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Journal of Applied Sciences and Environmental Management
World Bank assisted National Agricultural Research Project (NARP) - University of Port Harcourt
ISSN: 1119-8362
Vol. 9, Num. 3, 2005, pp. 53-57
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Journal of Applied Sciences & Environmental Management,
Vol. 9, No. 3, 2005, pp. 53-57
Mycotoxigenic and Proteolytic Potential of Moulds
Associated with Smoked Shark Fish (Chlamydoselachus anguincus)
* ESSIEN, J P; EKPO, M A; BROOKS, A A
Department of Microbiology, University of Uyo,
P. M. B. 1017 Uyo, Nigeria.
Code Number: ja05058
ABSTRACT:
Among the 33 moulds isolated from 20 samples of
wood-smoked Chlamydoselachus anguincus (shark-fish) 20 isolates were
capable of producing metabolites toxic to fertile Hubbard Golden Comet (Niger
chick) eggs. Aspergillus and Pencillium isolates were the
predominant moulds. Other toxigenic moulds isolated were Eurotium, Fusarium and Cladosporium species.
The protease producing potential of the isolates varied among the genera and
between isolates of the same species. The
existence and growth of these moulds on smoked fish is a pointer to the
potential health risk associated with the consumption of mouldy dry fish.
@JASEM
Preservation of fish by smoking is carried out after catching, and
smoked fish may be eaten without further cooking. From the processing centres
to the market centres smoked fishes are often contaminated with microorganisms
including moulds (Okafor and Nzeako 1985, Wu and Salunkhe 1978). Information on
the effect of wood smoke on the properties of food and microflora associated
with foods is gradually being generated (Wu and Sahunkhe 1978, Okafor and Nzeaka
1985, and Essien et al.; 1999). But reports on the role of moulds in the
spoilage of smoked foods and their mycotoxigenicity is scanty. In view of the
present day concern about mycotoxins and the kind and activities of moulds in proteinaceous
foods; the mycotoxigenic and proteolytic potentials of moulds associated with
smoked shark fish (Chlamydoselachus anguincus) was investigated.
MATERIALS AND METHODS:
Sample Collection: A total of 50 woodsmoked shark fish (C. anguincus) samples
were obtained from Ine Okoi (Fish processing centre) and Uyo main market
(Market centre) located respectively, in Oron and Uyo Local Government Areas of
Akwa Ibom State, Nigeria.
Isolation of Moulds Associated with Smoked
Shark Fish Samples:
Dilution plate and direct isolation techniques were used for the isolation of
moulds from the smoked fish samples. In the isolation process, malt extract
agar (Difco) and potato dextrose agar (Difco) plates were used. To prevent bacterial
growth 100 ppm oxytetracyline was added (Mossel et al 1975 and Pitt
1979).
Fish samples often surface sterilization
in sodium hypochloride with 1000ppm available chlorine were aseptically milled
and the fish powder were diluted in sterile distilled water with 0.05% Tween 80
at a ratio of 1/10 (v/w) (Jarvis 1978). During the isolation process 20g of the
sample was diluted in 180ml sterile distilled water and homogenized for 3
minutes in a top drive homogenizer. Petri dishes were inoculated using the spread-plate
technique and incubated in the dark at 25°C for 7 days. Afterwards petridishes
with visible colonies were selected and the mould contamination levels for 1g
samples were determined. The samples were examined with the naked eye and with
a stereomicroscope. The colonies with different morphological characters were
repeatedly inoculated on appropriate media for purification.
Identification of the mould isolates was made for penicillia
according to Pitt and Hocking (1985), aspergilli to Raper and Fennell (1965)
and other Deuteronomycetes, Ascomycetes and Zygomycetes to Pitt and Hocking
(1985) and Samson et al (1981).
Cultivation of Isolated Molds and
Production of Mycotoxins:
For the production of mycotoxins, spores of 106 (determined by means
of a hamocytometer) per ml from the sample were added to 50ml of potato
dextrose broth (Difco) in Erlenmeyer flasks (250ml) and incubated at 27°C
for one month. When moulds did not produce spores mycelia were used for
inoculation. After incubation the content of the flasks were sterilized by
autoclaving (121°C for 15 minutes) and 10ml of culture filtrate was
prepared from each of the isolates. The remaining material from the flasks was
extracted with chloroform with the aid of a high speed mixer (Gallenkamp 300 England). The extraction procedures was repeated 2 times using
100ml of chloroform for each extraction. The chloroform extracts were combined,
concentrated in a flask evaporator and diluted to 5ml with chloroform.
Assay of Mycotoxigenicity of Mould Isolates
The mycotoxigenicity of
the mould metabolite was assayed using the chicken embryo toxicity test (Horwitz
et al 1975, Wu and Salunkhe 1978, Essien 2000). The air sacs of groups
of ten fertile Hubbard Golden Comet (Niger chicks)
eggs were loaded with 0.02ml of the culture and chloroform extracts before
incubation. The rate of hatching was taken as the index of toxicity. Control
groups of eggs were injected with chloroform and sterilized potato dextrose
broth.
Determination of Proteolytic Activity of
Mould Isolates: The
Medium to detect protease produced by fungi contained nutrient agar (Difco) to
which gelatin was added as the protein source (0.4% final concentration) (Anagnostakis
and Hankin 1975). Inoculated plates were incubated for 7 days, afterwards a
saturated solution of ammonium sulphate was poured over the agar surface to
enhance visibility of the zones of proteolysis (SAB 1951).
The concentration of protease enzyme produced over
time by the moulds was estimated based on teh ability of the isolates to
produce coagulase enzymes that can clot milk (Elsie and Mansel 1987). In this
analysis 1ml of the cell free extract from 4 day old mould cultures and 1ml of
sterile cow milk were mixed in a test tube. Control experiments which
consisted of 2ml of milk only and 2ml of extract only in separate test tubes
were also prepared. The samples were then incubated at 27°C and
checked every 15 minutes interval until complete coagulation of the milk
samples occurred. The rate of clotting was determined using the Folman
equation (Berridge 1955 and Elsie and Mansel 1987) as follows:
T = K + t
C
where T = clotting time (minutes)
C = the concentration of enzyme
K and t are constants depending on the
enzymes and milk substrate respectively.
The protease enzyme concentration was
calculated by extrapolating the formula above, where
T = K
C+t
RESULTS AND DISCUSSION
Mould contamination levels were calculated for 1g
samples of the smoked fish from fish processing and market centres. The
results are presented in Table 1. The mould counts were higher in fish samples
from the market centre indicating a gradual loss in the preservative efficacy
of wood-smoking over storage time. The higher mould counts of smoked fish from
market centre may also be attributed to continuous contamination of the item
through frequent handling and to the general poor sanitary condition of many
Nigerian market centres. The fish samples from the market centre also harboured
more mould genera and species.
Of the 33 moulds isolated from the 20 smoked fish
samples. Only seven (21%) of the isolates were found in samples from the fish
processing centre in Ine Okoi and 26 (79%) from the Uyo market centre. Aspergillus
flavus was the dominant (27.2%) mould flora of the smoked fish. Aspergillus
niger, Penicillium expansum and P. viridicatum were
also frequently encountered in the smoked fish. These 4 mould species (A. flavus,
A. niger, P. expansum and P. viridicatum) comprised
72.7% of the total fungal isolates, while Cladoporium sp, Eurotium repens,
Fusarium roseum and Paecilomyces sp comprised the remaining 27.3%.
The effect of mould growth metabolites on
the hatchability of the eggs, is presented in Table 2. Toxicity was noticed in
either the culture filtrate or chloroform extract of 20 cultures and in some
cases in both fractions. The extent of toxicity depended on the species and
strains of the moulds. Only 8 of the 15 Aspergillus cultures were toxic
to chick embryo. Toxic aspergilli included 6 isolates of A. flavus and
2 isolates of A. fumigatus. None of A. nigerisolates found on the smoked fish produced toxic methabolites.
Among the mycotoxin producing aspergilli PC-31, MC-8, and MC-10 of A. flavus
and MC-32 and MC-33 of A. fumigatus demonstrated the highest level
of toxicity to chick embryos. Seven of the 9 Penicillium cultures from
the fish samples were also found to be mycotoxigenic. These include 3 isolates
of P. expasum (MC-23, MC-26 and MC-27) and 4 isolates of P. viridicatum
(PC-30, MC-24, MC-25 and MC-28). Their toxicity was observed mostly in
chloroform extracts of cultures and decreased with increase in dilution level.
The culture filtrate of mycotoxigenic penicilli had little toxic effect on
chick embryos. Similarly chloroform extract of Cladosporium isolate
MC-3, as well as MC-9 and MC-12 of Fusarium roseum exhibited mycotoxigenicity.
The mycotoxigenic potentials of the mould
genera implicated in the present study have earlier been reported by Davis (1981) and Heperken and Alperden (1988). The most significant
member of the mould flora, A. flavus, is known to produce aflatoxins,
while P. expansum and P. viridicatum produce penicillic acid and ochratoxin
respectively when cultured on organic substrates (Joffe 1965). Of the
remaining genera, Eurotium repens have been associated with the
production of sterigmatocystin. Fusaria species produces trichothecenes and zearalenone
(Davis and Diener 1978, Marasas et al 1979) while Cladosporium
species are known to produce epicladosporic acid and fagicladosporic acid (Joffe
1965).
Results on the protease producing potential of the
mould isolates (Table 3) showed that the moulds have weak proteolytic
potential. Their proteolytic activities however varies among the isolates.
Among the isolates, strains of P. expansum and P. viridicatum demonstrated
the most noticeable proteolytic activity, folowed by strains of A. nigerand A. flavus. Their ability to elaborate protease may be
responsible for their preponderance in the smoked fish samples analysed. It is
also an indication of their active role in the spoilage of smoked fish. On the
other hand Cladosporium sp, Eurotium repens and Fusarium roseum
exhibited a relatively low protease producing potential, and were not commonly
encountered in the food item.
The frequent existence of mycotoxigenic moulds
that can produce proteases in smoked fish, strengthened the possibility of high
level of contamination by moulds. This is an indication of the possible health
risk associated with the consumption of mouldy dry fishes. Therefore mouldiness
of smoked proteinaceous foods should be viewed with serious concern because of
the ability of the moulds to produce mycotoxins, some of which are very
dangerous and lethal to humans and animals even in small doses (Purchase
1974). Proper storage of smoked fish is also necessary because poor storage
methods and unhygienic handling of the items are known to predispose them to
microbial contamination.
Table 1. Microbial Flora of smoked Chlamydoselachus
anguincus.
Property
|
Processing
Centre (PC) n=12
|
Market centre (MC) n = 8
|
Total No. of isolates
|
Isolate code
|
(a)
Mould Count (102
cfu/g)
(b)
Mould Isolates
Aspergillus flavus
Aspergillus fumigatus
Aspergillus niger
Cladosporium sp.
Eurotium repens Fusarium roseum
Paecilomyces sp.
Penicillium expansum
Penicillium viridicatum
|
2.18
+
-
+
+
-
-
-
-
+
|
3.86
+
+
+
+
+
+
+
+
+
|
9
2
4
3
2
3
1
4
5
|
PC-5,-15,-31,MC-6,-8,-10,-11,-13,-14.
MC-32-33
PC-1,MC-4,-5,-16
PC-17,MC-3,-19.
MC-2,-18
MC-9,-12,-20
MC-22
MC-21,-23,-26,-27
PC-29,-30,MC-24,-25,-28.
|
Total
|
4
|
9
|
33
|
|
n = Number of samples, + = Present, - = Absent
Table 2: Percent hatchability of chick eggs loaded with mould growth
metabolites.
Mould
|
Strain Culture filtrate |
Chloroform extract
|
|
Undil. 1:10
|
Con. 1:10 1:100
|
A.
flavus
A.
fumigatus
Cladosporium sp
Eurotium
repens Fusarium roseum
P. expansum
P. viridicatum
|
PC-5
PC-31
MC-8
MC-10
MC-11
MC-14
MC-32
MC-33
MC-3
MC-18
MC-9
MC-12
MC-20
MC-23
MC-26
MC-27
PC-30
MC-24
MC-25
MC-28
|
10
0 20
0 40
0 20
60
80
0 30
0 50
60
60
60
40
70
80
70
80
50
40
60
50
|
0
0
0
0
0
10
0
0
0
0
0
0
10
0
0
0
0
0
0
0
|
0
10
0
20
0
0
0
20
0
0
20
0
0
10
20
10
0
0
|
40
30
60
10
20
40
20
30
20
40
30
|
Mean of
Hatchability of eggs loaded with chloroform was 95% and for sterile Potota dextose
broth was 100% (controls).
Table 3: Proteolytic
potential of moulds isolated from Chlamydoselachus anguincus
Mould
|
Isolate code
|
Milk clotting
time (minutes)
|
Concentration
of protease (ug/ml)
|
A. flavus
A.
fumigatus
A. niger
Cladosporium sp
E. repens
F. roseum
Paecilomyces sp
P. expansum
P.viridicatum
|
PC-5
-15
-31
MC-6
-8
-10
-11
-13
-14
MC-32
-33
PC-1
MC-4
-5
-16
PC-17
MC-3
-19
MC-12
-18
MC-9
-12
-20
MC-22
MC-21
-23
-26
-27
PC-29
-30
MC-24
-25
-28
|
555
570
615
555
615
570
570
555
555
705
090
600
615
630
615
690
705
690
690
690
615
630
615
690
615
570
540
555
570
555
570
615
615
|
0.0014
0.0016
0.0015
0.0015
0.0016
0.0013
0.0015
0.0014
0.0015
0.0015
0.0014
0.0016
0.0015
0.0014
0.0016
0.0014
0.0013
0.0011
0.0014
0.0015
0.0016
0.0014
0.0015
0.0015
0.0016
0.0017
0.0019
0.0018
0.0017
0.0016
0.0018
0.0016
0.0015
|
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