ASSESSEMENT OF MICROBIAL LOAD AND CHARACTERIZATION

OF FUNGI ISOLATED FROM SMOKED FISH IN OSIELE MARKET,

ABEOKUTA OGUN STATE.

WRITTEN BY

OLAYIWOLA RISIKAT ADEBOWALE

19/50/0671

A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF SCIENCE

LABORATORY TECHNOLOGY, SCHOOL OF SCIENCE AND TECHNOLOGY,

MOSHOOD ABIOLA POLYTECHNIC, ABEOKUTA, OGUN STATE.

IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF

HIGHER NATIONAL DIPLOMA (HND) IN MICROBIOLOGY

JANUARY, 2025.
 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the Study

Fishes are important source of food for human globally because man gets a lot of minerals,

vitamins, lipids and proteins from fishes and theirs products. However, availability of these

vital nutrients depends to a large extent on the methods of preservation such as salting,

roasting, drying, and freezing (Gram and Huss, 2001; Whong et al., 2003; Awe and Adejo,

2018). Fresh and smoked fishes of different types are of great demands by the consumers in

Nigeria because they are relatively cheaper source of animal protein. Fish and fish products

are important, not only from the nutritional point of view but also as a source of income and

revenue to the sellers and the government respectively. Fishes are perishable, high-protein

foods that typically contain a high level of free amino acids. Microbes metabolize these

amino acids, producing ammonia, biogenic amines such as putrescine, histamine, cadaverine,

organic acids, ketones and sulfur compounds (Dalgaard et al., 2006).

Fishes are regarded as omnivorous animal because they feed on plants and other small sea

animals of water bodies. Some of the varieties of fishes which are available in the world are

as follows: Siamese fighting fish, Goldfish, Guppy, Blowfish, Common carp, Snakehead

murrel, Nile tilapia, Ocean sunfish, Oscar, Wels catfish, Sucker-mouth catfish, Northern pike,

Freshwater angelfish, Asian arowana, Blue tang, Neon tetra, Swordfish, Common molly,

Stonefish, Barramundi, Giant oarfish, Bluegill, Mahimahi, Whale, shark, Rainbow trout,

Atlantic salmon, Basa, Zebrafish, Frilled shark, Giant, Megalodon, Burbot, and Garfish

(Mahendra et al., 2016).

The smoking processes of fish are of two forms viz. wet hot smoking and dry hot smoking.

Both processes are carried out at temperatures high enough to cook the fish. Wet hot smoking
usually takes about 1 - 2 hours and yields a moist, versatile product with about 40 - 55

percent moisture content, while dry hot smoking, which is usually preceded by the former

process, takes about 10 – 18 hours, sometimes days and yields fish with 10 - 15 percent

moisture content. In the tropical countries such as Nigeria, Akande and Tobor (1993) and

Olokor et al. (2007) documented that smoke-drying of fish is one of the oldest available local

forms of preservation methods essentially employed by most fishing communities. Smoke-

drying, apart from giving the product desirable taste and odour, preserves and prolongs the

shelf-life of fish products conveniently at ambient conditions through its anti-bacterial and

oxidative effects, lowering of pH, imparting desirable colouration, accelerating the drying

process and acting as antagonist to spoilage agents (Olokor et al., 2007; Sengor et al., 2004;

Eyo, 2012).

Smoking simply means a heating process that dries the fish to preserve it from spoilage

(Uzuegbu and Eke, 2000). Most dry fish consumed in Nigeria are smoked (Okonkwo, 2001).

Smoking of fish from smoldering wood for its preservation dates back to civilization (Olokor,

2007). The steps in the smoking process are necessary not only for safe preservation, but also

to produce good flavor and aroma (Ray and Ray, 2004). Hence smoked fishes are less prone

to microbial spoilage than fresh fish. However spoilage still occurs as a result of growth of

microbes due to partial dehydration during smoking.

Smoking reduced the total viable count significantly in all samples, but when the smoked

products are constantly exposed to the effect of humid environment, the possibility for an

increase in the moisture content of the smoke-dried product is inevitable thereby enhancing

the activity/proliferation of these microorganisms. Eyo (2001) stated that smoked fish

samples may have a relatively high water activity level which is a prerequisite for microbial

growth.
Fish spoilage is a complex process involving both non-microbiological and microbiological

processes. Non-microbiological deterioration is caused by endogenous proteolytic enzymes,

which are concentrated in the head and viscera and attack these organs and surrounding

tissues after death. Enzymatic spoilage is followed by the growth of microorganisms, which

invade the fish flesh, causing breakdown of tissues and a general deterioration of the product.

According to Nyarko, et al. (2011), smoked sardine from marketing centers had higher

microbial counts than those from smoking sites due to the better sanitary conditions of the

latter. Adegunwa, et al. (2013) reported that smoked fish collected at a camp location in

Odeda, Ogun State, Nigeria had higher microbial load than other locations as a result of

handling, frequent exposure, poor environmental and sanitary conditions. The disparities in

contamination levels between locations have been observed to be influenced by one or more

of the factors enumerated by Tatcher and Clark (1973) as follows: Source of the raw fish;

Temperature of food during storage and processing; Severity of freezing process in terms of

lethality to microorganisms; Additional contamination introduced by handlers;

Contamination after the fish had already been processed. Smoked fish is relished food item in

many dishes in Nigeria; therefore, there is need for corresponding concern for safety issues in

fish consumption (Riches, 2012).

Adebayo-Tayo et al. (2008) reported Aspergillus flavus, Aspergillus tereus, Aspergillus

fumigatus, Absidia spp., Rhizopus spp., A. niger, Mucor spp., Cladosporum spp., Penicillium

italicum, Penicilium viridatus, Candida tropical and Fusarium moniliformis as fungal

isolates found in smoked dried fishes sold in different markets in Uyo, Nigeria.

Akise et al. (2013) reported predominant fungi species isolated from three different

anatomical parts of smoke-dried fish under storage to include P. italicum, Penicillium

oxalicum, Mucor, Saccharomyces, Rhodotorula spp. and Aspergillus spp. Majority of the

fungi produce mycotoxins. Mycotoxins produced by Aspegillus species are known to produce
many types of toxins such as aflatoxins, ochratoxins and sterigmatocystine (Hashem, 2011.

Acute aflatoxicosis in humans has been reported in different parts of the world and was

characterized by symptoms like vomiting, abdominal pain, pulmonary oedema, convulsion,

coma, and death with cerebral oedema and fatty involvement of the liver, kidney, and heart

(Akinyemi et al., 2011).

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 fungus on the fish

species. Adebayo-Tayo et al. (2008) reported the presence of aflatoxins and other metabolites

in smoked fish due to A. flavus in smoked fish sold in Uyo, Akwa Ibom state, Nigeria and

confirmed that consumers could have been at risk of aflatoxin poison.

Samuel et al. (2015), Sani et al. (2016), and Osibona et al. (2018) reported to have isolated

fungi of different species from smoked fish. These fungal species can spoil food products or

produce mycotoxins or both (Anderson and Thrane, 2006). These smoked fish are exposed to

contamination by fungi through handling of fish during processing, smoking techniques,

moisture content and storage method. Also, fungal contamination can also be through the air

because these fishes are hawked in open trays. Most of these fungi are known to have public

health risks based on certain pathogenic attributes, causing varying degrees of health

problems to both animals and man. These mycotoxins may result in decreased productivity,

immune suppression and chronic damage to vital tissues and organs of animal and humans

(CAST, 2003). It also causes reduction in nutrient value, rotten odour, unpalatable taste and

many economic losses which include rejection of the fungal contaminated smoked fish at

international market (Hassan et al., 2011). Thus, this study tend to characterize fungi isolated

from commonly sold smoked fish in Osiele market, Abeokuta, Ogun State.
1.2 Statement of the Problem

The consumption of smoked fish is a widely practiced culinary tradition in various cultures

due to its unique flavor and extended shelf life. However, the smoking process does not

inherently guarantee the complete elimination of microbial contaminants, including fungi.

The presence of fungi in smoked fish can lead to spoilage, off-flavors, and potential health

risks due to the production of mycotoxins. Despite its popularity, there is limited information

regarding the types and characteristics of fungal species isolated from commonly sold

smoked fish products. Therefore, this study tends to characterize fungi isolated from

commonly sold smoked fish in Osiele market, Abeokuta, Ogun State.

1.3 Justification of the Study

The presence of fungi in smoked fish products can pose a significant health risk to

consumers. Certain fungal species can produce mycotoxins, which are toxic compounds that

can lead to serious health issues, including food poisoning and long-term chronic diseases.

By identifying the fungi present in smoked fish, this study will inform the public and

regulatory bodies about potential risks associated with specific products.

Fungi can contribute to spoilage and degradation of food products, affecting flavor, texture,

and overall quality. Understanding the types of fungi associated with smoked fish will help

manufacturers and retailers implement proper quality control measures, ensuring that

products meet safety and quality standards, thereby safeguarding their reputation and

customer satisfaction.

1.4 Aim and Objectives of the Study

The aim of the study is to characterize fungi isolated from commonly sold smoked fish in

Osiele market, Abeokuta, Ogun State.

The objectives of the study are:

i. To isolate and identify the fungal species present in smoked fish samples.

ii. To evaluate the total fungal count from the fish samples
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Fish Smoking

Fish is highly susceptible to deterioration without any preservative or processing measures

(Okonta & Ekelemu, 2005) and requires proper handling and preservation to increase its shelf

life, quality and nutritional value (Ye, 2009). The fishing industry despite its importance

suffers from enormous post- harvest losses which are estimated at 35–40% of landed weight

(Food and Agriculture Organization, 2004). FAO (2004) estimated that post- harvest losses

remain about 25% of the total world catch annually. These losses have a profound adverse

impact on fishing communities whose status and income often depend on post-harvest

activities. Such losses also have a detrimental impact on the socio-economic life of the

fishing communities and reduce the amount of animal protein available to large segment of

the population.

Smoking as a way of preparation of fish and meat has been known to the human kind since

the stone age (Adeyeye, 2016). Smoking represents set of the chemical, thermal, diffusive

and biochemical processes proceeding in preliminary salted product. Today, the technology is

applied in many forms to treat 40–60% of the total amount of meat products and 15% of fish.

Smoking is defined as the process of penetration of volatiles resulting from thermal

destruction of wood into the surface of meat or fish products (Adeyeye, 2016).

Smoke not only gives special taste, colour and aroma to food, but also enhances preservation

due to the dehydrating, bactericidal and antioxidant properties of smoke. The process of

smoking fish occurs through the use of fire. Wood contains three major components that are

broken down in the burning process to form smoke. The burning process is called pyrolysis,
which is simply defined as the chemical decomposition of wood by heat. The major wood

components are cellulose, hemicellulose and lignin (Alasalvar et al., 2011).

The major steps in the preparation of smoked fish are salting (by bath or injection of liquid

brine or dry salt mixture), cold smoking, cooling, packaging (air/vacuum or modified) and

storage. Smoking, one of the oldest preservation methods, combines the effects of salting,

drying, heating and smoking (Alasalvar et al., 2011). Typical smoking of fish is either cold

(28–32°C) or hot (70–80°C). Cold smoking does not cook the flesh, coagulate the proteins,

inactivate food spoilage enzymes or eliminate the food pathogens, and hence refrigerated

storage is necessary until consumption (Alasalvar et al., 2011), although dry cured hams are

cold smoked and require no refrigeration.

Finally, smoking is the oldest food preservation technique. However, Underwood and Shoop,

(2007) reported that convective smoking is being substituted by the use of smoke flavourings.

Several authors (Kostyra and Barylko-Pikielna, 2006; Varlet et al., 2007) have reported that

the treatment of salmon with smoke flavouring leads to changes in its physicochemical and

sensory attributes. According to Martinez (2009), the characteristics of the final product

depend on the composition of these flavourings and may be very similar to those achieved

with the traditional cold smoking method.

The advantages of smoking fish are manifold. Fish smoking prolongs shelf life, enhances

flavour and increases utilization in soups and sauces. It reduces waste at times of bumper

catches and permits storage for the lean season. It increases protein availability to people

throughout the year and makes fish easier to pack, transport and market especially in the rural

areas. However, there are several problems associated with the traditional processing

methods which predispose the artisanal catch to large-scale post-harvest losses (Varlet et al.,

2007).
2.1.1 Fish Smoking Methods

i. Hot Smoking

Hot smoking does have different main processes that: cooking; when the smoking is

performed at temperatures above 80°C, the flesh of the fish is cooked, the heat can destroy

the microbes that found inside and, on the fish, and inactivate enzymes in the gut and flesh,

drying: the smoke that produced from fire also generates heat that he to dry the fish and

smoking; the smoke is produced by burning wood containing a number of compounds, some

of which kill bacteria; the process has a preservative value. The smoking process can take the

form of wet hot smoking or dry hot smoking. Both processes are carried out at temperatures

high enough to cook the fish. Wet hot smoking usually takes about 1–2 h and yields a moist,

versatile product with about 40–55% moisture content but a limited shelf life of 1–3 days

(Arvanitoyannis and Kotsanopoulos, 2012).

Dry hot smoking, which is usually preceded by the former process, takes about 10–18 h,

sometimes days, and yields fish with 10–15% moisture content, sometimes even below 10%.

Fish smoked by this process have a shelf life of 6–9 months when stored properly. The fish

treated with hot smoking is ready to consume and has desired aroma. It is applied at the

temperature of 50–80°C for 20–60 min (Arvanitoyannis and Kotsanopoulos, 2012).

ii. Warm Smoking

It is fish smoking that carried out by applying a temperature between 25–50°C (Alasalvar et

al., 2011).

iii. Cold Smoking

Cold smoking treatment is that the flesh is not going to be cooked, the protein will not be

coagulated, does no inactive spoilage enzymes or destroy the food pathogen so that storing
the food inside the refrigerator until consumption is necessary (Alasalvar et al., 2011). Cold

smoking is performed below 25°C and the temperature used in the cold smoking process

varies depending on the species of fish but is usually between 15–23°C.

2.1.2 Chemical Composition of Smoked Fish

Smoking is one of the methods of preserving fishery products. Various studies on

preservation by smoking have been carried out, such as the Coban and Patir (2013) study on

smoking Oncorhynchus mykiss with the addition of clove oil, and Ficicilar and Genccelep

(2017) on Rainbow Trout. The quality of smoked fish products is influenced by raw

materials, smoking methods, smoking concentrations and raw materials for smoking sources

(Ficicilar and Genccelep, 2017). Indicators in assessing the durability of smoked fish

products can be seen fromthe values of TBA, TVB and pH as well as the value of biogenic

amines consisting of histamine, putrescine, cadaverine, tyramine, and tryptamine compounds.

The quality of smoked fish can be seen from the value of protein, vitamins and minerals it

contains. According to Adeyeye et al. (2018), the processing of raw materials will influence

on the nutritional composition of the final product. Several nutrients in the food will be lost

during the processing. Amino acids, vitamins, and minerals will usually be easily

decomposed due to heat so that the quality of smoked fish can be determined also from the

content of amino acids, vitamins, and minerals.

2.1.3 Microbiology of Smoked Fish

Traditionally processed fishery products are very susceptible to microbiological damage.

Microbiological damage can be caused by pathogenic bacteria or fungi. Damage to a product

depends on the initial bacterial count, sanitation and hygiene during processing, and

preservation methods and storage methods. Improper processing methods will cause the

growth of Salmonella bacteria in the product (Mailoa et al., 2013). According to Rukmayeni
et al. (2013), the smoking method using liquid smoke at a temperature of 30-60ºC can extend

the shelf life of smoked fish products. This can be determined by the number of bacterial

colonies during storage. Some pathogenic bacteria in smoked fish will not grow because of

the heat from the smoking process. The high temperature used can damage these pathogenic

bacteria as well as the presence of chemical compounds from the smoke that are useful as

antimicrobials (Felix and Kehinde, 2015).

Spoilage and pathogenic bactericidal activity of smoke are due to integrated effect of heating,

drying, salting and also as a result of deposition of polyphenolic constituents on fish.

Polyphenolic constituents like formaldehyde and acetic acid are found to show bactericidal

effect which can prevent fungal growth and can inhibit viral activities. According to Syam

(2018), the count of bacteria from smoked fish in improved smoking kiln; Total Plate Count,

Salmonella, Total coliforms, and Escherichia coli count were (1.936 × 106, 1.550 × 104, 2.00

× 104 and 0 cfu/g) than fish smoked in traditional smoking kilns (5.656 × 106, 2.100 × 104,

4.53 × 104 and 6.67 × 102 cfu/g). Smoking of fish product minimizes the number of

Salmonella, Total Coliforms, and E. coli found in the C. gariepinus fish as highest figures

were found in fresh samples 3.575 × 10 4, 1.80 × 105, and 4.00 × 104 cfu/g, respectively and

that higher temperatures during smoking resulted in more microorganisms being killed and

not able to multiply (Syam, 2018).

Microbiological study shows the microbiological changes of hot smoked rainbow trout fillets

treated with/without nisin and lysozyme during 42 days of storage at 4°C. The Total

Mesophilic Aerobic Bacteria in raw rainbow trout was determined as 4.40 log cfu/g,

indicating a good quality of fish. The smoking and brining process significantly reduced the

microbial load of rainbow trout (Oyeleye, 2020)..

Aerobic Mesophilic Bacteria and Lactic Acid Bacteria were found dominant flora in both

types of processed fish, with AMB reaching concentrations up to 9.5 and 7.8 Log10 (CFU/g)

in the smoked fish and smoked dried-fish samples, respectively and Enterobacteriaceae, E.

coli, B. cereus, C. perfringens, yeasts, and molds have been found in many samples, while

other bacteria like Salmonella spp., L. monocytogenes, and S. aureus were not detected

(Ghazali et al., 2014). The variation showed between the minimum and maximum values

indicates that it has important variability within the samples. This variation can be explained

by the fact that the samples were collected from various processors and sellers where the

quality of the raw material varied, as well as handling and hygiene practices. The density

AMB, Enterobacteriaceae, and E. coli was also significantly higher (p < 0.05) in SF than in

SDF (Ghazali et al., 2014). This may occur due to that of smoke d fish have more moisture

content than that of smoked dried-fish. The high load of Aerobic Mesophilic Bacteria is

likely due to a high contamination level of the raw material, and these microorganisms were

not fully eliminated during the smoking treatment. The post process handling and storage

conditions are also potential sources of renewed pollution of the processed fish (Jorgensen et

al., 2000). The detection of E. coli in ten samples suggested a contamination by fecal matter

from animal or human origin during post smoking handling.

The presence of B. cereus and C. perfringens beyond the permitted limits may arise a hazard

to consumer's health (Ghazali et al., 2014). Fish and fish products are exposed more for the

spoilage as of bacteria biomass get increased. Smoking with wood-smoke has antibacterial

activities that could effectively suppress bacterial growth. According to Nowsad, 2007), the

bacterial count significantly decreases as of increasing time of smoking at 20, 25, and 30

minutes.
2.1.4 Physico-chemical and Functional Changes during Smoking

Smoking as a way of preparation of fish and meat has been known to the human kind since

the Stone Age. Smoking represents set of the chemical, thermal, diffusive and biochemical

processes proceeding in preliminary salted product. Today, the technology is applied in many

forms to treat 40–60% of the total amount of meat products and 15% of fish. Smoking is

defined as the process of penetration of volatiles resulting from thermal destruction of wood

into the surface of meat or fish products (Jurtsenko, 2011; Larsen et al., 2008).

Smoke not only gives special taste, colour and aroma to food, but also enhances preservation

due to the dehydrating, bactericidal and antioxidant properties of smoke.

Smoked fish contains compounds that are favourable and also compounds that are hazardous

to human health. The advantage of eating smoked fish is that it has high protein content and

at the same time is low in saturated fat. On the other hand, eating too much of smoked fish

can increase the risk of stomach cancer (Jurtsenko, 2011).

Traditional way of fish smoking is a preservation method giving a characteristic flavour and

colour to the product. However, during this process undesirable compounds can be formed,

mainly the PAHs. These molecules are produced during pyrolysis of organic material that is

used for generation of the smoke, mainly wood. It has been established that the content of

carcinogenic PAHs in smoked fish mostly depends on smoking temperature and smoking

time. The levels of PAHs in raw and cold-smoked fish are lower than the levels in hot-

smoked fish samples (Jurtsenko, 2011).

Smoke dry salting is a curing method that retains the intactness of the fillet surface and its

colour, both of which are important consumer quality criteria (Birkeland et al., 2004).

Brining can help to maintain the final weight and improve the palatability of salmon flesh,
although it leads to the loss of some nutritional components (Jurtsenko, 2011; Larsen et al.,

2008).

Several authors have indicated that brining offers advantages over smoke dry salting in that it

stops the flesh becoming rancid through contact with the air (Andre et al., 2005; Jurtsenko,

2011) and increases the final weight of the product by encouraging the intake of water

(Gallart-Jornet et al., 2007).

Gallart-Jornet et al. (2007) showed that lean and fatty fish behave differently during the

salting procedure. Fat presents a physical barrier to NaCl and water transport and therefore

acts as a limiting factor to the diffusion of salt and water diffusion during salting step as a

consequence of its hydrophobicity. Doe (2008) recommended brine to be used with fatty fish

because immersion limits the oxidation processes affecting the flesh of the fish.

Finally, smoking is the oldest food preservation technique. However, convective smoking is

being substituted by the use of smoke flavourings (Jurtsenko, 2011; Underwood and Shoop,

2007). Several authors (Kostyra and Barylko-Pikielna, 2006; Varlet et al., 2007) have

reported that the treatment of salmon with smoke flavouring leads to changes in its

physicochemical and sensory attributes. Besides, depending on the composition of these

flavourings, the characteristics of the final product may be very similar to those achieved

with the traditional cold smoking method (Martinez et al., 2009).

2.1.5 Quality and Safety status of Smoked Fish

Traditional smoking techniques involve treating of pre-salted, whole or filleted fish with

wood smoke in which smoke from incomplete wood burning comes into direct contact with

the product; this had been found to contaminate smoked fish with PAHs if the process is not

adequately con- trolled or if very intense smoking procedures are employed (Guillen and

Sopelana, 2005; Gómez-Estaca et al., 2011).

PAHs constitute a large class of organic compounds, containing two or more fused aromatic

rings made up of carbon and hydrogen atoms and smoked fish is one source of PAH (Guillen

and Sopelana, 2005). When fish is smoked, roasted, barbecued or grilled, PAHs are formed

as a result of incomplete combustion or thermal decomposition of the organic materials

(WHO, 2006). Pyrolysis of the fats in the meat/fish generates PAH that become deposited on

the meat/fish. PAH production by cooking over charcoal (barbecued, grilled) is a function of

both the fat content of the meat/ fish and the proximity of the food to the heat source

(Kazerouni et al., 2001).

Several analyses of charcoal roasted/grilled common fish by several researchers (Guillen and

Sopelana, 2005; Akpambang et al., 2009; Linda et al., 2011) have proven the presence of

PAHs such as benzo [α] pyrene, anthracene, chrysene, benzo[α]anthracene, indeno [1,2,3-c,d]

pyrene. Several researchers (Borokovcova et al., 2005) reported that most of these PAHs

have been found to be carcinogenic while some are not. Emerole (2000) studied and screened

for the presence of PAH in local foodstuffs available in Nigerian market. He discovered that

appreciable amounts of benzo[a] anthracene and benzo [α] pyrene were found present in three

varieties of smoked fish and smoked meat (suya) purchased from a popular market in Ibadan,

Nigeria.

In a recent study carried out by Olabemiwo et al. (2011) to assess the PAH content of two

smoked fish species available in Western Nigeria, he found out that the sum of all PAHs in

the smoked fish (Claria gariepinnus and Tilapia guineensis) ranged from 0.497 to 0.814

μg/kg and 0.519 to 0.772 μg/kg, respectively. High levels of PAHs have been reported to be

associated with the dark colourations in intensively heated fish products. This was supported

by Ova and Onaran (2008) who reported that the PAH levels were significantly higher in the

fish skins than in the edible parts.

Adeyeye et al. (2015) also reported that traditional drum smoked samples had high BαP and

PAH levels (five out of six major PAHs (fluorene, anthracene, benzo[β]fluoranthene,

benzo[α]anthracene, benzo[α]pyrene and benzo[ghi]perylene exceeded the European Union

(EU) maximum permis- sible level of 5.0 μg/kg for BαP).

Studies have also reported the presence of aflatoxins: highly toxic com- pounds naturally

produced by Aspergillus flavus, Aspergillus parasiticus and some microsclerotial species of

Aspergillus section Flavi in fish and fish feed (Adebayo-Tayo et al., 2008; Almeida et al.,

2011; Barbosa et al., 2013). In addition, the hazardous effects of aflatoxin contaminated feeds

on fish health and production have been documented (Abdelhamid et al., 2007; Zaki et al.,

2008). In spite of the available data on aflatoxin contamination of various foodstuffs and

other livestock feeds, and the associated health implications, little or scanty data exist on the

occurrence of Aspergillus species and aflatoxins in fishes in Nigeria and many other

developing nations despite the fact that fishes are widely consumed in these countries.

Adebayo-Tayo et al. (2008) had reported the presence of Aflatoxin B1 and G1 in high

concentrations in fish and concluded that smoked dried fishes stored for sale in Uyo markets,

South-South, Nigeria were heavily contami- nated with aflatoxigenic fungi. Fafioye et al.

(2002) studied the fungal infestation of five traditionally smoked dried freshwater fish in

Ago- Iwoye, western part of Nigeria and isolated and identified 11 different fungal species of

which A. flavus was the most frequently encountered fungi on the fish species. Adeyeye et al.

(2015) reported the presence of Listeria mono- cytogenes in traditional drum smoked fish

samples from Nigeria and this poses risk to smoked fish consumers.

2.1.6 Chemical Hazards in Smoked fish

PAHs constitute the largest class of chemical compounds containing two or more fused

aromatic rings made up of carbon and hydrogen atoms, known to be genotoxic agents (EC,
2002). They are generally classified as relatively persistent organic environmental

contaminants formed during incomplete combustion processes which occur whenever wood,

coal or oil are burnt (Guillen and Sopelana, 2005), with combustion sources being the most

predominant (Simko, 2002). PAH contamination can signifi- cantly affect smoked fish

quality and safety.

The United States Environmental Protection Agency and EU identified PAHs as potential

carcinogens and listed them among prioritized pollutants (EC, 2006). These contaminants are

widespread in foodstuffs not only as a result of environmental pollution but also as a

consequence of some thermal treatments, which are used in the preparation and manufacture

of foods. Human exposure to PAHs occurs in three ways: inhalation, dermal contact and

consumption of contaminated foods (Silva et al., 2011), with diet being the major source as it

accounts for 88–98% of such contamination (Farhadian et al., 2011).

Food is one source of PAH. When food particularly meat, meat products and fish is smoked,

roasted, barbecued or grilled, PAHs are formed as a result of incomplete combustion or

thermal decomposition of the organic materials (WHO, 2006). Pyrolysis of the fats in the

meat/fish generates PAH that become deposited on the meat/fish. PAH production by

cooking over charcoal (barbecued, grilled) is a function of both the fat content of the

meat/fish and the proximity of the food to the heat source (Kazerouni et al., 2001).

Several analyses of charcoal roasted/grilled common food items have proven the presence of

PAHs such as benzo[α]pyrene, anthracene, chrysene, benzo[α]anthracene and indeno[1,2,3-

c,d]pyrene (Akpambang et al., 2009; Guillen and Sopelana, 2005; Linda et al., 2011). Most

of these PAHs have been found to be carcinogenic while some are not (Borokovcova et al.,

2005).
2.2 Fungi

A Fungi is any member of the group of eukaryotic organisms that includes micro-organisms.

Around 148,000 Species of fungi have been described by taxonomists. These organisms one

classified as a kingdom separately from the other eukaryotic kingdoms fungi. Fungi are

everywhere in forests and fields in soil, in our buildings and in the biotech co-operations

(Rama and Alisha, 2021). The kingdom fungi also called “Hidden” kingdom. It is the last

great unknown among the multicellular organisms. In 1753, Linnaeus coined the term fungi

as a plant class. Jahn and John (1949) and later Whittaker (1959) Used the term to define the

kingdom however; Moore (1980) was the first to apply a formal diagnosis to fungi as the

name of a Kingdom. Since the work of Whittaker (1957 and 1959) (Li et al., 2021).

For a time Margulis and Schwartz (1982 and 1988) claimed that the Fungi were deduced

from a lineage that noway had flagella. Still, the drift of Substantiation from molecular

polygenetic Studies demonstrated that the fungi evolved from a tanned line .Indeed, that the

chytrids were part of that line and that the Fungal Clade was part of a larger clade Called the

Opisthokonts (Creatures fungi in the Unikonta), a group that includes the choanoflagellates

and the metazoans, suggests that the Fungi and creatures are relatives (Abulude et al., 2013).

Fungi have evolved remarkable metabolic versatility for utilizing diverse nutrient sub states

for growth in their saprophytic Symbiotic and pathogenic lifestyle (Li et al., 2021). The

ability to reprogram Cellular metabolism according to the availability and quality of nutrients

in the environment is Fundamental for fungi to survive and thrive in diverse habitats. This

Unique decomposing ability has rendered fungi an indispensable part of the ecosystem and

popular Partners for establishing symbiotic relationships. Fungi are thallophytes that have no

green plant pigment. In many fungi the fruit bodies are the first to develop and subsequently

to Originate Spore bearing cells. The fungal body which is generally called thallus is either a
Single cell or a thread like Structure. That is called hyphae. The actual fungus body

(mycelium) is a relatively Uniform structure Composed of long cylindrical septate Colourless

or coloured hyphae sometimes provided with Clamp Connections. The hyphal walls are

smooth, and may be thin or thick and sometimes encrusted. Fungal groups can be related by

cell wall composition presence of both chitin (Vijayakumar et al., 2016).

In the middle of the 20th century the three major kingdoms of multicellular eukaryotes.

Kingdom plantae, Kingdom Animalia and kingdom Fungi were recognized as being

absolutely distinct the microscope made it possible to recognize and identify the great variety

of fungal species living or dead matter or live organic matter. Fungi (molds and yeasts) are an

important group of micro-organisms (Vijayakumar et al., 2016). Fungal contamination of

pharmaceutical products can cause not only serious economic losses to the manufacturer but

can also lead to serious health Problems to customers (patient).

In addition, some fungi form symbiotic relationship with the roots of trees and other plants.

The relationship, which is called mycorrhizal association, is mutually beneficial to both the

plant and Fungus. Fungi play a very important role in regulating natural processes. For

Example, it has been estimated that in a year several million leaves fall to the ground in each

acre (0.4ha) of a temperature deciduous forest. These leaves do not continue to pile up year

after year because various saprotrophic fungi break them down. For the reason, essential

nutrients in the leaves are recycled to the soil (Brett and Donald, 2011).

Fungi are also the major group of organisms responsible for wood decay. In the Middle Ages

in the Northern Europe, for bread making – The mold fungi, ergotamine a long–acting

alkaloid. Historically, fungi were classified as plants because they possess a cell wall and root

liked structures. But fungi do not perform photosynthesis, nor are they autotrophs. Their cell
walls are made up of different substance than plant cell walls, and eventually by the 20th

century. They began to be classified as their own kingdom (Garcia-Rubio et al., 2022).

2.2.1 Morphology of Fungi

Many fungi produce only single cells. Some fungi are dimorphic with yeast phases and

filamentous phases. The complexity of fungal morphology and life cycle is a double edged

sword to scientists (Saranraj and Anbu, 2017).

The cell wall in Fungi presents many modifications of structure and Composition The cell

wall is a characteristic Structure of fungi and is composed mainly of glucans, chitin and

glycoproteins. The cell wall is a skeleton with high plasticity that protects the cell seat from

different stresses, among which osmotic stand out (Basu et al., 2015). As the Components of

the fungal cell wall are not present in humans, this Structure is an excellent target for

antifungal therapy. The cell wall a hedge that's substantially composed of Carbohydrates

girding the Cell. The inner subcaste is a Chitin (a long-chain polymer of an N-acetyl

glucosamine) subcaste, Composed substantially glycan; the external subcaste is mostly

manoporoteins while the intermediate byer is a mixture of both the inner and outer byer. The

tube membrane is a semipermeable lipid bilayer between the cell call and the inside of the

cell (Jain, 2012).

The tube membrane is quite fluid and Flexible due to its Constituents of lipids. Sterols and

proteins yeasts cells are suitable to maintain proper membrane fluidity at different

temperatures, which is important during turmoil. The cytoplasm is that portion of cell

enclosed by the plasma membrane and excluding other membrane-bound organelles. The

mitochondrion is an organelle where aerobic respiration occurs. Mitochondria correspond of

a double membrane that's the position of the conversion of Pyruvate (a metabolic emulsion)

and he tricarboxylic acid cycle. The vacuole 3 a membrane-bound Structure that Stores
nutrients and is also where the all breaks down proteins. The endoplasmic reticulum is a

network of membranes and is usually where the cell manufactures proteins, lipids and

carbohydrates for membranes and secretion (Pietik et al., 2005).

2.2.2 Morphological classification of Fungi

2.2.2.1 Yeasts

Yeasts may be defined as a unicellular Fungus reproducing by budding or Fission and

sexually by Sport fermentation It is a type of fungi contain only Single cell. Yeast sizes very

greatly, depending on species and environment, typically 3-4um diameter. The term yeast is

often taken as a Synonym for saccharomyces cerevisiae but the phylogenetic diversity of

yeasts is shown by their placement in two phyla; the Ascomycota and Basidiomycota (Dix

and John, 2005).

2.2.2.2 Filamentous Fungi (Molds)

It is also called as filamentous fungi. A molds is a Fungus that grows in the form of

multicellular or filaments called hyphae. They can be found indoors and outdoors and are part

of our national environment. Molds are frequently decomposers and sometimes they act as

parasites (Li et al., 2021). Molds consist of long, branching filament of cells called hyphae.

These hyphae continue to grow of branch, to form fangled mass of growth Called mycelium.

Hyphae may be divided at intervals by transverse wall of cells known as septa (sing. Septum)

such fungi are called septate fungi. In contrast fungi to that lack septa are called as aseptate

fungi or coenocytic fungi (Ali, 2013).

2.2.2.3 Yeast like Fungi

They grow partly as yeasts and partly as chains of elongated budding Cells which are joined

end to end Forming Pseudo mycelium. A perfect example being candida in which the Bud
remains attached to mother cell and elongates followed by repeated budding Forming chains

of elongated cells pseudo hyphae (Treseder et al., 2005).

Yeast like Fungi may be basidiomycetes such as Cryptococcus neofermans or ascomycetes

such as Candida albicans. However, some species like Candida albicans also produce true

hyphae. The rapid screening test for C. albicans and C. dubliniebis. These species having

ability to form tube within two hours. When incubated in human serum at 37°C. This is

called germ tube (Garnier et al., 2017).

2.2.2.4 Dimorphic Fungi

This Fungi exist either as yeasts or as molds depending upon the growth conditions. The

fungi are also described as thermally dimorphic fungi. The yeast form also called parasitic

phase occurs in host tissues and vitro (in culture) at 37°C, while filamentous form called as

saprophytic phase occurs in soil at 22-25°C. Most of the Fungi causing Systemic infections

belong to this category. e.g. Histoplasma capsulatum, Sporothrix schenckil (Ravimannan et

al., 2016).

Blastomyces dermatitidis, Paracoccidioides brasiliensis and Coccidioides immitis.

Dimorphic is therefore frequently used as general reference for fungi being suitable to switch

between incentive and filamentous cells, but not necessary limiting further shapes

(Ravimannan et al., 2016).

2.2.3 Cultivation of Fungi

Media Containing high carbohydrate Source, nitrogen, source are required for growth of

Fungi at PH range of 5 to 6, and a temperature range from 15 to 37°C (Atanda et al., 2013).

There are Two types of cultural Media: -Natural media and Synthetic media.
 i. Natural media are composed of natural Substrates, Similar as herbaceous or a woody

stems, seeds, leaves, sludge mess wheat origin, and oatmeal etc. natural media are

generally easy to prepare but they have the disadvantage of their unknown

Composition. Some examples include coin meal agar, potato dextrose agar, V-8 juice

agar, and dung agar (Atanda et al., 2013).

ii. Synthetic media these are the media in which only pure chemicals in definite

Concentrations are used. Due to their known Chemical compositions, these media are

useful for nutritional and metabolic Studies E.g. Czapek-Dox medium. Synthetic type

media contain defined amounts of carbohydrate, nitrogen and Vitamin sources.

Sabouraud‘s dextrose agar (SDA) with antibiotics Chloramphenicol which inhibits

contaminating bacteria and cycloheximide which suppress Contaminating Fungi) has

been routinely mycotic agents from clinical specimen (Atanda et al., 2013).

2.2.4 Reproduction of fungi

Reproduction is the formation of new individuals having all the characteristics typically of

the species. Fungi reproduce either sexually or asexually (Makun et al., 2010).

2.2.4.1 Sexual Reproduction

Fungal sexual reproduction differs from sexual reproduction in animals or plants in many

aspects. The process of sexual reproduction typically consists of three distinct phases. The

fusion of two protoplast of different cells (male and female) called plasmogamy‖. The fusion

of two nuclei brought together by plasmogamy is called karyogamy. Karyogamy results in

the fusion of these haploid nuclei and the formation of a diploid nucleus. The cell formed by

karyogamy is called Zygote. The differentiated sex organs called gametangia. Female

gametangia are called oogonium; male gametangia are called antheridia (Makun et al., 2010).
Gametangia may be developed terminally or sub terminally, in an intercalary position on

main or branch hyphae, as terminal or lateral appendages to non-mycelial thallus, or form the

entire thallus (Vijayakumar et al., 2016).

i. Nuclear cycle

Fungi have a cycle of haploid and diploid structure. The diploid phase begins with

karyogamy and ends with meiosis. In the majority of fungi, there is no distinct alternation of

haploid and diploid thalli (Brandt and Park, 2013).

ii. Heterokaryosis

All the nuclei are not of the same kind. Mutation freely converts a homokaryotic mycelium

(containing similar nuclei, derived from a single parent nucleus) into heterokaryotic. The

phenomenon of the existence of different kinds of nuclei in same individual is known as

heterokaryosis (Li et al., 2021).

iii. Parasexuality

In some fungi, true sexual cycle comprising of nuclear fusion and meiosis is absent. The

fungi derive the benefits of sexuality through a cycle known as parasexual cycle. This process

in which plasmogamy, karyogamy, haploidization take place, but not at specified points in

the thallus or the life cycle. This is demonstrated by Pontecorvo and Roper (1952) in

Aspergillus and Penicillium. Since then parasexual cycle has been discovered not only in

several members of Deutromycetes but also in fungi belonging to Ascomycetes and

Basidiomycetes (Vijayakumar et al., 2016).

2.2.4.2 Asexual reproduction

Fungi which are medically important mostly reproduce by this method. They produce

different asexual spores by mitosis which may be vegetative spores or aerial spores. The

formation of spores in fungi is called sporulation (Saliou et al., 2016).

1. Vegetative spores- It include:

a. Blastospores: These are formed by budding from parent cell, as in yeasts.

b. Arthrospores: These are formed by fragmentations of the ends of hyphae which

results in production of rectangular or cuboidal thick-walled spores called

Arthrospores.

c. Chlamydospores: These are thick walled resting spores developed by rounding up

and thickening of hyphal segments (Saliou et al., 2016).

2. Aerial spores: The aerial hyphae are covered with a fibrous layer made of hydrophobin, a

family of secreted proteins that form a hydrophobic layer on hyphae and spore surface.

The aerial spore included:

a. Conidiospores or conidia: These are most common in Ascomycota and anamorphic

fungi. These are the spore which are borne externally on sides or tips of special

hyphae called conidiophores. The conidiospores of a large number of fungi are borne

inside structures called conidiomata. In lower fungi some genera form true

conidiospores. When these spores are small in size and occur singly, they are called

micro conidia However, when they are large in size, multicellular and septate are

called macro conidia. The spores forms within the sporangiophores (Saliou et al.,

2016).

b. Phialospores- They are modified conidia borne at tip of a flask shaped specialized

conidiphore called phialide. c) Sporangiospores- These are spores formed within a

sac-like sporangium borne on the ends of specialized hyphae called sporangiophore.

3. Vegetative reproduction: It is a type of reproduction which involves the somatic portion

of the fungal thallus where new individuals are formed without the production of seeds or

spores by meiosis or syngamy. Vegetative reproduction tale place by the following:

a. Fragmentation: Fragmentation is common in filamentous fungi. In fragmentation,

the mycelium breaks into smaller fragments by accident or some external force. Each

fragment can develop into a new mycelium under favorable conditions. e.g. Spirogyra

reproduce by cutting themselves into fragments (Sandle, 2014).

b. Budding: Budding is the production of small outgrowth bud from a parent cell. As

the bud is formed, the nucleus of the parent cell divides and one daughter nucleus

migrate into the bud. The bud increases in size and after that it detaches from parent

cell and forms a new individual. E.g. Yeasts (Sandle, 2014).

c. Fission: In unicellular fungi like the fission yeast, the single cell multiplies by fission.

Here, the parent cell elongates and divides transversely into daughter cells. First, the

nucleus divides, followed by the division of the cytoplasm and wall formation thus,

dividing the parent cell into two. The two daughter cells separate and live

independent. e.g. Sacchromyces pobbe (Sandle, 2014).

d. Rhizomorphs: Rhizomorphs are a special morphological adaptation root-like

structure found in fungi. It is complex organs produced by fungi. They were

occasionally visible emaciating from ectomycorrhizal roots. In contrast to roots, they

tended to look bright white when young. As Rhizomorphs senesced, they darkened

and become fragmented and eventually disappeared. Rhizomorphs can live an average

of 11 months under favorable conditions, they resume growth to give rise to new

mycelia and also they remain dormant but with the onset of favorable conditions the

Rhizomorphs resume growth and may also give rise to fruiting bodies. E.g. Honey

fungus (armillaria mellea) (Avendano-Hernandez and Sanchez, 2013).

2.2.5 Nutrition

Some fungi having a nutritious value. Fungi are heterotrophic fungi. They use complex

organic compounds as a source of carbon. Fungi do not photosynthesize. Fungi perform an

essential role in the decomposition of organic matter and have fundamental roles in the

nutrient cycling and exchange. Mushrooms are the best example of the nutritious fungi.

Mushroom have many varieties like white button mushroom, oyster mushroom, porcine

mushroom, Enoki Mushroom, reishi mushroom. There are ten thousand types of Mushroom

present in the world but only 25% mushrooms are edible (Stajich et al., 2009).

They contain Beta-glucan Vitamins B, Potassium, selenium, Vitamin D, lipids, amino acids,

carbohydrates, water etc. mushroom are reach in proteins. The component in Mushroom may

exert antioxidant, anti-inflammatory, anti-cancer effects. They a type a fungus that contains a

substance called Ergosterol. Ergosterol can be transform into vitamin D with exposure to

ultraviolet light. The Oyster Mushroom that one helps fight against HIV and cancer. White

button mushroom are world most commonly eaten mushroom It has to effective in a

preventing breast and prostate cancer in both animal and human cells, maitake mushroom has

anti-cancer, antiviral, immune system enhancing effects and may also help to control both

high blood pressure and blood sugar level (Stajich et al., 2009). The micro molecules are

body builder and provide energy for metabolic process. The parasitic fungi lives in or on the

living body of a plant or animal and absorbs organic molecule as a nutrients through cell wall

from the tissue of that host. Fungi thrive in surroundings that are wettish and slightly acidic

and can grow with or without light. They vary in their oxygen demand most fungi are

obligates aerobes taking oxygen to survive. Other species similar, as the chytridiomicota that

live in the rumen of cattle, are obligate anaerobes in that they only use anaerobic respiration

because oxygen will disrupt their metabolism or kill them. Provocations are intermediate

being facultative anaerobes. This means that they grow stylish in the presence of oxygen
using aerobic respiration but cancer survive using anaerobic respiration. When oxygen isn‘t

available. The alcohol produce from yeast fermentation is used in wine and bear product. It is

also used in bakery Industries (Stajich et al., 2009).

2.2.6 Contamination/Spoilage

The two types of fungi that are important in food spoilage are yeasts and molds. Fungi

growing on foodstuffs render them useless and sometimes even toxic. Fungi are common

contaminates of dairy products, which provide a favorable for their growth. Since people

began producing and storing food products, spoilage and food losses and waste become

important issues for human with regards of food safety and security. Fungal contamination of

dairy foods can occur at different stages, from dairy farms to dairy processing units and

consumer‘s homes. Cereals and bakery products serve as a valuable source of nutrients in the

diet of many people. Yeasts is used in the dough which releases CO 2 and bread becomes

spongy. Usually the mold spoilage of bread is due to post processing contamination (Aguado

et al., 2016).
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 STUDY SITE

The smoked Clupea harengus samples were purchased from Osiele market, Abeokuta, Ogun

State and microbiological analysis were carried out at the Microbiology Department of

Science Laboratory and Technology, Moshood Abiola Polytechnic, Abeokuta, Nigeria.

3.2 Sampling Size, Collection and Processing

The samples were purchased from the market and kept inside a polyethylene bag to be carried

to the Laboratory for further analysis. A total of 6 hot smoked Shawa retailers were

patronized per market, Clupea harengus was purchased at random from 3 retailers per market

for every trial (R1, R2, R3). The samples from each 3 retailers were used for analysis for two

weeks, making a total of 48 samples, for the experiment. All samples obtained were ground

with the use of an electric blender and fine fish samples were obtained.

3.3 Preparation of Media

Nutrient Agar, Potato Dextrose agar, Brillant Green agar and Mannitol salt agar were

aseptically weighed, distilled water was transferred into the conical flask and the media were

sterilized inside the autoclave for 15 mins at 121 oC, allowed to cool and poured into the

plates respectively. Thiosulphate citrate bile salts sucrose agar and Salmonella-shigella agar

were weighed, distilled water transferred and the media homogenized and made sterile with

the use of magnetic stirrer and hot plate to boiling points for 10 mins under frequent agitation

and allowed to cool to about 45oC before it was poured into the inoculated plate.
3.4 Cultivation and Enumeration of Bacteria

Fine grinded smoked catfish sample (1g) was aseptically weighed and was transferred into a

MacCartney bottle containing (9mls) of sterile distilled water and shaken thoroughly to make

10-1 dilution. Serial dilutions were carried out using sterile syringe that delivered the required

volume 1ml accurately to make decimal dilutions of 10-2 to 10-10. Before every 1ml dilution

transfer, the diluents were shaken well for proper mixing of the sample with the diluent using

pour plate methods, 0.1ml of inoculum was transferred into sterile labelled petri dishes.

About 20mls of sterilized molten nutrient agar, potato dextrose agar, Brillant Green Agar,

Mannitol salt agar, Thiosulphate citrate bile salts, sucrose agar, cooled to about 45 oC, was

poured into the inoculated petri dishes and allowed to gel. Some plates were also prepared as

control to check on the sterility of the diluents. The plates were then incubated at 37 oC for 24

hours.

3.5 Purification of Isolates

Nutrient agar (20mls) was transferred to sterile petri-dishes and allowed to solidify after

which they were dried in a hot air oven at 30 oC. This was done to get rid of moisture on the

cover of the plates and on the agar itself suspected colonies of Vibrio cholera,

Staphylococcus aureus, Escherichia coli, Salmonella spp etc were purified by streaking on

nutrient agar plates and were subjected to Gram staining and other Biochemical tests. Other

microbiological activities carried out were gram staining reaction and other biochemical tests

such as that of the citrate, Kligeler iron agar, sulphide indole and sugar utilization.
 CHAPTER FOUR

RESULTS

4.1 Coordinate and Location Description of the Sample

The sample, which was identified as FOSPd6, was taken from Panla fish at Osiele Market.

The sampling location's coordinates are 3.02376°E and 7-79884°N". There was an open

waste disposal, stagnant water and a pack of firewood.

Table 4.1

Sample Code Sample Fish type Longitude and Sample Description/

location latitude Location Activities
Osiele market Panla 3.02376°E and Open waste disposal,
7.79884°N stagnant water and a
pack of firewood
FOSpd5 Osiele Market Panla 3.4505°E and Commercial activities,

7.1928°N crowded environment,

bush and open waste

disposal

FOSSe3 Osiele Market Shawa 3.4505°E and Open environment

7.1933°N

FOSSe6 Osiele Market Shawa 3.4513°E and Commercial activities

7.1911°N
4.2 Fungi Load

Table 4.2 shows the total fungal counts (TFC) of fungi isolated from smoked fish samples

collected at Osiele Market in Abeokuta, using Potato Dextrose Agar as the growth medium.

The results reveal significant variations in fungal contamination levels across different fish

types and market locations.

Panla fish from the location designated as "OsieleFOSPd6" exhibited the highest fungal load,

with a TFC of 2x10-7 – 2 x107 times, indicating heavy fungal contamination. This level of

fungal growth may reflect suboptimal handling or storage practices at this market. In contrast,

the same fish type (Panla) from the location marked as "Osiele" had a considerably lower

fungal count of 5×10-3 – 5 x103, suggesting more favourable handling or environmental

conditions at this site.

In the case of Shawa fish, the contamination levels also varied between locations. At

"OsieleA," the fungal count was 6×10−5 – 6x 105 which was significantly higher than the

count recorded at "OsieleB," where the TFC was 6×10 −7. This discrepancy highlights

differences in the degree of fungal contamination between the two locations, potentially due

to variations in hygiene, smoking techniques, or storage conditions.

The high fungal counts in certain locations, such as "OsieleFoSPd5," raise concerns about the

potential risks of mycotoxin production, which could pose health hazards to consumers.

Conversely, the relatively low fungal loads observed in other locations indicate better control

over fungal growth during processing and storage.

Table 4.2

Isolation code Fish type Colonies Potato dextrose Agar (TFC) (cfu/ml)

FOSpd6 Panla 16 2x10-7 →2 x107

FOSpd5 Panla 10 5x10-3 →5 x103

FOSSe3 Shawa 15 6x10-5 →6 x105

FOSSe6 Shawa 12 6x10-7 →5 x107

4.3 Frequency and Percentage of Occurrence

The data in Table 4.3 represents the frequency and percentage distribution of fungi isolated

from smoked fish (Shawa and Panla) sold at Osiele market in Abeokuta. It provides an

overview of the occurrence rates of fungal isolates based on their codes and the prevalence

within the two fish types.

For Shawa fish, the isolate coded FOSSe6 10-5C had the highest frequency of occurrence,

appearing six times, which accounted for 66.6% of the total fungal isolates. The isolates

FOSSe3 and FOSSe6 were observed only once each, contributing equally to the remaining

33.2% of the isolates (16.6% each). This indicates that FOSSe6 10-5C is the dominant fungal

species associated with Shawa fish in this market.

In contrast, for Panla fish, FOSPd6 10-5C was also the most frequently occurring isolate,

recorded 6 times, constituting 90% of all fungal isolates. The isolates FOSPd6 and FOSPd5

occurred 6 and 5 times, respectively, representing 5.2% and 4.8% of the total fungal counts.

These figures suggest that FOSPd6/10-5C overwhelmingly dominates the fungal population

in Panla fish, with the other isolates playing a minor role.

This distribution pattern highlights a significant disparity in the dominance of fungal isolates

between the two fish types, with FOSPd6/10-5C being particularly prominent. The high

frequency of this isolate, particularly in Panla fish, may indicate specific environmental or

storage conditions that favour its growth, or it could reflect the biological characteristics of

the fish type itself.

4.3 Frequency of distribution of fungi isolated from smoked fish at Kuto market in

Abeokuta

Isolated code Frequency of Percentage of Frequency of Percentage of

occurrence occurrence occurrence occurrence

(Shawa) (Shawa) (Panla) (Panla)

FOSSe6 6 66.6 13 5.2

FOSSe3 3 16.6 12 4.8

FOSPd6 6 66.6 225 90

Total 6 100% 250 100%

4.4 Cultural and Morphological Characteristic of Fungi Isolate

The data in Table 4.4 provides detailed descriptions of the cultural and morphological

characteristics of the fungal isolates, along with the suspected identities of the fungi based on

these observations.

The isolate coded FOSSe6 10-5C exhibits a rapid growth rate, forming colonies that initially

appear white and fluffy but later turn dark due to conidial production. The colonies develop a

granular to powdery texture and cover the Petri dish within 72 hours. The reverse side of the

plate displays a yellowish to pale brown colour, with black pigmentation appearing as the

colonies mature. Microscopic examination reveals septate hyphae, globose to subglobose

vesicles densely covered with phialides, and round, dark conidia arranged in basipetal chains.

These characteristics are consistent with Aspergillus niger.

The isolate FOSPd6 forms colonies that appear white with a yellow reverse colour.

Microscopically, the hyphae are observed to be septate and hyaline, indicating a transparent

structure. Based on these observations, the suspected fungus is identified as a species of

Penicillium.

Lastly, FOSPd3 produces colonies that are creamy, smooth, and pasty in texture, typically

white to off-white in colour with a moist appearance. Microscopically, the presence of

hyphae, pseudohyphae, and budding yeast cells is noted, with the yeast cells appearing round.

These features strongly suggest the isolate is Candida albicans.

The identification of these fungal isolates provides insights into the potential risks associated

with fungal contamination in smoked fish. Aspergillus niger and Penicillium species are

known for their ability to produce mycotoxins, which could compromise food safety, while
Candida albicans is an opportunistic pathogen that may pose health risks in immune

compromised individuals.
Table 4.4 Cultural and morphological of fungi isolates

Isolate code Cultural Characteristic Morphological and Suspected fungi

Microscopic Characteristic
FOSPd6 Initially white and fluffy, turning dark due to Hyphae appear septate, Aspergillus niger
conidial production, Granular to powdery, vesicle Globose to
Rapid, covering the Petri dish within 72 subglobose, densely covered
hours, reverse colour is Yellowish to pale with phialides, conidia
brown on the underside of the plate and Black Round, black or dark brown,
pigmentation visible macroscopically as rough-walled, in basipetal
colonies mature. chains.

FOSSe6 Colony appear white, yellow reverse colour Hyphae appear septate and Penicillium sp
hyaline.
FOSSe3 Colonies are creamy, smooth, and pasty, often Hyphae, pseudohyphae, and Candida albicans
white to off-white in colour, moist texture, yeast cells are observed,
round yeast cells are
observed, often budding.
Table 4.1 : Shows the description

Sample Code Sample Location Fish Type Long/Lat Sample

Description/Location
Activities
KSP1E Kuto Market Panla N7O8!18.01920,E3021’1.19940 No open waste disposal or
stagnant water and the
seller exhibit unhygiene
practice throughout the
market activities
KSS1E Kuto market shawa N7O8’19.499, E3O21’0.88660 Presence of waste
disposal
KSS1D Kuto market Shawa
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