaka. A statistically significant relationship between the type of samosas and spring rollsand the level of contamination by microbial parameters was observed.Beef samosas were substantially predominant in average counts of most microbial parameters, with chicken samosas mostly coming second. In contrast, vegetable samosas had the minimum average of these contaminations. InListeriaspp. andE. colicontamination of frozen items, chicken samosa was the worst. Likewise, the category of spring rolls also had a significant association with their contamination levels by different microorganisms. In most cases,while chicken spring rolls showed the highest, vegetable spring rolls had the least microbial loads compared to other corresponding rolls.Chicken spring rolls were approximately two times more overgrown with APC andB. cereusthan vegetable spring rolls. Since samosas and spring rolls are supposed to be processed following similar and standard manufacturing procedures, remarkable fluctuations in allmicrobial loads within them can be rationalized by their wide range of ingredients (Tables 4 and 5) and variable food safety and hygiene issues practiced during preparation of these foods.

Table 3Distribution of mean and range of microbial counts (lg(CFU/g)) in different samosa- and roll-based ready-to-cook frozen food samples.

Table 4Ingredients of animal-based ready-to-cook frozen foods used in this study.

Table 5Ingredients of plant-based ready-to-cook frozen foods used in this study.

The presence of yeast and mold was satisfactory for most vegetable items in this study. This parameter’s most outstanding mean value was 3.36 (lg(CFU/g)) for beef samosa, while no fungus was found in roti chapati. Animal-originated samples had a greater unsatisfactory rate; more than 20% of chicken nuggets were unsafe for consumption due to fungal contamination. Both yeast and mold cause various degrees of deterioration and decomposition of foods.They can cause hazards to human health because of their ability to produce toxic metabolites known as mycotoxins [23].

The percent of unacceptable chicken-based RTC frozen items due to increased growth of APC centered around 40% –60% (Table 6).The unsatisfactory status due to the increased growth of APC, yeast and mold,S. aureus,B. cereus, andVibriospp. was not significantly affected by the types of these chicken-based frozen foods. However,in terms ofListeriaspp. andE. coli, the unacceptable rate was significantly related to the kinds of chicken products.

Table 6Frequency percentage of positive and unsatisfactory chicken-based ready-to-cook frozen samples according to different microbial parameters.

The relationship among the types of RTC frozen samosa and roll samples and their unsatisfactory status caused by different microorganisms are presented in Figs. 1 and 2. The types of samosas were not significantly related to their poor food safety status, caused by all kinds of microbial growth exceptS. aureus. A hundred percent vegetable samosa was safe in terms ofS. aureuscontamination,whereas about 60% of each chicken and beef samosa remained unsuitable for human consumption. Based on the food safety limits of most microbial parameters, more than half chicken and beef samosa samples were unacceptable for consumption. ConsideringListeriaspp. contamination, about 3/4 chicken samosa was unacceptable, followed by about 2/3 forB. cereusandE. coli. The high contamination byB. cereusandE. coliindividually caused 3/4 beef samosas unsafe for consumption; that was the highest unsatisfactory rate of samosas caused by any microorganism.B.cereus,Listeriaspp., andE. colihad the worst similar impact on the quality of all 3 samosas. Excessive contamination by each of these microbes individually led more than half samosas to be unsafe for consumption. The overgrowth ofB. cereusandListeriaspp. made approximately 78% of chicken spring rolls unsatisfactory for intake. Again, about 2/3 chicken spring rolls were considered unsafe according to the safety limits of APC,Vibriospp., andE. coli. Accordingly, microbes likeB. cereus,Vibriospp., andE. colimade 2/3 shrimp spring rolls unsuitable for consumption. The food quality of vegetable spring rolls was also low. More than half of these rolls were unsatisfactory due to increased contamination byE. coli(73% ),B. cereus(63% ), andVibriospp. (63% ). However, the types of frozen spring roll were not significantly associated with the unacceptable level of these products caused by any of the microbial parameters studied.

Fig. 1 Relationship between types of ready-to-cook frozen samosa samples and their unsatisfactory status caused by different microorganisms.

Fig. 2 Relationship between types of ready-to-cook frozen roll samples and their unsatisfactory status caused by different microorganisms.

The frequency percentage of positive and unsatisfactory plant-based frozen samples according to different microbial parameters is presented in Table 7. The safety status of plant-based RTC frozen foods in terms of APC was not significantly related to the types of these frozen foods. Interestingly, the unsatisfactory rate of allplant-based frozen items was constantly 0% in the case of yeast and mold andSalmonellaspecies. On the other hand, the categories of the frozen items contaminated byS. aureus,B. cereus,Listeriaspp.,Vibriospp., andE. coliwere significantly different in regard of food safety concerns. More than half vegetable spring roll samples were unacceptable due to containing a more unusual number ofS. aureus. Respectively, about 63% and 53% vegetable spring rolls and samosas were not suitable for consumption due to their excessive contamination withB. cereus. Besides,contamination withListeriaspp. was responsible for about 54% and 62% unacceptable vegetable spring roll and samosa,respectively. Furthermore, the poor consumption safety in about 73% and 54% vegetable spring rolls and samosas, respectively,was attributed to the hazardous load ofE. coli.

Table 7Frequency percentage of positive and unsatisfactory plant-based ready-to-cook frozen samples according to different microbial parameters.

In terms ofSalmonellaspp., all plant-based items, along with chicken wings and strips, were safe. Percentages of unsafe chicken sausage and spring rolls were the highest, 28.50% and 33.33% ,respectively.Salmonellacan enter the food chain at any stage of food processing; therefore, consequences for humans after consuming the contaminated end products depend on the food-processing conditions [24]. Meat contamination bySalmonellacan occur via cutting board, grinder, mincers, blinders, and contaminated water used to clean equipment and faulty monitoring devices [25].

The mean distribution of microbial quality parameters (lg(CFU/g))of animal- and plant-based RTC frozen food samples are presented in Table 8. While the magnitude of contamination by every studied microbial parameter was distributed between animal- and plantbased groups, animal-based frozen items significantly had unusually higher growth of all pathogens than that of plant-based items. Other than contamination by APC and yeast and mold, both animal- and plant-based samples were the worst affected byB. cereus, followed byE. coli.

Table 8Mean distribution of microbial quality parameters (lg(CFU/g)) of animal- and plant-based ready-to-cook frozen food samples.

The results in this study demonstrated that animal-originated chicken samples like nuggets, wings, sausage, and cutlets have a greater microbial count than plant-derived samples like paratha, dalpuri, roti chapati, and aloo-puri. These noticeable differences of microbial contamination among chicken items may be credited to their unique ingredients, manufacturing procedures, and the quality of safe food handling practices maintained during productions [5].These high numbers of APC cannot make any food unsafe alone,but they indicate poor handling, storage, or inadequate general hygiene [26]. Sultana and colleagues found almost all tested frozen food samples collected from different supermarkets of Dhaka had higher APC counts whereStaphylococcusspp. were the most prevalent [11]. Chicken-based samples tested in that study were highly contaminated that almost supports our findings.B. cereuswere prevalent in both animal- and plant-based frozen food samples that might be responsible for causing foodborne intoxication [27].Species ofBacillusand related genera have long been taxing to food producers because of their heat resistant endospores [28,29].Besides, other undesirable microorganisms such asE. coli,S. aureus,Salmonellaspp.,Vibriospp.,Listeriaspp., and yeasts and molds have long been reported as food spoilage and foodborne pathogens [30].Fungi are common contaminants of meat and meat products. They either cause spoilage or produce mycotoxins, making the infected meat harmful for consumption [31].

Naturally, animal-originated foods contain a relatively higher number of diverse bacteria and fungi than those of plant origin. Raw or undercooked meat and poultry, raw or lightly cooked egg and egg products, and unpasteurized milk are the most perishable foods. They favor the replication of microorganisms because of high concentrations of rapidly available nutrients and high-water activity [32].On the contrary, plant-based products are primarily spicy and contain many phytochemicals that intrinsically prevent food spoilage microorganisms from overgrowing [33]. At the manufacturing level,proper hygiene practices restrict the microbial count and intoxication in foods at the benign level. The microorganisms detected in this present study demand careful analysis of contamination’s root causes and sources, including raw materials, air, water, equipment, and even food-safety knowledge of employees at critical processing steps in the food production chain [5,34].

This study shows that all animal-originated ready-to-cook frozen foods are contaminated with several common pathogenic indicator microorganisms. At the same time, it is a good sign that plant-based products are relatively safe. As RTC frozen food items usually require heating before consumption that potentially kills pathogens, the level of contamination reported in this study may not directly affect the consumer’s health. However, the possibility of microbial intoxication and deterioration of overall nutritional quality of these foods due to extended growth of microorganisms should be considered carefully.The moderate number and fewer types of samples analyzed in this study limits the authors to throw any general comments about the overall microbial quality and safety of RTC frozen foods sold in Dhaka. Nonetheless, the authors believe this report will encourage authorities and manufacturers to improve and monitor food hygiene practices at every stage of food production, including raw material handling and processing, finished product packaging, and food storing at the proper freezing temperature as well.