Evaluation of the Hygienic and Microbiological Quality of Dried and Smoked Fish Sold in the Various Markets of the City of Abéché (Chad)

Fish is a vital source of animal protein and micronutrients for many communities (rural and urban). It could be the cause of serious Toxi-infection with health consequences if handled in unsanitary conditions. The goal of this study aims to assess the hygienic and microbiological quality of dried fish and smoked fish sold in the various markets in the town of Abéché. A total of 30 samples of dried and smoked fish were collected from the vendors surveyed. Germs sought were counted in the laboratory according to the standard method of microbiology. There most of the respondents are female with 96.67%; 93.33% of them are married. The average age of respondents is between 26 and 35 and more than half (56.57%) are uneducated. The average load of dried fish in the three markets is 4.60 X 107 CFU/g for FAMT; 7.33 X 105 CFU/g for CT; 2.87 X 104 CFU/g for CTT; 1 CFU/g for Staphylococcus aureus. On the other hand, for smoked fish, the average varies between 6.81 X 107 CFU/g for FAMT; 2.28 X 106 CFU/g for CT; 1.56 X 105 CFU/g for CTT and 1 CFU/g for yeasts and molds. Enumeration of staphylococci in the three (3) samples gave high values in the range of 14 to 90 CFU/g. Additionally, the level of contamination by total mesophilic aerobic flora is 74.67% for dried fish and 36.33% for smoked fish. Therefore, it is necessary to follow up when fish processing to reduce the level of contamination by flora microbial.


Introduction
Fish is a source of animal origin which African populations often use to cover their needs and ensure their food security (Word Fish center, 2005). In the African countries as in many countries in the world, fishing and aquaculture play an important role in the supply of animal protein for food. At Chad, aquaculture is still underdeveloped and concerns only fish farming. However, the Fishing is the third activity in the rural sector (FAO, 2002). Fishing in Chad is practiced in all the surface waters of the country, namely the rivers (the Chari and the Logone) and the Lakes (Lake Chad, Lake Fitri, Lake Lé ré and the Toupouris Lakes) (LCBC, 2007). In addition to a source of good quality protein for human consumption, fish is also a significant source of income for the population (Femon, 2013). It also constitutes a source of vitamin D, B and A (K. Diè nè ba, 2005). Fish meat contains little cholesterol but a lot of mono and polyunsaturated acid whose preventive and protective role against cardiovascular disease has been demonstrated by several studies (Corraze and Kaushik, 1999;Regost et al., 2021;Cahu, 2004;Bourre, 2004 and. By virtue of this dietary quality, the fish has therefore been recommended in food for the prevention of diseases cardiovascular (Dicko et al., 1990). Since the recurring drought that the country has experienced, a significant number of wetland herders (Lake Chad, Lake Fitri and Mayo kebbi) with lost their cattle are converted into sinners. Fishing is practiced by different categories of fishermen, especially professional fishermen and seasonal fishermen who experience this activity by selling fish to resellers who export it to major urban centers notably Abé ché for fishing in Lake Fitri and Lake Chad. However, despite the fish is a vital source of animal protein and micronutrients for many rural community, it can also be the cause of serious Toxi-infection with health, political and economic consequences, (WHO 2003). In Abé ché , the majority of sellers buy fish carried away by lack of river and this can lead to a source microbial contamination; the transformation does not take place at their level. Although this sector is artisanal, it has assets and deserves follow-up for its improvement. It's in this perspective that we initiated this study. Hence, the objective of evaluating the hygienic quality and microbiological characteristics of dried and smoked fish sold in the markets of the city of Abé ché .

Description of the City of Abé ché
The study took place in the city of Abé ché , the provincial capital of Ouaddaï , in the department from Ouara located in the east of the country towards the Goz-beida road. It extends between 13° 48'584'' of north latitude and 20°58'139'' east longitude. The study area is under the influence of a climate intertropical with a dry season of 9 months and a rainy season of 3 months. The diet of these two (2) seasons is defined by the fluctuations between the masses of northern dry areas (harmattan) and the masses of maritime wetlands of the South-West (the monsoon). Average rainfall annual is about 300 mm. The temperature of the region is variable according to the periods. There average annual temperature in Abé ché is around 28°C with a variation in the cold season (December to February) between 16 and 35°C in the dry season (April and May) between 25 and 41°C. The city has three markets spread over the seven (7) districts. The market of Taradona is located in the second district. In this walk the fish are dominant and sold at the edge of the paved road. This locality has two sites: the market of Taradona and the Ridjil market where there is a lack of dried and smoked fish by the sellers. In these two sites, the fish are displayed in the open air and sold in detail. Of Moreover, these markets are not urbanized and most of the sellers occupy places by chance. Subsequently, the former livestock market is located in the sixth district of the city of Abé ché . Finally, the central market that is located in the center of the city between first and second boroughs. In this market, fish are sold in a minority.

Period and Type of Study
This is a descriptive cross-sectional study, which was conducted from June 10 to July 27, 2022 in the city of abé ché . The analyzes were carried out at the microbiology laboratories and INSTA chemistry. This study was focused on the evaluation of the hygienic quality and of dried and smoked fish sold in the markets of the town of Abé ché .

Preparation of Culture Media and Diluents
The preparation of the culture media consists first of all in calculating the medium in millilitres. This calculation is made according to the quantity of distilled water in ml. The mass of dehydrated media was weighed on a scale by referring to the indications mentioned on the box. These backgrounds were then mixed with a quantity of distilled water and then brought to the boil with stirring constant with a magnetic stirrer. After sterilization in an autoclave at 121°C for 15 minutes with the exception of the Hectoen media, these media were cooled between 45 to 50°C. A volume of 15 to 20 ml of medium was poured into previously sterilized Petri dishes. The germs sought were total mesophilic aerobic flora, total coliforms, coliforms thermotolerants, Staphylococcus and Salmonella.  1 ml is taken from the 10-4 and 10-5 dilutions and introduced aseptically into the boxes of Disposable petri dishes. 15 ml of PCA medium (Plate Count Agar) melted and cooled to water bath at 45°C. The mixture is homogenized by circular movements of the boxes. After solidification, 5 ml of PCA was added. This second layer prevents the invasion of the box by germs which can make it difficult to read. The boxes have been then incubated at 30°C. After 24 to 72 hours of incubation, counting is done by comp Stage colonies on plates with 30 -300 colonies.

Enumeration of Total and Thermo-tolerant Coliforms (ISO 4832: 2006).
A volume of 1 ml was taken from the 10-2 and 10-3 dilutions and introduced into the Petri dishes at single use, then 15 to 20 ml of sterilized MCK medium is added. The mixture was homogenized se by circular movements with the hand. After solidification, 5 ml of EMB was added. Inoculated plates were incubated at 44°C for thermotolerant coliforms and 37°C for total coliforms. The counting was carried out after 24 to 48 hours of incubation.

Search and Enumeration of Presumed
Pathogenic Staphylococcus (NF standard V08-057-1, November 1994) 0.1 ml of the stock solution and 0.1 ml of the 10-2 dilution were inoculated on the surface in Petri dishes in which the Chapman medium was previously poured. The boxes were in cubed at 37°C for 24 hours. After incubation at 37°C for 24 hours, the colonies do The amples obtained were subcultured into the nutrient broth tubes for the coagulase test. A positive coagulase test result concluded the presence of Staphylococcus aureus.

Detection and Enumeration of Salmonella (ISO 6579/A1 standard: July 2007)
The search for and counting of salmonella require several culture media (mi places Rapport, Hectoen) and take place in several stages. ÿ pre-enrichment: it consists in incubating at 37°C the ground material of 25 g of fish in 225 ml of EPT for 24 hours. ÿ selective enrichment: 1 ml of the pre-enriched solution was inoculated into the test tubes containing 9 ml of Rappaport Vassiliadis Soja (RVS) broth respectively. The pipes are then incubated for 24 hours at 37°C. ÿ isolation: After enrichment, 0.1ml of the enriched solution was taken and inoculated on the Hectoen selective medium by the streaking method using a sterile glass Pasteur pipette. The plates were incubated for 24 hours at 37°C.

Research and Enumeration of Yeasts and Molds (ISO 21527-1 standard; July 2008)
Sabouraud medium was used to search for these germs. Indeed, 0.1ml of the 10-2 and 10-3 dilutions 10-1 , were inoculated into the selective medium contained in Petri dishes. There Colonies are read after 3 to 5 days of incubation at 25°C. They are in the form whitish rounded.

Method of Calculation and Interpretation of Results
After the incubation period mentioned in the standard specific to each germ, we proceed the counting of characteristic colonies for each dish containing less than 300 colonies and 15 colonies minimum or any other number indicated in the standard. The number N of germs present in the sample analyzed is considered as an average weight of successive dilution and given by the following formula: C1+C2= sum of the characteristic colonies on the two dishes retained V= volume of inoculums applied to each box d1= dilution rate corresponding to the first dilution retained n1 = number of dishes retained at the first dilution n2 = number of dishes retained at the second dilution.

Statistical Method
The data collected was analyzed using the XLSTAT (6.1.9.) statistical software. Descriptive made it possible to have the socio-economic parameters (mean, standard deviation, percentage and frequency). The analysis of variance (ANOVA) made it possible to make the comparison between the mean of the parameters evaluated. The Newman-Keuls test (for multiple comparison) was used to determine the level of significance between the variable (types of fish sold) at the 5% threshold.

Origin of Dried and Smoked Fish
Dried and smoked fish arrive in Abeche directly from N'Djamena, Lac-Tchad and Hello. The sellers send their money to the wholesalers and they in turn package the fish in hermetically sealed boxes and transported to Abé ché by lorries of transportation. At the local level, the fish were retailed to end consumers by the sellers.

Profile of Respondents
A total of 30 sellers were surveyed for a month on information relating to different variables and hygienic practices of dried and smoked fish in the three (3) markets in the town of Abé ché .  Figure 1 represent the rate of sellers according to survey sites. This figure shows that the Taradona market presents more fish sellers with 65.52% followed by the former livestock market with 31.03% and the large market represents a low rate of sellers (3.45%).  Table 3 represents the socio-professional characteristics of the respondents, including gender female presents 96.67% unlike male 3.33%. The age of respondents is located between 22 and 45 years old, on average 33.55% and most respondents are married with 93.33%. By elsewhere, more than half of the respondents have not been to school, on the other hand those who are educated have a low rate (3.33%). In addition, the majorities of respondents sell smoked fish (56.67%) and keep much more in boxes (93.33%). Le tableau 3 repré sente les caracté ristiques socio-professionnelles des enquê té s, dont le sexe fé minin pré sente 96,67% contrairement au sexe masculin 3,33%. L'âge des enquêtés est situé entre 22 à 45 ans, moyennement 33,55% et la plupart des enquêtés sont marié s avec 93,33%. Par ailleurs, plus de la moitié des enquêtés n'ont pas été scolarisés par contre ceux qui sont instruit pré sentent un faible taux (3,33%). De plus, la majorité des enquê té s vendent les poissons fumé s (56,67%) et conservent beaucoup plus dans des cartons (93,33%).

Microbiological Analysis
The different germs identified and counted are recorded in tables 1.2 and respectively for the three markets in the town of Abé ché .
Smoked fish: 6,81. x 10 7 UFC/g pour FAMT ; 2,28. X 10 6 UFC/g pour CT ; 1,56 x 10 5 UFC/g for CTT et 3 UFC/g for yeasts and molds. The figure shows that the unsatisfactory microbiological quality is 74.67% for the dried fish and 36.33% for smoked fish. On the other hand, 5.33% are satisfactory for dried fish and 46% for smoked fish.

Assessment of Survey Results
The survey found that the majority of dried and smoked fish sold in Abé ché come from N'djamena, Lac-Chad and Salamat. Our results corroborate those of (Abdoullahi and al.;2016) who reported that the dried fish of Chad come from the region of Sa lamat. Table 2 shows that the majority of respondents are mama women. children (93.33%) and who have not been to school (56.67%). (Kokou Abotchi 2010;Oulaï et al 2007) reported a similar result by carrying out the same studies in Togo and Côte of ivory. The lack of education does not allow these women to resort to the hygiene of foodstuffs which could have harmful consequences on the health of consumers adders. The conservation of dried and smoked fish is mostly done in coaches. tones (93.33%). These results are in agreement with the observation of (Seid et al., 2021) that the fish are transported to the market, on board tractor-trailers, packaged in boxes and bags. Storage in boxes avoids losses, their spoilage and also retain their nutritional and organoleptic value. The boxes used for the conservation of these fish are boxes whose provenance is doubtful. These are sometimes boxes used for the transport of chemical products which could lead to travel gives. It emerges from this study that the most consumed fish in the town of Abé ché are smoked fish. Our results are similar with the work of (Mananga et al 2019) at Brazzaville (Congo) who reported that the same type of smoked fish is consumed the most.

Assessment of the Microbiological Quality of Fish
The level of contamination by total mesophilic aerobic flora is 74.67% for dried fish and 36.33% for smoked fish with respective averages of 4.60 x 107 and 6.81 x 107 CFU/g. Our results are well above the averages reported by (Abdoullahi et al 2016); (JC Micha et al 2018), respectively 5.5 x 105 to 2.2 x 106 CFU/g in the city of N'djamena (Chad) 188.07 x 105 CFU/g for dried fish and 117.60 x 105 CFU/g for smoked fish. The high level of contamination of our samples could be explained by non-compliance with hygiene rules at the place of processing and sale. The fish are sold in the open air, around public roads, next to rubbish and next to dirty stagnant water. These could expose them to dust, proliferation of flies and other insects. These can lead to a risk of microbial contamination of dried and smoked fish. Non-compliance with the rules of good hygienic practices by food processors and sellers leads their contamination by coliforms and total flora Hadjer (2014). Faecal coliforms are present in both types of processed fish (dried and smoked) with respective averages of 1.43 x 105 CFU/g for dried fish and 1.22 x 105 CFU/g for fish smoked with non-satisfaction rates of around 74.67% and 36.33%. (J C. Micha and al 2018) reported opposite averages to ours respectively 90.80 CFU/g for dried fish and 23.73 CFU/g for smoked fish. Both results suggest that dried fish are more contaminated than smoked ones. This difference is due to the ensure that the processing of fresh fish into dried fish is done in an unhealthy environment (worn or rolled materials, in the open air, on the ground, within reach of reptiles, flies and other insects) and by the customer who often handles them with his bare hands. According to (Tidjani et al 2013); and Attamar (2018), several bacteria responsible for Toxi Collective Food Infections (TCFI) can belong to the flora of the digestive tract and be conveyed by the hands of the wearer (E. coli for example). Fecal coliforms are bad markers of the hygienic conditions of the manipulator. Their presence in the food usually shows fecal contamination E. coli has not been identified in the samples but the strong presence of fecal coliforms suggests the presence of E. coli, because this bacterium represents about 80% of fecal coliforms. That could indicate a risk to consumers. However, the pathogen such as Salmonella was not isolated in our samples, contrary to the results obtained by (Abdoullahi et al 2016), Oku and Amakoromo (2013). They reported the presence of Salmonella and Staphylococcus aureus in their study. This difference could be explained by the fact that the fishing was carried out in unpolluted waters and also by the smoking treatment for smoked fish. Our results showed a low level contamination of smoked (3.33%) and dried (6.66%) fish by yeasts and mold. According to the AFNOR standard (1996), the fungal flora for dried fish must not exceed 103 CFU/g and 102 CFU/g for smoked fish. These values are therefore at above that recommended by the standard. Our results are reduced to those of (Abotchi 2010); (Abdoullahi et al., 2016) who reported a high rate of contamination of dried and smoked fish in their study respectively 92.5% for smoked fish and 5.9 x 103 CFU/g for dried fish. This difference would be due to the sample size and also to the environment in which the fish were processed. The spread of fish on mats, tarps, rusty materials, wood and storage on polluted soil, would be the exogenous cause of mold contamination. According to Jeantel and al., (2006), the behavioral attitude would be at the origin of post-contamination of the fish. dried and smoked which would be subject to the proliferation of fungi. On the contrary, according to (Bourgeois et al., 1980), the high contamination of smoked fish can be explained by the great capacity of yeasts and molds to grow on substrates with low activity of the water. From a general point of view, non-compliance with hygiene rules and exposure of products in an unhealthy environment would be the source of microbial contamination in some of our samples.

Conclusion
The realization of this work allowed us to evaluate the living organisms in fish dried and smoked fish sold in the markets of the town of Abé ché . Dried fish are more contaminated than smoked fish due to non-compliance with Ischikawa rules. Failure to observe good processing hygiene practices leads to contamination of fish after drying and smoking. In Abé ché , the fish come from N'djamena and Salamat and sold in the various markets of the city d'Abé ché , where the Staphylococcus aureus was found at the former cattle market unlike to other markets. The level of contamination of the total mesophilic flora is 74.67% for dried fish and 36.33% for smoked fish with a respective average of 4.60 x107 CFU/g. However, despite the fact that the fish suffer contamination from microbial, they are a source of animal protein, micronutrients and a source significant income for the population. Therefore, the study suggests the mastering of condition conservation, processing and proper use in order to avoid or reduce the risk of microbial contamination. have appeared to influence the work reported in this paper.

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Data availability statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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