Macrobrachium vollenhovenii continues to receive tremendous attentions
as it supports very important local fisheries in Nigeria and other regions of
Africa (Jayachandran, 2001; Akintola
et al., 2009b). Also, the safety and quality of the prawn at different
biomes had been evaluated for microbes and reported to be suitable for consumption
(Akintola et al., 2009a).
Ice against traditional smoking for fish products is becoming more popular.
However, irregular supply of electricity in the country hampers the use of ice
as a means of preserving fish products in spite of the high profile of ice globally
as the cheapest and commonest method for prawn preservation (Kirschnik
et al., 2006).
Prawns offer quality protein, low saturated fat, may contain Omega-3 (Ω-3)
fatty acids and contribute to cardiovascular stability of adults as well as
childrens growth and development (FDA, 2007). Rapid
spoilage due to higher water content and other issues mentioned in Abu-Bakar
et al. (2008) are important concern and affect shelf-life of the
The shelf-life depends on the numbers and types of microorganisms, mainly bacteria,
initially present and their subsequent growth as well as natural sources (Abu-Bakar
et al., 2008). Shelf-life reported for whole M. rosenbergii stored
on ice, included 3 days (Nip and Moy, 1988), 8 days
(Lindner et al., 1988), 7-10 days (Kirschnik
et al., 2006) 14 days (Abu-Bakar et al.,
Lalitha and Surendran (2006) reported reduction in
bacterial load after 5 days due to the inability of some of the bacterial species
to survive and/or grow at low temperatures. Studies on the impact of ice on
the qualities and safety of other freshwater prawn of genus Macrobrachium
are rare in spite of their widely acknowledged status as species of ecological
and economic importance to many fisheries.
The objective of this study was to evaluate the impact of ice in controlling
the population of microbes in Macrobrachium vollenhovenii and to determine
the shelf-life. In this light microbial composition of freshly collected M.
vollenhovenii were investigated against the prawns exposed to two ice treatments.
MATERIALS AND METHODS
This study was carried out within the period of 12th March to 12th June, 2010
in the Laboratories of the Fisheries and Microbiology Departments of the Lagos
State University, Ojo.
Collection of sample: Live Macrobrachium vollenhovenii with mean
weight of 30±25 g were purchased from fishers from Badagry Creek at the
Topo landing station in hours of 7.00-8.00 a.m. Samples were packed into a sterile
Thermocool® boxes containing chlorinated water (5 ppm) and transported within
1 h to the laboratory in the Department of Fisheries, Lagos State University.
The whole prawns were then washed in deionized water and after which the water
in the Thermocool® box was drained. The samples were confirmed killed after
20-25 min of no contact with water.
Samples were aseptically distributed randomly into two groups of prawn stored
with Direct Contact with Ice (DCI) in ratio of 1:1 (w/w) in Thermocool®
box labelled A while the samples without direct contact with ice were placed
0.006 mm thick polythene bags, 150 g prawn/bag following Kirschnik
et al. (2006) and thereafter placed in the Thermocool® box labelled
Melted ices in each box were replaced after 12 h and drained regularly through
the tap on the Thermocool® box. Five prawns were withdrawn for microbial
analyses the first day and thereafter every two days of storage (day 0, 2, 4,
6, 8, 10) from the two experimental set up under room temperature of 27-30°C
while the fresh prawns served as control.
Microbiological analysis: Twenty five gramme of the prawn muscle tissues
were aseptically taken and crushed in a mortar with a pestle. Prawn homogenates
were prepared by adding 9 mL of sterile distilled water to 1 g of the crushed
samples. Thereafter, 0.1 mL each of the homogenized solutions was inoculated
using spread plate method into nutrient and MaConkey agar and incubated at 25°C
for 24 to 36 h. The total viable count was determined on the nutrient agar and
coliform on the MaConkey agar. The dilution out was 10-4 using pour
method. After incubation the colonies were counted using colony forming unit
and cfu g-1 was calculated.
Characterizations and identification of isolate: In order to identify
each isolate, pure cultures were examined for cultural and morphological characteristics
based on their colour, shape and pigmentation, by Gram staining. The bacterial
isolates were identified using Bergeys Manual of Systematic Bacteriology
(Ludwig and Klenk, 2001).
Statistical analysis: Analysis of variance was performed using statistical
Package for Social Scientist (SPSS, 2006) version 15.
One way ANOVA was conducted to test differences in the mean values between fresh
prawns and each of the treatments in five replications. p<0.05 was considered
Flora on fresh prawn: The total aerobic bacterial counts on freshly
caught wild whole freshwater Macrobrachium vollenhovenii ranged from
3-5 log10 cfu g-1. A total of seven bacterial species
were isolated from the fresh prawn (Table 1). The Isolates
were mainly Gram-negative bacteria with Escherichia coli being the most
dominant (61%). Indole and H2S producing bacteria: Escherichia
coli and Pseudomonas aeruginosa and Proteus mirabilis and
Salmonella sp. collectively constituted 29% of the isolates. Only two
Gram-positive rod and cocci, Bacillus cereus and Staphylococcus sp.
Bacteria flora on prawn stored in ice: The impact of the two icing treatments
on the African river prawn were alike significantly (p<0.05) in terms of
ability to cause the absence of bacteria which were present in the freshly caught
prawn (Table 1 and Fig. 1). Proteus sp.
and Enterobacter aerogenes were not detected in the two ice treatments
by day 6 whereas, both treatments inhibit the presence of Salmonella sp.
at 8 days storage, respectively.
The population of Pseudomonas aeruginosa increased significantly (p<0.05)
after 6 days with population counts of 106-107 cfu g-1
while the H2S and the other indole producing bacteria, Escherichia
coli were not detected. This suggested the dominant role of Pseudomonas
aeruginosa in promoting spoilage of freshwater prawn. Although, in this
study Staphylococcus sp. showed psychrophillic attributes and icing treatments
were not able to reduce the population of the organism. Population of Gram-positive
rod and coccus Bacillus cereus and Staphylococcus sp. increased
with storage days in ice.
|| Microflora in fresh Macrobrachium vollenhovenii and
succession during ice storage
|Values are not significantly different between treatments,
||Changes in bacterial counts (±SD) on Macrobrachium
vollenhovenii during 10 days storage in two ice treatments
The total aerobic bacterial counts on fresh Macrobrachium vollenhovenii
were within values (about 5 log10 cfu g-1) previously
reported for farmed Macrobrachium rosenbergii (Leitao
and Rios, 2000) but below 7.00 log10 cfu g-1 specified
by the International Commission on Microbiological Specification for Foods (ICMSF,
1986). The total bacteria counts were not significantly different (p>0.05)
The percentage composition of indole and H2S producing bacteria
were considerable lower in the fresh prawn nevertheless, they have been implicated
as agents of spoilage in food (Leitao and Rios, 2000).
The isolation of two Gram-positive rod and cocci, is similar to findings of
Lalitha and Surendran (2006) with lower population of
Gram-positive bacteria in farmed fresh Macrobrachium rosenbergii.
The presence of Escherichia coli in the fresh prawn and not reported
for farmed M. rosenbergii in Brazil (Leitao and Rios,
2000) shows the influence of prevailing environment on the bacteria composition
in muscle of freshwater prawn as averred by Gomez-Gil et
al. (1998). Bello-Olusoji et al. (2008)
reported the presence of Salmonella sp. and Escherichia coli
in M. vollenhovenii and stated that they are habitat selective. Salmonella
sp. and Escherichia coli are indicator microorganisms of fecal pollution
which are not indigenous to the aquatic environments (Papadopoulou
et al., 2007). Staphylococcus sp. is reported to be part of
the normal human and animal microflora and may be found in aquatic systems polluted
by sewage (Papadopoulou et al., 2007). Incidence
of human activities particularly the slaughtering and processing of pork in
the banks of Badagry Creek was observed by Akintola et
The impact of the two icing treatments on the African river prawn were alike
significantly (p<0.05) in terms of ability to cause the absence of bacteria
which were present in the fresh prawn. Proteus sp. and Enterobacter
aerogenes were not detected in the two ice treatments by day 6. Whereas,
both treatments inhibit the presence of Salmonella sp. at 8 days storage,
respectively indicating differential abilities of bacteria to withstand icing
conditions as reported by Miyamato-Shinogara et al.
(2000). Proteus sp., Enterobacter sp. and Salmonella sp.
are reported to not being able to tolerate cold storage (Nimrat
et al., 2008).
Nimrat et al. (2005) note that Staphylococcus
sp., Bacillus cereus and Pseudomonas aeruginosa are present in
the spermatophores of fresh black tiger shrimp (Nimrat et
al., 2008) showed strong resistant to ice storage conditions in this
study. The significant increase (p<0.05) in the population of these group
after 6 days while the H2S and indole producing Escherichia coli
were not detected suggested dominant role of Pseudomonas aeruginosa in
promoting spoilage of freshwater prawn. The most prolific organisms during the
ice storage of fish were found in significant numbers at the end of storage
(Kirschnik et al., 2006). Psychrophillic Pseudomonas
sp. is a primary cause of spoilage irrespective of initial microflora on
prawns stated Abu-Bakar et al. (2008).
According to Papadopoulou et al. (2007), Staphylococcus
sp. are not able to propagate in competition with fishes natural microflora.
Population of Gram-positive rod and coccus Bacillus cereus and Staphylococcus
sp increased with storage days in icing in view of their psychrophilic nature
(Lalitha and Surendran, 2006).
Ice treatments as a means of ensuring food safety and quality of prawns needs
further investigation since the population of Staphylococcus sp, Bacillus
cereus and Pseudomonas aeruginosa were not controlled by this means.
These organisms have been implicated as promoting food borne diseases (Le
Loir et al., 2003; Archer, 2004) and are
of health concerns as regards food safety globally. Both Staphylococcus sp.
and Bacillus cereus are diarrhoeal and emetic toxins producers that cause
serious illness and fatalities in human consequent to consuming food contaminated
with the toxins.
Further study need to be explored as regards the status of Staphylococcus
sp. in relations to the type of strains particularly, Staphylococcal Enterotoxins
(SE) producing types in the freshwater prawn. Le Loir et
al. (2003) stated the need for better understanding of the interactions
between S. aureus and the food matrix and of the mechanisms of SE production
in foodstuffs. Bacillus cereus is a common pathogen in foods. However,
the cfu numbers may not be sufficient to draw conclusion about the status of
the safety and quality of the food substance. Attention should rather be placed
to the toxicity and the level of cereulide in the food.
A wide range of pathogens isolated in this study are related to human and animal
interface particularly Staphylococcus sp., Salmonella sp. and
Escherichia coli. This indicated the importance of defining this interface
as a critical control point in the management of food safety in prawn from the
wild. Also, it implies the need to define and enforce acceptable limits of interaction
between human and aquatic environments. The practice of using ice as a means
of achieving safe and quality prawn is adequate when spatial and temporal distance
is short between source of capture and consumption. The population of the psychrophillic
Pseudomonas sp. festered with increasing days of storage, the prawns
are however safe when not consumed beyond the 7 days of storage with ice.
The authors would like to acknowledge the assistance of Opeyemi, Adeoye and
Orji, Elizabeth Ifeyinwa of Microbiology Department, Lagos State University
for helping with data collection.