Microbial Quality and Chemical Safety of Modern Convective Smoked Guinean Barracuda Fish from Lagos State, Nigeria  

Adeyeye S.A.O.1 , Oyewole O.B.1 , Obadina A.O.1 , Omemu A.M.2 , Abayomi S.O.1
1 Department of Food Science and Technology, Federal University of Agriculture, Abeokuta, Nigeria
2 Department of Hospitality and Tourism, Federal University of Agriculture, Abeokuta, Nigeria
Author    Correspondence author
International Journal of Aquaculture, 2015, Vol. 5, No. 33   doi: 10.5376/ija.2015.05.0033
Received: 15 Sep., 2015    Accepted: 01 Nov., 2015    Published: 04 Jan., 2016
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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 Adeyeye S.A.O., Oyewole O. B., Obadina A.O., Omemu A.M., And Abayomi S.O., 2015, Microbial Quality and Chemical Safety of Modern Convective Smoked Guinean Barracuda Fish From Lagos State, Nigeria, International Journal of Aquaculture, 5(33): 1-10

Abstract

Safety of street-vended smoked fish has become public health issue in Nigeria in recent times. This study therefore was carried out to assess the microbial quality and chemical safety of modern convective smoked Guinean barracuda fish from Lagos State, Nigeria. Fresh fish (100) samples were collected from twenty major processing centres from Lagos State, Nigeria and the samples were smoked with modern convective kiln. The fresh and smoked fish samples were analysed for proximate, quality and rancidity indices (PV, TBA, TVB-N, TMA and FFA), polycyclic aromatic hydrocarbon (PAH), heavy metals and microbiological (coliform, fungi, Listeria monocytogenes, Salmonella paratyphi and Staphylococcus aureus). The results revealed that the mean moisture, protein, fat, crude fibre, ash and carbohydrate contents (%) of smoked fish samples ranged from 8.34-9.78, 57.43-63.71, 13.24-17.47, 1.36-1.92, 1.89-2.89 and 8.29-12.12 respectively. The vales of PV, TBA, TVB-N, TMA and FFA were in the range of 7.23-9.53 meq. peroxide/kg, 1.11-1.51,  1.00-1.51 mg Mol/kg , 15.36-17.91 mgN/kg and 2.03-2.49 mgN/kg respectively. The results of PAHs showed the presence of sixteen (16) PAHs compounds in the smoked fish samples.  However, only few of the sixteen PAHs compounds detected are above the 5.0 μg /kg B (α) P maximum permissible levels stipulated by EU Regulation 1881/2006. The concentrations (μg/g) of Hg, Pb, Cd, and Cr in the smoked fish samples were in the range of 0.0016-0.0024, 0.0012-0.0023, 0.0015-0.0023, and 0.0629-0.0856 respectively. The microbiological analysis revealed the presence of L. monocytogenes and S. aureus in the smoked fish samples. In conclusion, study showed that smoked Guinean barracuda fish may constitute higher risks (chemical) to consumers if adequate precaution is not taken but the samples are microbiological safe.

Keywords
Smoked fish; Microbial quality; Safety; PAHs; Heavy metals

1 Introduction
Fish represents an important part of total fish captures and is an important sector in the national strategies of fight against poverty, food security and food safety (Ahmed, 2011). Fish and fishery products constitute an important food component for a large section of world population, more so in developing countries, where fish forms a cheap source of protein (Amusan et al., 2010; Odu and Imaku, 2013). In the last two decades there has been an increase in awareness about the nutritional and health benefits of fish consumption (Amusan et al., 2010; Odu and Imaku, 2013). The Food and Agriculture Organization (1994) asserted that fish contributes about 60% of the world’s supply of protein and that 60% of the developing world derives more than 30% of their annual protein from fish (Amusan et al., 2010). However, in Nigeria, fish constitute 40% of the animal protein intake (Olatunde, 1998; Amusan et al., 2010). Fish is highly perishable, therefore a considerable effort has been directed to extend the shelf-life of fish using preservation and processing techniques, such as refrigeration, freezing, canning, smoking, salting, drying and frying. Smoked fish constitute a major source of animal protein for a vast majority of the population in Nigeria, particularly the rural population (Eyo, 1992). The drying effects during smoking, together with the antioxidant and bacteriostatic effects of the smoke, allow smoked products to have extended shelf-life (Eyo, 2001).
 
In Nigeria, smoked fish is gradually taken over most of the major markets and streets as people are now accustomed the consumption of smoked fish as a ready-to eat food. School children and adults are now involved in the consumption of fried fish especially to accompany other starch-based foods such as garri, pap, etc (Adeyeye et al., 2015).
 
The smoked fish has become so popular in Nigerian dishes and there is need for corresponding concern for safety issues in smoked fish consumption (Riches, 2012; Adeyeye et al., 2015). Although several researchers have worked on quality and safety of smoked fish, there is drought of data on the quality and safety of street-vended fried fish in Nigeria. Da Silva et al. (2008) examined the microbial safety and quality of smoked blue catfish (Ictalurus furcatus) steaks treated with antimicrobials and antioxidants during 6 weeks ambient storage. Fafioye et al. (2002) studied the fungal infestation of five traditionally smoked dried freshwater fish in Ago-Iwoye, Nigeria and isolated and identified eleven different fungal species of which Aspergillus flavus was the most frequently encountered fungi on the fish species. Adebayo-Tayo et al. (2008) reported the presence of aflatoxin and other metabolites in smoked fish due to Aspergilus flavus in smoked fish sold in Uyo, Akwa Ibom State, Nigeria and confirmed that consumers could have been at risk of aflatoxin poison.
 
Certain heavy metals such as lead, cadmium, mercury and chromium have been detected in smoked fish and have also been recognized to be potentially toxic within specific limiting values (Smirjakova et al., 2005). There are also strong pressures on chemical safety for smoked, barbecued and fried fish products from the EU institutions. Thus the Codex Alimentarius Commission on contaminants in food, at its 29th session from 16 to 20 April 2007 established a reflection on reducing levels of Polycyclic Aromatic Hydrocarbons (PAHs) in food dried and smoked (EC, 2007). In addition, the EU Regulation 1881/2006 requires a formal setting new stricter rule on the content of PAH in smoked products. The presence of PAHs, especially benzo[a]pyrene, in smoked fish has previously been reported (Simko et al., 2002) but little information is available concerning the influence of the smoking and frying processes.
 
This study therefore was carried out to assess the microbial quality and chemical safety of modern convective smokedGuinean barracuda fish from Lagos State, Nigeria.
 
2 Materials and Methods
2.1 Sampling procedure
Fresh Guinean barracuda fish (100) samples were collected from twenty major processing centres from Lagos State, Nigeria by purposive sampling method in frozen sterile containers (Ziploc). The fresh fish samples were taken to the Fish Hatchery Unit, Federal University of Agriculture, Abeokuta, Nigeria for smoking and laboratory analysis.
 
2.2 Proximate analysis 
The proximate composition of all the samples were carried out in triplicates according to the standard method AOAC (2000).
 
2.3 Physico-chemical analysis
Kent pH meter (model 7020, Kent Ind. Measurement Ltd., Surrey, U. K) equipped with a glass electrode was used to measure the pH of the flesh in triplicates, employing 10g of smoked fish homogenized in 10ml of distilled water. The rancidity (quality) indices of all the samples were carried out in triplicates according to the standard method AOAC (2000). All chemicals used in this study were of the analytical grade unless stated otherwise.
 
2.4 Determination of PAHs by Gas Chromatography (GC)
To start the cold extraction 2g of each sample was weighed into a clean extraction container and 10 ml of dichloromethane was added, thoroughly mixed and allowed to settle. Themixture was carefully filtered into clean solvent and rinsed into extraction bottle, using filterpaper fitted into Buchner funnel. The extract was concentrated to 2ml and then transferredfor clean up and separation (this involved further purification of the extract prior to gaschromatographic analysis). To achieve this, 1cm of moderately packed glass wool wasplaced at the bottom of 10mm ID × 250mm long chromatographic column. Slurry of 2gactivated silica in 10ml dichloromethane was prepared and placed into the chromatographiccolumn. To the top of the column 0.5cm of sodium sulfite was added.The column was rinsed with additional 10ml of dichloromethane. The column was pre-elutedwith 20ml of dichloromethane and this was allowed to flow through the column at a rate ofabout 2minutes until the liquid in the column was just above the sulfite layer. Immediately, 1ml of the extracted sample was transferred into the column. The extraction bottle was rinsedwith 1ml of dichloromethane and added to the column as well. The stop-cork of the columnwas open and the eluant was collected with a 10ml graduated cylinder.Prior to exposure of the sodium sulfite layer to air, dichloromethane was added to thecolumn in 1-2ml increments. Accurately measured 10ml of the eluant was collected andlabeled aliphatic. The concentrated aliphatic fractions were transferred into labeled glassvials with rubber crimps caps for GC analysis. 1 μL of the concentrated sample was injectedby means of syringe through a rubber septum into the column. Separation occurs as thevapour constituents’ partition between the gas and liquid phases. The sample componentswere automatically detected as they emerge from the column (at a constant flow rate) by theFlame Ionization Detection (FID) detector whose response was dependent upon thecomposition of the vapor.
 
2.5 Heavy metal analysis
Heavy metal, such as Cu, Cd, Hg and (Pb) in fresh and smoked fish samples were determined by AOAC (2000) method using atomic absorption spectrophotometer. All chemicals used in this study were of the analytical grade unless stated otherwise.
 
2.6 Microbiological studies
The presence of pathogens in smoked fish samples was investigated in the microbiology laboratory. The microbiological procedures recommended by the International Commission on Microbiological Specifi­cation for Foods (ICMSF, 1986) were applied. Culture media were those of Oxoid, Biolife and Difco.For each sample, 25g were weighed out and transferred to a sterile blender with 225ml of 0.1% peptone and mixed tho­roughly for 2minutes to prepare fish homogenate. The samples were then analyzed as follows.
 
2.6.1 Total viable bacterial counts
Appropriate dilutions of the fish homo­genate were prepared and inoculated onto sterile petri dishes. Plate count agar (Oxoid) media were then poured. Plates were incubated at 37°C for 48hours and colonies were then counted and repor­ted as total colony count/ml. A second set of plates was incubated at 37°C for 48hours in a carbon dioxide incubator or under anaerobic conditions using a gas pack anaerobic jar. Colonies were then counted and reported as anaerobic total bacterial count. In case of spore formers count, the food homogenate was boiled first at 75–80°C and then rapidly cooled. Ap­propriate serial dilutions were prepared and inocu­lated onto the surface of sterile and dried plate count agar media. These were incu­bated finally at 37°C for 48hours.
 
2.6.2 Detection of Escherichia coli
One ml of each of the decimal dilutions of the fresh and smoked fish homogenate was plated on poured Eosine Methylene Blue Agar (Oxoid) and then incubated at 37°C for 24hours. Counts were calculated from the number of growth on the plates. The colonies with green metallic sheen were counted as Escherichia coli.
 
2.6.3 Detection of Staphylococcus aureus
A sample of 0.1ml of the fresh and smoked fish homogenate and dilutions was inoculated on Baird-Parker (Difco) agar plates and incubated at 37°C for 48hours. Co­lonies appearing to be black and shiny with narrow white margins and surrounded by clear zones were identified by coagulase test reactions. The coagulase test was carried out by first inoculating typical colonies in brain heart infusion broth (Dif­co) and incubating at 37°C for 24hours. From the resulting cultures, 0.1 ml was then added to 0.3ml of rabbit plasma in sterile tubes and incubated at 37°C for 4 hours. The formation of a distinct clot was evidence of coagulase activity.
 
2.6.4 Detection of Salmonella paratyphi.
Samples of fresh and smoked fish homogenate and dilutions were inoculated in Salmonella-shigella agar (Oxoid) and incubated at 37°C for 24hours. For identification, 2–3 suspected colonies were inoculated into tryptone broth for indole test, triple sugar iron agar slant (Oxoid), urea broth and lysine iron agar. These were incubated at 37 °C for 24hours. Salmonella species is indole nega­tive, on triple sugar iron it produces acid (yellow) and alkaline (red) with or without gas and hydrogen sulfide, is urea negative, and on lysine iron agar shows an alkaline (purple) reaction throughout the medium. Serological tests were then carried out.
 
2.6.5 Detection of Listeria monocytogenes.
A sample of 0.1ml of the fresh and smoked fish homogenate and dilutions was inoculated on Brilliant Listeria Agar (Oxoid) plates and incubated at 37°C for 24hours. Co­lonies appearing were counted and reported asListeria monocytogenes.
 
2.6.6 Enumeration of fungi
Appropriate dilutions of Sabouraud dext­rose agar plates (Oxoid) were poured over 1ml of the fresh and smoked fish homo­genate and dilutions. Plates were incubated at 25°C for 3 days and then colonies were counted and reported as fungal count/ml.
 
2.7 Data analysis
The data obtained were subjected to descriptive statistics using IBM SPSS version 21.0 software. One way analysis of variance (ANOVA) was performed followed by Duncan’s Multiple Range Test (p<0.05) to find the difference between means. Significant level was set at P<0.05.
 
3 Results                                                                                                                                                               
The moisture, protein,  fat, crude fibre, ash and carbohydrate contents (%) of fresh fish samples (Table 1) were in the range of 75.83-78.84, 16.56-17.89%, 2.67-4.96, 0.12-0.15, 0.14%-0.17 and 0.94-1.19  respectively while that of the smoked fish (Table 2) ranged from  8.34-9.78, 57.43-63.71, 13.24-17.47, 1.36-1.92, 1.89-2.89 and 8.29-12.12  respectively. PV, FFA, TBA, TVB-N and TMA of fresh fish samples (Table 3) were in the range of 6.97-9.14meq. peroxide/kg, 0.93-1.11, 0.85-1.03mgMol/kg, 13.08-16.23mgN/kg and 1.83-2.46mgN/kg respectively while that of the smoked fish (Table 4) were in the range of 7.31-9.53meq.peroxide/kg, 1.11-1.51, 1.00-1.51mgMol/kg, 15.36-17.91mgN/kg and 2.03-2.49mgN/kg respectively.

 

Table 1 Proximate composition of fresh Guinean barracuda fish samples from 20 different processing centres

 

Table 2 Proximate composition of modern convective smoked Guinean barracuda fish samples from 20 different processing centres

 

Table 3 Quality indices of fresh Guinean barracuda fish samples from 20 different processing centres

 

Table 4 Quality indices of modern convective smoked Guinean barracuda fish from 20 different processing centres

 

The concentrations of different PAHs in smoked fish samples are shown in Table 5. The concentrations of sixteen (16) PAHs compounds analyzed in the smoked fish samples revealed as follows. The concentrations of Acenaphthene, Fluorene, Phenanthrene, Anthracene and Fluoranthene were 2.16 μg /kg, 3.51 μg /kg, 5.94 μg /kg, 4.63 μg /kg and 4.26 μg /kg respectively. The concentrations (μg/kg) of Pyrene, Benzo (a) anthracene, Chrysene, Benzo (b) fluoranthene, Benzo (a) pyrene and benzo [ghi] perylene were 2.78, 4.39, 4.13, 3.42, 3.1 and 2.97 respectively. The concentrations of Indeno (1, 2, 3-cd) pyrene and Dibenzo (a,h)anthracene were 3.68  and 2.91 respectively.

 

Table 5 Concentration (μg /kg) of polycyclic aromatic hydrocarbons in modern convective smoked Guinean barracuda fish from Lagos State, Nigeria

 

The results of the concentrations μg/g of Hg, Pb, Cd, and Cr in the smoked fish samples analyzed are presented in Table 6. The concentrations of Hg, Pb, Cd, and Cr in the smoked fish samples were in the range of 0.0016-0.0024, 0.0012-0.0023, 0.0015-0.0023 and 0.0629-0.0856 respectively.

 

Table 6 Heavy metals profile (Concentration (μg/g) of modern convective smoked Guinean barracuda fish from 20 different processing centres

 

The results of the microbiological study (Table 7 and Table 8) indicated that TVC of smoked fish samples were in the range of 1.0 x 103-4.3 x 103cfu/g. Staphylococcal count of smoked fish samples (Table 8) were in the range of 1.4 x 102-3.8 x 102 cfu/g. S. paratyphi and E. coli were not detected in smoked fish samples.

 

Table 7 Microbial quality (cfu/g) and pH of fresh Guinean barracuda fish from 20 different processing centres

 

Table 8 Microbial Quality (cfu/g) of modern convective smoked Guinean barracuda fish from 20 different processing centres

 

4 Discussion

The PV values were below the recommended value of between 20 and 40mgeq.peroxide/kg for rancid taste to begin (da Silva et al., 2008). The FFA values are below the threshold for rancidity detection in smoked fish. The thiobarbituric acid value (TBA) is used to assess the degree of fish spoilage especially in fatty fish. The TBA test measures a secondary product of lipid oxidation, malonaldehyde (da Silva, 2002; da Silva et al., 2008). The TBA values of smoked fish samples were also low. The TBA (1.73 to 2.91mg TBA/kg) values were within the range specified by USFDA. TVB-N is related to protein breakdown and is an index of fish spoilage (da Silva, 2002). The legislative standard for TVB-N include: 20mgN/100g for fresh fish, 30mgN/100g stale fish and 40mgN/100g for fish that is unfit for human consumption but can be used for animal feed (FAO, 1992; da Silva, 2002; da Silva et al., 2008). In this study, TVB-N of smoked fish samples are within the range of legislative standard for TVB-N which is 20mgN/100g for fresh fish (da Silva, 2002; da Silva et al., 2008). This suggests that the level of protein decomposition or breakdown in all the samples is low. The trimethylamine value (TMA) of 2.03-2.49mgN/kg for fried fish samples are within the range of <3mgN/100g for fresh fish, >8mgN/100g for spoiled fish and > 5mgN/100g for doubtful quality specified (da Silva, 2002; da Silva et al., 2008).

 

The result of PAH analysis revealed the presence of sixteen (16) PAHs compounds analysed in the smoked fish samples. Only few of the concentrations of sixteen (16) PAHs compounds are above the 5.0μg /kg B (α) P maximum permissible levels set by EU Regulation 1881/2006 (Amos-Tautua et al., 2013). Levels of the four heavy metals investigated in all the fried fish samples are generally below the maximum permissible levels set by World Health Organization (Brain and Allen, 1993) for Pb (0.3 ppm); Cd (0.2 ppm), Hg (0.2ppm) and Cr (0.5ppm) and hence pose no risk to consumers (Amos-Tautua et al., 2013).

 
The TVC values obtained for the smoked catfish samples were within the range of specified microbiological limits recommended by ICMSF (1986) for fish and fishery products, the maximum recommended bacterial counts for good quality products (m) is 5 x 105 (5.7log10CFU/g). The Staphylococcal count values obtained for the fried fish samples were low below the specified recommended value for all fish. S. aureus was isolatedfrom fried fish samples and this can be attributed to post processing contamination. The Staphylococcus aureus isolatedfrom smoked fish samples was within the safety level which is equal to or greater than 104/g recommended by (FDA, 2001). S. paratyphi was not detected in smoked fish samples and this conforms with the specified microbiological limits recommended by ICMSF (1986) for S.paratyphi count for fish and fishery products which is the presence of the organism, that is zero tolerance. In all cases, the absence of Salmonella paratyphi and E. coli suggests Good Manufacturing Practices (GMP) and non faecal contamination of the products.
 
5 Conclusion
In conclusion, this study showed that modern convective smoked fish samples are of good quality. However, the presence of certain polycyclic aromatic hydrocarbons in some samples above the recommended permissible levels may subject consumers to higher risk (chemical) if care is not taken but the samples are microbiological safe.
 
Acknowledgement
The authors acknowledge the contribution of Mrs. E. Adeniran, the technologist in Food Microbiology laboratory of Dept. of Food Science and Technology, Federal University of Agriculture, Abeokuta, Nigeria for her assistance.
 
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