First Insight in Fungi Diversity and Mycotoxins Contaminating Smoked Fish Sold in Yaounde Retails Markets in Cameroon

Research Article

J Bacteriol Mycol. 2024; 11(1): 1214.

First Insight in Fungi Diversity and Mycotoxins Contaminating Smoked Fish Sold in Yaoundé Retails Markets in Cameroon

Olivier Ziem2,3; Gwladys Ekwe Priso1; Amandine Plidikoua1; Moise Ntah A Ayong1; Balbine Adande1; Modeste Lambert Sameza1; Francois-Xavier Etoa3; Francioli Koro Koro1*

¹Department of Biochemistry, Faculty of Sciences, University of Douala, Cameroon

²Department of Animal Biology and Physiology, Faculty of Sciences, University of Douala, Cameroon

³Department of Microbiology, Faculty of Sciences, University of Yaounde I, Cameroon

*Corresponding author: Franciolo Koro Koro Department of Biochemistry, Faculty of Sciences, University of Douala, P.O. Box 12 574, Douala, Cameroon Email: korokorogozion@yahoo.fr

Received: January 10, 2024 Accepted: February 20, 2024 Published: February 27, 2024

Abstract

Mycotoxins are one of the major food poisons for the human’s liver. Their production is generally related to improper food storage condition, mainly cereals and groundnuts. In the present study we assessed the moulds diversity and mycotoxin contamination of smoked fish, one of the most eaten foodstuffs in Cameroon. For this purpose, 1000 specimens of smoked fish were randomly collected from 10 retail markets in Yaoundé and pooled into 50 composite samples. Moulds were isolated by dilution, suspension and culture methods. Identification of fungal was assessed by phonotypical characterization. Three different mycotoxins (Aflatoxin B1, Ochratoxin A and fumonisin B) were tested on fish. Semi-quantitative immuno-chromatographic assay was used for (AFB1) and Fumonisin B (FB), ELISA assay was used for Ochratoxin A (OTA). The identification of moulds flora associated with smoked fish revealed that they belonged to three genuses namely Aspergillus, Penicillium and Absidia. Only species belonguing to Aspergillus genus appear to produce Aflatoxins. Three types of mycotoxin were detected (AFB1, OTA and FB) with occurences of 76%, 18% and 6% respectively at levels above the reference maximum admissived limits.

Keywords: Aspergillus; Fungi isolates diversity; Smoked fish contamination; Mycotoxins; Retails markets; Cameroon

Abbreviations: AFB1: Aflatoxin B1; FB: Fumonisin B; OTA: Ochratoxin A; ISO: International Standardization Organization; rpm: rounds per minute; PDA: Potato Dextrose Agar; OGA: Oxytetracyclin Glucose Agar; NF: Normes Francaises; V/V: volume/volume; CEA: Coconut Extract Agar; UV: Ultraviolet; ppb: part per billion; ELISA: Enzyme-Linked Immunosorbent Assay; HPLC-SM: High Performance Liquid Chromatography couple with Mass Spectrometry

Introduction

Fish in all their forms, remain one of the most used foods in the world, with an average consumption level of 20.1 kg per capita [1]. In Cameroon, fish products contribute up to 25.5% of protein diet of the population [2]. In African countries including Cameroon, smoking fish remains the most suitable method for fish preservation. However, several studies have revealed the presence of pathogenic bacteria such as Staphylococcus aureus, Salmonella spp., Escherichia coli pathotypes, Listeria monocytogenes in smoked fish [3-8] and fungi which can produce mycotoxins under certain conditions such as high temperature and humidity rate [9]. Tropical climate and poor crop storage conditions are frequently responsible of fungal growth and mycotoxin production [10]. Mycotoxins are chemical compound mainly produced by fungi belonging to the Aspergillus, Penicilliums and Fusariums genus [11] which have negative impact on human and animal health [12]. Consumption of mycotoxin contaminated food may induce acute or chronic affections, including non-communicable diseases [13]. According to International Agency for Research on Cancer chronic exposure to Aflatoxin B1 (AFB1) or its precursors has been associated with genotoxicity and hepatocellular carcinoma [14]. Concerning Fumonisin B (FB), studies revealed it association with oesophageal cancer incidence in South Africa and China [15,16]. Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius are the most known species and have been subject of several research studies that have demonstrated their aflatoxin production capabilities [17-20]. In addition to these three species, aflatoxin production capabilities have been discovered more recently in the following Aspergillus species: Aspergillus tamarii, Aspergillus ochraceoroseus [21], Aspergillus pseudotamarii [17], Aspergillus bombycis [22] and Aspergillus rambelli [23]. The occurrence of mycotoxins in Cameroonian food commodities such as maize, peanuts, beans and soybeans has been reported by many authors [24-26]. But there is a lack of data about fungal and mycotoxin contamination of smoked fish. Given the economic and nutrition importance of smoked fish and the increase of gastrointestinal cancers in Cameroonian population, the present study aims to dose mycotoxin and identify fungi contamining and producing aflatoxin on smoked fish sold and intended to human consumption in Cameroon.

Material and Method

Sample Collection

A total of 1000 specimens of smoked fish were randomly collected from ten markets (Figure 1) of Yaoundé urban council during a 6 months period. The different market has been chosed by their capacities of selling many types of smoked fish consumed in Cameroun. Sampling was performed according to ISO 2859-1 Standards [27]. Smoked fish were collected using individuals steriles plastics bags and transported to the laboratory under refrigerated boxes. The specimens were then pooled into 50 composite samples and grouped by species and by markets. All composite samples were grounded and divided into 3 aliquots. One aliquot for moulds isolations, the second aliquot for mycotoxins detection and the third aliquot for humidity rate assessment.

Figure 1: Location map of different markets and their smoked fish collection sites in Yaoundé.

    

    
    

    

    Figure 1: Location map of different markets and their smoked fish collection sites in Yaoundé.

Humidity Rate Assessment

Humidity rate of composite smoked fish samples was measured using a numeric thermohygrometer (traceable reference 620-2273) following manufacturer’s instruction.

Mould Isolation and Identification

Ten grams of each composite sample were added to 90 ml of sterile Buffered Pepton Water (Biokar) and homogenized during 1min using a stomacher (LB 400 VWR) to obtain a 1/10 dilution. The futhers decimals dilutions were prepared in BPW as described by ISO 6887-1: 2017 [28] and inoculated in Potato Dextrose Agar (PDA Biokar) medium and Oxytetracyclin Glucose Agar (OGA Biokar) medium according to NF V 08-59: 2002 standard [29]. All plates were incubated for 5 days at 25°C ± 1°C. After the incubation period molds culture were isolated in OGA and PDA plates for identification assays. The identification of fungal isolates was performed according to standard taxonomic systems based on morphological observation of mycelium, thallus and spore using the dichotomous keys [30]. The microscopy observation with 100x magnification after coloration with lactophenol blue based on the shape of conidiophores and conidia dimension completed the identification algorithm.

Assessment of the Capacity of Fungal Isolates to Produce Aflatoxin

The ability of fungal isolates to produce aflatoxins was performed according to Pane B. Ouattara-Sourabie [31]. Fungal isolates were plated on Coconut Extract Agar (CEA) and incubated at 25°C ±1°C during 7 days. After incubation period, fluorescence under Ultraviolet (UV) radiation was assessed using wood lamp at wavelength of 365 nm. The fluorescence and characteristic color of fluorescence were used to evaluate the ability of each isolate to produce aflatoxins [32-35]. Aflatoxin diffusion areas shows a blue radiation around the isolate under UV light.

Mycotoxins Assays

Three different mycotoxins (Aflatoxin B1, Ochratoxin A and fumonisin B) were tested on smoked fish samples. Semi-quantitative immuno-chromatographic assay was used to detect Aflatoxin B1 (AFB1) and Fumonisin B (FB) while ELISA assay was used to detect Ochratoxin A (OTA). Briefly, the AFB1 assay was based on competitive inhibition of colloid gold immunochromatography principle. 2g of composite sample were grounded into centrifuge tube and 2 ml of 70% methanol-water solution mixture (V /V=7:3) were added to obtain a detection limit of 10 ppb. The mixture was shaked for 5 min and centrifuged at 4000 rpm for 5 min at room 25°C. Then 0.1 ml up-layer clear liquid was added to 0,4 ml of deionized water and mixed. 3 drops of this mixture were dropped on the sample collecting region of the test card. The negative result showed a red T line on the test card and positive result showed an invisible red T line on the test card.

Concerning the detection of OTA, 5 g of composite sample was introduced into 50 ml centrifuge tube with 10 ml of 70% methanol-water solution mixture (V/V=7 :3), Then shaked and centrifuged at 4000 rpm for 5 min to get supernatant. 1 ml supernatant was dried at water bath at 50°C. For the test procedure, 500 μl of sample buffer was added into centrifuge tube and vortexed until fully dissolve the dry residue on well and 100 μl of sample was dropped into sample region of card test the results were readed after 5 min. A negative result showed a T line on the test card and a positive result showed an invisible T line on the test card.

For FB test, 50 g of composite sample was grounded; 3 g were added into 3 ml of 80% methanol and mixed for 15 minutes at room temperature. After centrifugation for 10 minutes at 2000 rpm, 50 μl of the obtained supernatant were added to 150 μl of dilution buffer provided by the FB measurement ELISA kit (Euro Proxima) according to the manufacturer’s instruction.

Data Analysis

The frequencies and market distributions of fungal isolates were determined using proportion and rates. The results were presented in tables and figure. Mycotoxins dosage and humidity rate was assessed in triplicate.

Results

Smoked Fishes’ Identification

Smoked fish samples collected in different market of the city of Yaoundé belonged to 3 families namely, Claroteidae, Clupeidae and Scombridae. Claroteidae family was represented by one genus (Chrysichthys) and one specie (Chrysichthys sp). The Clupeidae family was represented by two genera (Ethmalosa and Sardinella) represented respectively by one specie each (Ethmalosa fimbriata and Sardinella maderensis). Scombridae family was represented by one genus (Scomberomorus) and one specie (Scomberomorus tritor).

All smoked fish samples species were collected in different markets according to their availability and their quantity (Table 1).

Table 1: Distribution of smoked fish’s specimen collected from different markets.





  

    
Smoked Fishes Specimen Species 

    
Number of Specimens    Collected by Markets

    
Total 

  

  

    
Mokolo market

    
Central Market

    
Melen Market

    
Mfoundi Market

    
Mvog Betsi    Market

    
Nsam Market

    
Ekounou Market

    
Etoudi Market

    
8th    market

    
Essos Market

  

  

    
Ethmalosa fimbriata

    
40 

    
40 

    
20 

    
40 

    
20 

    
20 

    
20 

    
20 

    
20 

    
20 

    
260 

  

  

    
Sardinella maderensis

    
40 

    
40 

    
20 

    
40 

    
20 

    
20 

    
20 

    
20 

    
20 

    
20 

    
260 

  

  

    
Chrysichtys sp

    
40 

    
20 

    
20 

    
40 

    
20 

    
20 

    
20 

    
20 

    
20 

    
20 

    
240 

  

  

    
Scromberomonus tritor

    
40 

    
20 

    
20 

    
40 

    
20 

    
20 

    
20 

    
20 

    
20 

    
20 

    
240 

  

  

    
Totals 

    
160 

    
120 

    
80 

    
160 

    
80 

    
80 

    
80 

    
80 

    
80 

    
80 

    
1000 

  










Table 1: Distribution of smoked fish’s specimen collected from different markets.

Relative Humidity of Smoked Fish Samples

The average relative humidity rate of smoked fish samples (Table 2) was 7.70±2.9 % for Ethmalosa fimbriata, 8.60±3.6% for Sardinella maderensis, 14.60± 3.4% for Chrysichthys sp and 18.9± 4.3% for Scomberomonus tritor.

Table 2: Relative Humidity rate of smoked fish samples species sold in Yaoundé markets.





  

    
Fish    Species

    
Relative    Average Humidity

      Rate    (%)

    
Limited Relative    Humidity Value for Fungal Growth (%)

  

  

    
Ethmalosa fimbriata

    
7.70±2.9

    
12.0

  

  

    
Sardinella    maderensis

    
8.60±3.6%

  

  

    
Chrysichtys sp

    
14.6±3.4

  

  

    
Scomberomonus tritor

    
18.9±4.3

  










Table 2: Relative Humidity rate of smoked fish samples species sold in Yaoundé markets.

Fungal Contamination of Smoked Fish Samples

18 composites samples (36%) amongst 50 composites samples were contaminated by fungal flora. Scomberomonus tritor was the most contaminated smoked fish sample with 12 composites contaminated samples (69,23%) followed by Chrysichtys sp 7 composites contaminated samples (60 %) and Sardinella maderensis 3 composites contaminated samples (25 %) (Table 3).

Table 3: Fungal contamination of smoked fish species sold in Yaoundé markets.





  

    
Fish    Species

    
Number    of

      Specimen    Collected

    
Number    of Composite Sample

    
Number of Composite    Samples Contaminated by Fungus

    
Occurence    of Fungal Contamination (%)

  

  

    
Ethmalosa fimbriata

    
300

    
15

    
0

    
0

  

  

    
Sardinella    maderensis

    
240

    
12

    
3

    
25

  

  

    
Chrysichtys sp

    
200

    
10

    
6

    
60

  

  

    
Scomberomonus tritor

    
360

    
13

    
9

    
69,23

  

  

    
Total

    
1000

    
50

    
18

    
36

  










Table 3: Fungal contamination of smoked fish species sold in Yaoundé markets.

Fungi Identification

22 fungi isolates were obtained from 50 smoked fish composites samples. These isolates were grouped into 3 genera namely Aspergillus, Absidia and Penicillium base to their cultural characters (Figure 2). Aspergillus genera counted for 90% of isolates and was represented by 4 species namely Aspergillus flavus, Aspergillus niger, Aspergillus candidus and Aspergillus fumigatus, Absidia and Penicillium genera were represented respectively by one specie each (Absidia sp and Penicillium sp) (Figure 3).

Figure 2: Cultural characters of fungi isolates on culture media.

    

    
    

    

    Figure 2: Cultural characters of fungi isolates on culture media.

Figure 3: Distribution of Fungi species isolated from smoked fish sold in Yaoundé markets.

    

    
    

    

    Figure 3: Distribution of Fungi species isolated from smoked fish sold in Yaoundé markets.

Figure 4: Aflatoxin producing isolates identification under UV light.

    

    
    

    

    Figure 4: Aflatoxin producing isolates identification under UV light.

Distribution Fungi Species According to Smoked Fish Species

The distribution of fungi species according to fish species indicated that only 3 species of smoked fish were contaminated by molds (Table 4). Scomberomonus tritor was the most regulary infested smoked fish species followed by Chrysichtys sp and Sardinella maderensis. No fungal specie was isolated from Ethmalosa fimbriata.

Table 4: Distribution of fungi species according to smoked fish species.





  

    
Moulds Species Isolated 

    
Smoked Fishes Species 

  

  

    
Ethmalosa

    
Sardinella

    
Chrysichtys sp

    
Scomberomonus Tritor

  

  

    
Fimbriata

    
Maderensis

  

  

    
Aspergillus flavus

    
- 

    
++ 

    
++++ 

    
++++++ 

  

  

    
Aspergillus candidus

    
- 

    
- 

    
+ 

    
++ 

  

  

    
Aspergillus niger

    
- 

    
+ 

    
+ 

    
+ 

  

  

    
Aspergillus fumigatus

    
- 

    
- 

    
- 

    
++ 

  

  

    
Absidia sp

    
- 

    
- 

    
- 

    
+ 

  

  

    
Penicillium sp

    
- 

    
- 

    
+ 

    
- 

  

  

    
Note: - : no isolate; + : one isolate ; ++ : two    isolates; +++ : three isolates ; ++++ : four isolates ; +++++ : five isolates 

  










Table 4: Distribution of fungi species according to smoked fish species.

Aflatoxin Producing Isolates Among Fungi Isolates

Only Aspergillus species isolates among the identified fungi species isolated from smoked fish were able to produce Aflatoxin. Amongst these, 9 isolates (75%) of Aspergillus flavus, 02 isolates (100%) of Aspergillus fumigatus, 3 isolates (100%) of Aspergillus candidus and 01 isolate (33,33%) of Aspergillus niger (33,33%) were able to produce Aflatoxin (Table 5).

Table 5: Percentage of Aflatoxin producing isolates amongst fungi species.





  

    
Fungal Isolate 

    
Number of Isolates Tested 

    
Number of Isolates Positives to    CEA Test 

    
Percentage of Aflatoxin Producing    Isolates (%) 

  

  

    
Aspergillus flavus

    
12 

    
9 

    
75 

  

  

    
Aspergillus niger

    
3 

    
1 

    
33,33 

  

  

    
Aspergillus fumigatus

    
2 

    
2 

    
100 

  

  

    
Aspergillus candidus

    
3 

    
3 

    
100 

  

  

    
Absidia sp

    
1 

    
0 

    
0 

  

  

    
Penicillium sp

    
1 

    
0 

    
0 

  










Table 5: Percentage of Aflatoxin producing isolates amongst fungi species.

Mycotoxin Dosage in Smoked Fish Samples

Fifty composite aliquots representing all smoked fish species sample were analysed for the detection of mycotoxins. 17 aliquots (34%) were contaminated by mycotoxins (Figure 5). AFB1 was the main contaminant with 13(76 %) of aliquots followed by OTA 03 (18 %) and FB 1(6%).

Figure 5: Distribution of mycotoxins detected in smoked fish samples sold in Yaoundé markets.

    

    
    

    

    Figure 5: Distribution of mycotoxins detected in smoked fish samples sold in Yaoundé markets.

Discussion

In this study, thirty-six per cent (36%) of smoked fish sample were contaminated by fugal flora. This result which remains high is similar with Olajuyigbe et al. [36] study who obtained a contamination rate of 37% on retailed fishery products collected in Lagos markets. However, this result is different from Akwuobu et al. studies [37], who obtained an isolation rates ranging from 67.6% to 84.8%. This relative high contamination of smoked fish samples by fungal flora could result from lack of good hygiene practices and poor storage of smoked fish samples. It can also be due to relative humidity rate of fish during storage conditions. In fact, humidity is a factor favoring the proliferation of fungi on fish samples [38]. In other hand, the differences amongst isolations rates observed for the different smoked fish species could be explained by the differences in the nature of smoked fish specimen and the differences of processes associated with the smoked fish specimen. Scomberomonus tritor was the most contaminated fish sample with a fungal contamination rate of 69.23%. This result could be explained by the average humidity rate of this specimen. In fact, the humidity rate assessment of smoked fish samples showed that Scomberomonus tritor specimen has the highest humidity rate (18.9%) above the humidity rate limit for fungal growth (12%).

The present study revealed that fungal genera associated with contamination of smoke fish samples sold in Yaounde market were Aspergillus, Penicillum and Absidia (Figure 3). This result is similar with a few exceptions to that of Akwuobu et al. [37] studies who detected the genera Aspergillus, Penicillium, Mucor, Rhizopus, Absidia and Candida as the main contaminant species of smoke-dried fish sold in Makurdi market. According to Pitt and Hocking [39], Aspergillus is one of the most dominant contamination species found on dried food in tropical and subtropical regions [40]. The genera Aspergillus counted for 90% of fungi isolates with the proportions of 53% for Aspergillus flavus, 12.5% for Aspergillus niger and Aspergillus candidus and 12% for Aspergillus fumigatus. The dominance of Aspergillus sp as main contaminant of dried fish samples are an indication of its ubiquitous nature [41], its strong spore reproduction, its mycelium diffusion ability and its ability to adhere and survive in high protein nutrient sources such as dried fish [42]. Aspergillus species usually grows faster than Penicillium but takes longer to sporulate [43]. Penicillium sp was also isolated in significant numbers (18.18%) in our study. Similar finding has been documented by Rafli et al. [44], Essien et al. [45]. Essien et al. [45] for example have reported that Penicillium sp. constitutes the second most contaminants of dried fishes.

Assessment of the ability of fungal isolates to produce Aflatoxin showed that only Aspergillus spp isolates were able to produce Aflatoxin. This result is similar with that of Job et al. [46] study in Jos metropolis. In their study, they showed that among the mold’s isolates, only Aspergillus flavus strains presented aflatoxigenic producing potentials. In fact, Aspergillus familie’s members incuding Aspergillus flavus, A. parasiticus and Aspergillus ochraceus, Fusarium species [47] and Fusarium moniliforme [48] are recognized as the most common fungi mycotoxins producer. The detection of potentially aflatoxin-producing mold strains among of Aspergillus fumigatis, Aspergillus niger and Aspergillus candidus isolates in this study remains intriguing. But this result could be explained by an adaptation of these species to local conditions. But this requires further studies.

The detection of mycotoxins such as Aflatoxin B1, Ochratoxin A and Fumonisin B above acceptable limits in 34% of smoked fish sample indicates a dangerous sanitary risk associated with smoked fish consumption in Cameroon. Nowadays, food safety is an increasing preoccupation for consumers and of public health utilities [49].

In Cameroon, gastrointestinal cancers occurrence has raised over the last decade and among the risk factors associated with the increase of the prevalence of gastrointestinal tract cancers, contamination of foodstuffs by mycotoxins and particulary Aflatoxin B1 is often incriminated [50]. It is well documented that aflatoxin B1 is the most abundant genotoxic and carcinogenic mycotoxin [51], which could induce gastrointestinal and metabolic disturbances in contaminated foods such as smoke-dried fish [52]. Indeed, the detection of aflatoxins and ochratoxin A in smoked fish samples could also reflect the presence of Aspergillus and Penicillium species [37]. However, it would be interesting in future to measure these mycotoxins using a quantitative method such as HPLC-SM.

Conclusion

This preliminary study showed for the first time that smoked fish consumed in Yaoundé-Cameroon are heavily contaminated both by aflatoxin B1-producing fungi and by mycotoxins such as aflatoxin B1, Ochratoxin A and Fumonisin B, above acceptable limits.

Author Statements

Acknowledgements

The authors are grateful to the Head of Biochemistry department of the Faculty of sciences of the University of Douala for laboratories facilities and Pr Modeste SAMEZA for his contribution for Moulds identification.

Authors’ Contributions

Gwladys Ekwe Priso and Olivier Ziem are PhD students of the University of Douala and university of Yaoundé 1. This preliminary project did not receive external grant funding. The cost for all aspect of this research was covered by the funds from the research grant granted by the Cameroonian government to researchers of the state universities.

Francioli Koro koro conceived and designed the experiments. Olivier Ziem, Moïse Ntah, Gwladys Ekwe Priso, performed the experiments. Francioli Koro koro, Olivier Ziem, Moïse Ntah, Gwladys Ekwe Priso, Amandine Plidikoua, Moise Ntah A Ayong, Balbine Adande, Modeste Lambert SAMEZA analysed the data. Olivier Ziem and Gwladys Ekwe Priso wrote the first draft of the paper and designed figures. All authors provided critical input. Francioli Koro Koro and Francois-Xavier Etoa supervised the research, edited and approved the final manuscript.

Conflict of Interest

The authors have no conflicts of interest to declare.

Ethical Consideration

This study does not involve any human or animal testing.

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Citation: Ziem O, Priso GE, Plidikoua A, Ayong MN, Adande B, et al. First Insight in Fungi Diversity and Mycotoxins Contaminating Smoked Fish Sold in Yaoundé Retails Markets in Cameroon. J Bacteriol Mycol. 2024; 11(1): 1214.