Mycotoxigenic and Proteolytic Potential of Moulds Associated with Smoked Shark Fish ( Chlamydoselachus anguincus ) *

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 25C 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 10 (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 27C 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 (121C 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. JASEM ISSN 1119-8362 All rights reserved J. Appl. Sci. Environ. Mgt. 2005 Vol. 9 (3) 53 57 Full-text Available Online at http:// www.bioline.org.br/ja Mycotoxigenic and Proteolytic Potential of Moulds... Essien, J P; Ekpo, M A; Brooks, A A 54 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 27C 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 woodsmoking 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. niger isolates 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, MC25 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. niger and 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

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 andNzeako 1985, Wu andSalunkhe 1978).Information on the effect of wood smoke on the properties of food and microflora associated with foods is gradually being generated (Wu andSahunkhe 1978, Okafor andNzeaka 1985, andEssien 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 andPitt 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 0 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 10 6 (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 0 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 0 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 0 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 andElsie andMansel 1987) as follows: 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 woodsmoking 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. niger isolates 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 andDiener 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. niger and 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.
of eggs loaded with chloroform was 95% and for sterile Potota dextose broth was 100% (controls).

Table 2 :
Percent hatchability of chick eggs loaded with mould growth metabolites.

Table 3 :
Proteolytic potential of moulds isolated from Chlamydoselachus anguincus