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AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA
ISSN Print: 2151-7517, ISSN Online: 2151-7525, doi:10.5251/abjna.2012.3.9.360.364
© 2012, ScienceHuβ, http://www.scihub.org/ABJNA
Micriobiological quality of smoke-dried mangrove oysters (Crassostrea
gasar) sold in Port Harcourt, Nigeria
Odu1, N.N, Njoku1, H.O. and Mepba2 H.D
1
Department of Microbiology, University of Port Harcourt, Nigeria
Department of Food Science and Technology, Rivers State University of Science &
Technology, Port Harcourt, Nigeria.
[email protected]
2
ABSTRACT
The microbiological quality of smoke-dried oysters (Crassostrea gasar) sold in Port Harcourt, Nigeria was
investigated. The microbial load and pathogens in smoke-dried oysters from four major fishing ports:
Kalabari (Ka), Bille (Bi), Okrika (Ok) and Nembe (NB) that supply the Port Harcourt market were evaluated
and studied. Highest total viable counts were recorded in oysters from Bille and Nembe fishing ports and
values ranged from 7.18-7.2log10 cfu/g and 6.7-6.9log10 cfu/g, respectively, while lowest counts were
obtained in oysters from Okrika fishing port (5.75-5.9log10 cfu/g. The bacteria pathogens isolated were in the
order of Staphylococcus aureus (45%), Bacillus cereus (25%), Micrococcus virans (15%), Klebsiella
spp.(10%) and E.coli (5%). Among the predominant fungal isolates were Aspergillus niger, Aspergillus
flavus, Penicillium spp, Neurospora spp. and Saccharomyces spp. Thus bacteria of aerial origin and storage
fungi were isolated from smoke-dried oysters from the four major fishing ports that supply the fish market in
Port Harcourt, Nigeria. The microbial load and pathogens in the smoke-dried oysters could be attributed to
poor handling practices and faulty storage temperature by the fish vendors.
Key words: Microbiological quality, oysters, fishing ports, bacterial isolates, pathogens.
INTRODUCTION
Molluscan bivalves such as oysters are seafoods that are
widely consumed. They are excellent sources of protein but
are highly perishable as fresh seafoods. Their short shelf
life thus pose serious practical problems of their storage
and distribution (Frazier and Westhoff, 2000). Oysters are
consumed raw (ingested whole) in some parts of the world
thereby leading to the transmission of pathogenic
organisms (CDC, 2006). Thus various bacteria that are
aetiologic agents of shellfish-related food infection such as
salmonellosis, shigellosis, Vibrio and Hepatitis A virus had
been isolated from oysters (CDC, 2006). Additionally,
septicaemia had been reported among consumers of raw
oysters from the Gulf of Mexico (FAO/WHO, 2005).
Smoke-dried oysters account for over 75% of the animal
proteins in the diets of people in the Niger Delta region of
Nigeria.
Traditional curing methods such as sun-drying and
smoking are used in preserving seafoods that cannot be
sold by fishermen (Eboh et al., 2006). Smoking was
originally used for meat preservation whereby surface
bacteria were killed by the heat of the smoke
and regrowth prevented by dehydration and inhibitory
chemicals such as formal dehyde in wood smoke (Potter
and Hotchkiss, 2000). Smoked-dried oysters have unique
flavour and are considered as delicacies in the Niger Delta
region. They are eaten as snacks or with fermented tubers
without subjecting them to further cooking. Although this
post-harvest technology had remarkably reduced wastage
and improved the keeping and eating qualities of seafoods
(FAO, 1999; Adebayo-Tayo and Ogunjobi, 2008) the public
health implication is underscored. The present study
evaluates the microbial load and pathogens in smoke-dried
oysters from local fishing communities in the Niger Delta
region of Nigeria.
MATERIALS AND METHODS:
Samples source and preparation: Smoke-dried
mangrove oysters (Crassostrea gasar) were purchased
from Creek Road Market, a popular seafood market in Port
Harcourt, during the months of April-June, 2010. The
oysters had been harvested and traditionally smoke-dried
at four fishing ports in Rivers State; (3 locations) and
Bayelsa State (1 location). Among the fishing ports in
Rivers State were: Kalabari (Ke boko Borikiri), Bille (Tuma
Borikiri) and Okrika (Anchichukama Borikiri). In Bayelsa
State was Nembe (Toniboko Borikiri). All samples were
transported to the laboratory in sterile polythene bags and
analysed within 2 hours of purchase.
Microbiological Analysis: Triplicate samples (25g each)
were asceptically transferred to a sterile stomacher (Model
BA 6021, Seward Medical, UK) and homogenised with 225
ml sterile 0.1% peptone water (Oxoid CM 509) for 2 min.
Serial ten fold dilutions of homogenates were made by
transferring 1ml to fresh sterile diluent. Aliquots of
appropriate dilutions were transferred separately to plates
of surface-dried Nutrient Agar (Oxoid CM 3), for total
bacteria count, Mannitol salt Agar (Oxoid) for
staphylococcal count, Salmonella-Shigella agar (Oxoid) for
salmonellae count. A pre-enrichment step described by
APHA (2001) was followed for isolation of salmonellae. All
Agric. Biol. J. N. Am., 2012, 3(9): 360-364
o
(15%), Klebsiella spp. (10%) and E.coli (5%). Among the
predominant fungal isolates were Aspergillus niger,
Aspergillus flavus, Penicillium spp, Neurospora spp, and
Saccharomyces spp (Table 3).
plates were incubated 37 C for 48 hours. The 5-tube MPN
method (Feng et al., 2002) was adopted for the
enumeration of total coliforms, faecal coliform and E.coli.
o
The tubes for total coliforms were incubated at 37 C for 24
o
hrs while those for faecal coliform were incubated at 45.5 C
for 24-48hrs. Positive tubes showing acid and gas
production were streaked on Eosin Methylene Blue Agar
o
and incubated at 37 C for 24 hrs. Estimation of bacterial
numbers was by the plate count and the Most Probable
Numbers (MPN) technique describe by Frazier et al., 1999.
-1
For isolation of bacterial types, 0.1 ml of the 10 dilution of
the sample was spread on appropriate media and
incubated. Typical colonies on the media were picked,
streaked out to obtain pure cultures and finally maintained
on Nutrient Agar slopes in the refrigerator. Pure isolates
were characterized and identified following morphological
and biochemical tests such as motility, catalase, oxidase,
gram stain, spore stain, indole production, citrate utilization,
Methyle
Red (MR), Voges Proskauer (VP), urease,
coagulase and carbohydrate fermentation test. The isolates
were identified following the method of Cowan and Steel
(2003).
Yeast and moulds were characterised and
identified using the schemes of Frazier and Westhoff
(2000).
RESULTS
Kalabari
Bille
Okrika
Nembe
8
7
Log Cfu/g of Meat
6
5
4
3
2
1
The microbial load of smoke-dried oysters from the various
fishing ports are expressed graphically in Fig 1. Highest
total viable counts were recorded in samples from Bille and
Nembe fishing ports and ranged from 7.18-7.2log10 cfu/g
and 6.7-6.9log10 cfu/g, respectively while lowest total
bacterial counts were obtained in samples from Okrika
fishing port (5.75-5.9log10 cfu/g. Smoke-dried oysters from
Kalabari and Bille fishing ports had highest staphylococcal
counts (3.49-3.59log10 cfu/g and 3.49log10 – 3.6log10 cfu/g,
respectively. The staphylococcal counts in samples from
Okrika and Nembe ranged from 3.3log10 cfu/g – 3.4log10
cfu/g and 3.3log10 cfu/g-3.5log10 cfu/g, respectively. Smokedried oysters from Kalabari fishing ports had significantly
(P≤ 0.05) higher fungal counts than samples from Bille,
Okrika and Nembe fishing ports. Mean total fungal counts
in smoke-dried oysters from Bille, Okrika and Nembe
ranged from 3.33-3.31 log10 cfu/g.
Data in Table 1 give the mean total/faecal coliform counts
per gram of samples from the four fishing ports. Total
coliform counts for smoke-dried oysters from Kalabari, Bille,
Okrika and Nembe fishing ports ranged from 14.2 ± 1.0 to
24.6 ± 1.6 coliforms/g while < 3 faecal coliforms/gram were
isolated on samples from each of the fishing ports. No
significant difference (P>0.05) was observed in the total
coliform counts (MPN/g) of samples from Kalabari and
Nembe fishing ports. Smoke-dried oysters from Bille
fishing ports had a significantly (P≤ 0.05) higher total
coliform count than samples from other fishing ports.
Table 2 presents data on the morphological and
biochemical characteristics of bacterial isolates in smokedried oysters from the four fishing ports. The bacterial
pathogens isolated were in the order of Staphylococus
aureus (45%), Bacillus cereus (25%), Micrococcus virans
0
NA
MSA
SDA
Fig. 1 Histogram distribution showing Microbial Load of Smoke dried
Oysters from fishing ports in the Niger Delta, Niger
DISCUSSION:
Highest total viable counts were obtained in smoke-dried
oysters from Bille, Nembe and Kalabari fishing ports (Fig
1.) The high bacterial counts on smoke-dried oysters from
these fishing ports could be attributed to the frequent
contamination of water with sewage in addition to the
endogenous microflora of the oysters.
Molluscs are
transported live to the point of sale or processing where the
flesh can often be contacted by hand. Although
contamination may occur at this stage, the significant public
health problems associated with shellfish arise from their
ability to concentrate viruses and bacteria from the
surrounding waters (Adams and Moss, 2005). The initial
microbial load on ready-to-eat foods is important, however,
factors such as, processing, storage and display may
influence the microbiological load of ready-to-eat foods at
the point of sale (Angelidis et al, 2006; Beuchat and Ryu,
2004). Although smoke-drying reduces water activity and
destroys bacteria through the agency of heat, postprocessing contamination can and do occur especially
during handling and transportation of processed foods to
the point of sale (Mepba et al., 2002). Besides, the high
relative humidity of the riverine communities, the exposure
of the smoke-dried oysters and the unacceptable direct
contact with hands of purchasers and hawkers alike during
haggling for price (Mepba et al., 2008) could significantly
contribute to the microbial load of the oysters.
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Agric. Biol. J. N. Am., 2012, 3(9): 360-364
Table 1. Total/faecal coliform counts (MPN/g)
LOCATIONS
Kalabari
TOTAL COLIFORMS (MPN/g)
24.6b ±1.6
FAECAL COLIFORMS (MPN/g)
2.0a ± 0.01
Bille
24.0a ± 7.0
2.0a ± 0.01
Okrika
14.2bc ±1.0
2.0a ± 0.01
b
Nembe
2.0a ± 0.01
41.0 ± 2.0
Values are means ± S.D. of triplicate determinations. Means in the same columns not followed, by the same superscripts differ significantly (P≤ 0.05) using Duncan’s New
Multiple Range Tests (DNMRT).
Oxidase
Citrate
Urase
Voges
proskauser
Indole
Glucose
Lactose
Maltose
-
-
+
-
+
-
-
-
+
-
A
A
-
-
Staphylococcus aureus
+
+
+
+
-
-
-
-
-
+
-
-
A
-
-
Bacillus spp.
Bi, Ka, NB, Ok.
Cocci
+
-
-
+
-
-
-
+
-
+
-
-
A/G
-
-
Micrococcus spp
Bi, NB, Ka, Ok.
Rods
-
+
-
+
-
-
-
-
-
+
-
-
A/G
A/G
+
Enterobacter spp
Bi, NB, Ka, Ok.
Rods
-
-
-
-
-
-
+
-
-
+
-
-
A/G
A/G
+
Klebsiela spp
Bi, NB, Ka, Ok.
Rods
-
+
-
+
-
-
-
-
+
-
+
-
A/G
A/G
+
E.coli
H2s
Coagulase
+
Rods
Methyl red
Catalase
Cocci
Bi, NB, Ka, Ok.
Motility
Ka, Bi, Ok, NB.
LOCATION
Gram stain
Spores
Cell
morphology
Table 2. Morphology and biochemical characteristics of bacterial isolates from smoke-dried oysters
IDENTITY OF GENUS
Ka = Kalabari, Bi = Bille, Ok = Okrika, NB = Nembe, A = Acid, A/G = Acid and Gas.
Table 3. Morphological characteristics of predominant fungal isolates from smoke-dried oysters from fishing ports in the niger delta.
LOCATION
COLOUR
MICROSCOPIC DESCRIPTION
IDENTITY OF ORGANISM.
Bi, Ka, NB, Ok
Yellowish-green mycelium
Brush-like condidia, septate branching
conidiphore was smooth/rough walled.
Penicillium spp.
Ka, Bi, NB, Ok.
Effuse black colony
Simple septate and branched conidia in
chains.
Aspergillus niger
NB, Bi, Ka, Ok
Greenish mycelium
Conidiophores with vesicles, unbranched,
conidiohores in chains
Aspergillus flavus
NB, Ok, Ka.
Pink
Septate hyphae with conidiophores
arranged at the tips
Neurosphora spp.
Ok, NB.
Dirty, cream
Spheroid with budding cells and one
ascus
Saccharomyces spp
Bi = Bille, Ka = Kalabari, NB = Nembe, Ok = Okrika
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Agric. Biol. J. N. Am., 2012, 3(9): 360-364
Data in Fig 1. also show that the staphylococcal counts
in oyster samples from Okrika and Nembe fishing ports
ranged from 3.29-3.36log10 cfu/g and 3.18-3.5log10 cfu/g,
respectively. Staphylococcus aureus has been isolated
in fish (Sokari and Anozie 2005), and in street foods
(Mepba et al., 2008). Other ready-to-eat foods such as
salads and sandwiches have also been implicated in
foodborne illness outbreaks. These foods are often
prepared by hand and this direct contact may lead to an
increased incidence of contamination with potential
foodborne pathogens, such as Staphylococcus
(Christiansen and King, 2007; Colombari et al., 2007;
Nguz, 2007). The presence of small numbers of
Staphylococcus aureus in food is not uncommon but not
a health threat because they are eliminated by cooking
or pasteurization (Jay et al., 2005). Additionally, large
numbers, typically > 106g-1 are required for production
of enough toxin to cause illness. Thus contamination is
necessary but is not alone sufficient for an outbreak to
occur (Adegoke, 2010).
(ICMSF, 1986). Christianson et al., (2008) reported
about 5-6log cfu/g of coliforms and E.coli on meat- and
cheese-based filled baguettes and salads containing
meat in Johannesburg, South Africa. It has been
suggested that several factors may contribute to the
presence of E.coli in ready-to-eat foods like smoke-dried
oysters, including poor handling practices by food
handlers, or cross contamination from food contact
surfaces or high storage temperatures as in the tropics
(Fang et al., 2003).
The predominant bacteria pathogens isolated from the
smoke-dried oysters were of aerial origin (Table 2). The
incidence of potential foodborne pathogens including S.
aureus (ca. 3log cfu/g); B. cereus (ca. 2log cfu/g), E.coli
(< 3.cfu/g) on the ready-to-eat smoke-dried oysters was
relatively low.
Studies have suggested that the
presence of S. aureus on ready-to-eat food may be as a
result of improper handling, cross contamination and
poor temperature control (Synder, 2009; Christison et
al., 2008). The predominance of Bacillus spp. could be
attributed to the display of the dried oyster meat without
any form of packaging. Being frequently displayed
uncovered the meat became liable to contamination to
bacteria of aerial origin. Some strains of Bacillus (e.g. B.
cereus and Staphylococcus aureus are known
enterotoxin producers (Bryant, 2007). The inherent
danger in the association of B. cereus and S. aureus
with or without their metabolic products in various foods,
without further heat treatment is the possible outbreak of
serious foodborne illness.
Smoke-dried oysters from Kalabari fishing ports had
significantly (P ≤ 0.05) higher fungal counts than the rest
of the tested oysters (Fig. 1). Post-processing fungal
contamination of oysters from the fishing ports was
expected given the poor handling practices often
exhibited by the fishermen and the high relative humidity
of the storage environment. Besides, the flesh of
molluscs such as cockles, mussels, oysters and clams
differs from that of free swimming fish by containing
appreciable amounts (about 3.0%) of carbohydrate in
the form of glycogen (Adams and Moss, 2005). Though
many of the same organisms are involved, spoilage is
therefore glycolytic rather than proteolytic.
Coliforms are still considered indicators for assessing
general hygienic status of food contact surfaces, the
contamination of oysters in the present study could be
attributed to poor handling practices and improper
storage temperatures (Jackson et al., 2005).
The high total coliform counts (Table 1) of smoke-dried
oysters from the fishing ports could be attributed to poor
handling practices by the fishermen. Besides, the seas
around coasts are influenced by inputs of terrestrial and
fresh water microorganisms and by human activities.
For instance, the seas in the Niger Delta has become
convenient dump for sewage and other waste products
(Eboh et al., 2006). Many shellfish used for food grow in
polluted waters and majority feed by filtering out
particles from large volumes of sea waters. These
waters contaminated with sewage pose the risk that
enteric organisms from infected individuals may be
present and concentrated by the filter feeding activities
of shellfish. However, a large infective dose of
enteropathogenic E.coli (EEC) is required for either the
enterotoxigenic or invasive illness to occur. Thus foods
must be highly contaminated or inadequately preserved
to allow for prolific growth (Adams and Moss, 2005).
Among the predominant fungi isolated from the smokedried oysters were Penicillium spp., Aspergillus niger,
Aspergillus flavus and Neurospora spp. (Table 3). The
high humidity and warm environmental temperatures in
the coastal areas of the Niger Delta predispose to
growth of storage fungi such as penicillia and aspergilli
that could result in mycotoxin production in the smokedried oysters. It has been suggested (Jay et al., 2005)
that conditions of high humidity and warm temperature
predispose to high levels of a flatoxin in foods, often
exceeding the upper limit initially established by the
Food and Agriculture Organization (FAO)and World
Health Organisation (WHO) of 30 μgkg-1.
Although aflatoxin may be considered amongst the most
caranogenic of natural products for some animals, it is
still not clear whether it is a carcinogen for man (Adams
and Moss, 2005).
Results from the present study show that E.coli counts
did not differ significantly (P≤ 0.05) and were > 3.0 cfu/g
of meat which did not exceed the maximum
recommended E.coli count for good quality product
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Agric. Biol. J. N. Am., 2012, 3(9): 360-364
common commercially available fish species in Oron
Local Government, Nigeria.Food Chem. 97:490-497.
CONCLUSION:
Bacteria of aerial origin and storage fungi were isolated
from smoke-dried oysters from the four major fishing
ports that supply the fish market in Port Harcourt,
Nigeria. Good hygienic practise aimed at minimizing the
microbial load of these ready-to-eat foods must be
considered. Proper packaging to prevent direct handcontact and contamination by bacteria of aerial origin,
control of relative humidity and warm temperature that
favour fungal growth is advocated.
Fang T.J., Wie, Q., Liao, C., Hung, M. and Wang, T. 2003.
Microbiological quality of 180C ready-to-eat food product
sold in Taiwan.Int. J. Food Microbiol. 80:241-250
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Rome, Italy.
FAO/WHO. 2005. Risk assessment of Vibrio vulnificus in raw
oysters. Interpretative summary and technical report .
Food and Agriculture Organization of the United
Nations/World Health Organization, Geneva, Switzerland.
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