Microbial Quality of Frozen Chicken Parts from three Import Countries into the Kumasi Metropolis of Ghana

Background: There is an upsurge in the consumption of chicken meat leading to a high influx of imported frozen chicken parts into the Ghanaian markets with little information on their microbial qualities. This study examined the microbial quality of imported frozen chicken parts from three major import countries (USA, the Netherlands and Brazil) into the Kumasi Metropolis. Methods: A total of 45 chicken meat parts of 15 thighs, wings and backs from wholesale cold stores market in the Kumasi Metropolis were randomly sampled for laboratory examinations. A ten-fold serial dilution was performed on each homogenized chicken parts to determine microbiological quality using Plate Count Agar [1], MacConkey Agar (MCA), Mannitol Salt Agar (MSA) and Desoxycholate Citrate Agar (DCA) for the total viable count (TVC), total coliform count (TCC), Staphylococcu s and Salmonella spp counts respectively incubated at 37 o C for 48 hours. Sabouraud Dextrose Agar (SDA) was used for fungal counts. We identified bacterial and fungal isolates using appropriate laboratory and biochemical tests. Descriptive data analysis was carried using SPSS-IBM version 16. Results: Mean TVCs of 5.93, 5.98 and 6.14 log 10 cfu/g were recorded for frozen chicken meats from the USA, the Netherlands and Brazil respectively. Means TCCs of 6.14, 5.93 and 5.98 log 10 cfu/g were obtained for chicken meats from Brazil, USA and the Netherlands respectively. Staphylococcus spp. ( 35.4%), E. coli ( 26.2%) , Salmonella spp. ( 24.6%), and Klebsiella spp . (13.8%) were isolated with Aspergillus spp (33.3%), Rhizopus spp (27.3%), Penicillin spp (24.2%), and Cladosporium spp (15.2%). Chicken thighs, backs and wings recorded 46.2%, 29.2% and 24.6% bacterial contaminants in this order. Bacterial isolates of 49.2%, 28.8% and 22.0% were recorded in frozen chicken meat products from Brazil, the Netherlands USA respectively. Conclusion: The results suggest that imported frozen chicken meats into the Ghanaian market have moderate quality with potential pathogens such as E. coli and Salmonella spp .


Introduction
Poultry production is dominated by chicken in many parts of the world due to the relative ease of its production [2] and serve as a major source of animal protein [3]. Meanwhile, chicken meat is known to cause many zoonotic foodborne infections in the world [4]. Global food production and consumption systems, therefore, required that food quality and safety be scrutinized and examined for public health safety [5,6]. Poultry and poultry meat are however prone to contaminations with potentially pathogenic microorganisms such as Salmonella, Campylobacter, S. aureus, E. coli and Listeria [7].
In Ghana, local broiler meat production is estimated at 35,000 tons, less than 25% of local demand [8]. Meanwhile, there is an upsurge in chicken meat consumption with a corresponding increase in demand for frozen chicken products [9,10]. An expected 14,000 to 158,000 tons is therefore required local demand [8] and this led to an influx of imported frozen chicken parts. Moreover, market prices of local broiler meat are 30-40% margin higher than imported frozen chicken meat making local poultry production uncompetitive [8, 11,12]. Furthermore, consumers prefer imported frozen chicken products because these come pre-cut parts of legs-quarters, thighs, breasts and wings and are affordable [13,14].
Due to International Trade Liberation, the Ghanaian markets are inundated with frozen chicken products from major import nations such as the United States of America (USA), the Netherlands and Brazil whose microbial qualities cannot be guaranteed [15]. The Kumasi Metropolitan Assembly and the Food and Drugs Authority of Ghana frequently ceased expired frozen chicken during routine surveillance at the Asafo market. However, there is a paucity of data on the microbial quality of pre-cut and packaged imported chicken meat products in the Kumasi Metropolis of Ghana [16].
This study, therefore, assessed and compared the microbial quality of imported frozen chicken parts from three major import countries (United States of America, the Netherlands and Brazil) into the Kumasi Metropolis of Ghana.

Study Area
The study site was the Asafo Market in the Kumasi Metropolis (Figure 2). The market is a major hub for wholesale cold stores and is a repertoire for about 95% of all imported frozen meats and fishes from the Tema harbour of Ghana. Poultry production in the metropolis accounted for 28.07% of the total poultry production in Ghana [17]. Ashanti Region is located in the middle belt and the third largest of the 16 administrative regions in Ghana. Kumasi Metropolis has an estimated population of 1,730,249; 36.2% of the regional population [17]. The region has 10.2% of the total land area of Ghana with a population of 4,780,380. The region lies between longitudes 0.15W and 2.25W, and latitudes 5.50N and 7.46N and has 27 districts with Kumasi the regional capital.

Study Design
This was a descriptive cross-sectional study designed to assess the microbiological quality of imported chicken parts sold in wholesale cold stores at the Asafo Market in the Kumasi Metropolis of Ashanti Region, Ghana.

Study Population and Size
A total of 30 licensed importers with large wholesale cold stores for frozen chicken at the Asafo market were involved. Three chicken parts (thighs, wings, and backs) were each collected from the three import countries (United State of America, Brazil and the Netherlands). A sample size of 45 comprising of 5 each of pre-cuts chicken thighs, wings, and backs from each import country was collected.

Sampling Technique
Multistage sampling; purposive, stratified and simple random techniques were employed. Firstly, wholesale import cold stores with frozen chicken meats from the three import countries (USA, Brazil and the Netherland) were purposively selected. Stores were further stratified into three import countries from which at least four cold stores were randomly sampled from each group.
Five newly arrived cartons of frozen chicken parts (chicken thighs, wings, backs) were then purchased from each import country for the study (table 1). These were transported under the ice to the laboratory for analysis.

Sampling Procedures
Aseptic sampling techniques were used to sample one kilogram of frozen chicken parts (thighs, wings and back) from each carton into sterile plastic envelope bags, sealed, and stored under the ice for the microbiological analysis.

Sample Preparation
A 25g portion of each chicken parts were excised aseptically into a sterile stomacher bag containing 225ml of sterile buffered peptone water and homogenized. Each homogenate was then used for serial dilution.

Serial Dilution and Culturing
A ten-fold serial dilution was performed on each homogenate for cultivation and identification of microbial contaminants as previously described [18,19]. Meanwhile, fungal isolates were enumerated and identified by morphological characteristics on Sabouraud Dextrose Agar (SDA) incubated at room temperature [20].

Identification of Fungi
The fungal isolates were identified by their morphological characteristics on Sabouraud Dextrose Agar (SDA) and microscopic examination using lactophenol cotton blue staining technique as previously described [20, 22].

9 Statistical Analysis
Data obtained from the microbial analysis were entered into Microsoft excel 2007 spreadsheet.
The means microbial counts were computed (cfu/g) and transformed into log10cfu/g. The data was subsequently imported into IBM-SPSS software version 16.0 and analyzed. Descriptive statistics were used to summarize the distribution of various variables into tables and figures. The mean counts of the three chicken parts and the country of import were compared using one-way analysis of variance (ANOVA) at a 95% confidence interval (P<0.05). Table 2 shows the mean log cfu/g counts of TCC, TVC, and Salmonella and Staphylococcus aureus for chicken meats from the three import nations. Chicken meat parts from Brazil had means TCC of 6.14 mean log10cfu/g whereas the Netherlands was 5.98 mean log10cfu/g. Chicken meats from Brazil recorded TVC of 6.14 mean log10cfu/g whereas the least was from the USA. The highest counts for Salmonella species and Staphylococcus aureus were from the USA. There were no significant differences (P > 0.05) in mean counts for chicken parts in the USA, the Netherlands and Brazil.      Table 5 shows the number of bacteria isolated on chicken parts from the three countries of import. Chicken thighs had the highest numbers of isolates (46.2%), followed by backs (29.2%) and wings (24.6%).

Fungi Isolates
In Table 6, mean log cfu/g for fungal counts for chicken products were 2.03, 2.06 and 1.97 for the USA, the Netherlands and Brazil respectively.   (9). Out of the 45 chicken sampled, a total of 25 (55.56%) had fungal growth of which 28%, 32% and 44% were isolated from the USA, the Netherlands and Brazil respectively 4.0. Discussion

Microbiological Quality
In this study, means total aerobic plate count of >5 log10cfu/g were reported for imported frozen and Nepal [31] respectively.
Mean total coliform counts and Salmonella counts of >4log10cfu/g were reported in this study consistent with 4.97 log10cfu/g and 6.5 log10cfu/g reported in previous studies [32,33]. The means counts in our study were higher than 3.14 to 3.80 log10cfu/g reported for chicken meat products from supermarkets in Ghana [28]. Higher coliform counts of 7.0 log10cfu/g and 32.2 log10cfu/g were also reported in previous studies contrary to this study [34,35]. In this study, total Staphylococci spp. of >4log10cfu/g were reported in agreement with previous studies [35,36] but higher than 1.99 log10cfu/g reported in market meat elsewhere [37].
We suspect that the differences in bacteria counts for chicken meats in this study and previous studies might be linked to technologies used, hygienic conditions during slaughtering, processing, packaging, storage and distribution [37][38][39][40]. A study using ISO 22000:2005 certified poultry processing plant reported remarkable reductions in bacteria counts in the four sequential processing steps [37,39]. The final washing, freezing and packaging are the critical control point identified to control microbial contaminations [37,39]. These processes might differ for poultry processing plants for each import country and hence the differences.

Bacteria isolates
Klebsiella spp., Salmonella spp., Staphylococcus aureus, and Escherichia coli were microbial contaminants isolated in this study. These bacteria are common contaminants of poultry meats [37]. Microbial contaminations occurred largely at evisceration due to faecal material from the cloaca of chicken carcasses [41]. Moreover, frozen chicken meat products imported into Ghana are subjected to several freeze-thaw cycles during transportation over long distances, through transit from ports to cold storage facilities providing favourable conditions for microbial proliferation. The presence of these bacteria is an index of the hygiene quality of the chicken meat.
Several studies also reported similar microbial contaminants consistent with this study [42,43].
Chicken products from Brazil recorded the highest bacterial isolates relative to the Netherlands and USA. We suspect the differences could result from differences in technologies employed in poultry husbandry and processing of the meats [40]. Also, the animal health, rearing conditions, quality of slaughtering, processing, packaging and storage conditions [44,45]. Although largescale slaughterhouses is a rapid and highly automated process, notwithstanding, considerable opportunities exist for contamination [46,47]. Hence, the performance of the slaughter process is very critical and has a direct consequence on the microbial quality of the chicken meat products [48]. This could have accounted for the observed differences in numbers of the bacteria isolates.
We obtained 26.2% and 24.6% prevalence for E. coli and Salmonella of which more than 50% were recorded for frozen chicken meat products from Brazil. These values are much lower than 66.7% and 91.7% reported for frozen chicken meat at retail outlets in Ghana [16]. These values in our study were fairly consistent with 27.82% and 13.64% obtained for Escherichia coli and Salmonella spp. but inconsistent with 18.52% and 1.84% for Staphylococcus aureus and Klebsiella spp. reported in Nigeria [43]. The presence of 35.4% S. aureus in chicken meat products of this study is inconsistent with 17.9% obtained by Kozačinski and colleagues in Croatia [32]. The presence of 24.6% Salmonella of this study is fairly similar to 20% reported in broiler chicken [49] but much lower than 91.7% in chicken drumstick [16].
The presence of S. aureus, E. coli and Salmonella points to possible contamination at the plants coupled with transportation and storage challenges [50] and raises microbial safety standard credibility. The mere presence of these bacteria in raw meat may not necessarily pose a health threat to consumers since the meats would be subjected to cooking. Compared with the permissible limits stipulated by [23-25] for coliforms counts, S. aureus, E. coli and Salmonella spp. counts for chicken breast, backs and thigh samples examined were slightly higher and unacceptable.

Fungal Isolates
Fungal contaminants, Aspergillus, Penicillium, Cladosporium and Rhizopus were isolated, consistent with previous studies [50]. Aspergillus and Rhizopus species were reported in chicken meat retailed in Lahore City, Pakistan [51] in agreement with this study. Candida and Cryptococcus spp. reported in poultry meat (drumstick and breast) retailed in Local Iraqi Markets [52] were not reported in our study. The presence of 33.3 % Aspergillus spp. and 24.2 % of Penicillin spp. in our study are in agreement with 35.7 % and 21.4% reported for Aspergillus spp.
However, Neocosmospora spp. and Mucor spp. reported by [53] were not found in this study.
Fungal contaminations in chicken meat products may occur due to improper processing and temperature fluctuation during storage and transportation [22,50]. The presence of fungal contaminants in chicken meat products poses a health threat to consumers. Some fungi under certain environmental conditions release secondary metabolites such as mycotoxins which could cause serious disorders in humans and animals [54]. Mycotoxins such as aflatoxins, ochratoxins, etc. produced by fungi like Aspergillus, Fusarium and Penicillium [55,56] in food could cause severe health problems such as allergic reactions, cancer, and organ damage [57]. Although, there is a paucity of information on the acceptable limits for fungal contaminants in water and food, the presence of mycotoxigenic fungi pose public health concerns.

Conclusion
The results of this study highlighted the microbial quality of pre-cut imported frozen chicken meats into the country stored in major cold stores in the Kumasi metropolis. The presence of potential foodborne pathogens is linked to possible contaminations at slaughterhouses and transportation from ports to cold storage facilities over long distances with several freeze-thaw cycles. Therefore regular monitory by regulatory authorities together with stakeholders is critical to ensure frozen chicken imported into the country are safe for public health good.