Processes for preserving and processing fish: Characterization and impact on microbiological quality in Lokossa, South-West Benin

 Corresponding author: Martinien Hospice Mahussi Assogba Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Processes for preserving and processing fish: Characterization and impact on microbiological quality in Lokossa, South-West Benin Martinien Hospice Mahussi Assogba *, Elie Linton and Issaka Youssao Abdou Karim Laboratory of Animal Biotechnology and Meat Technology, Department of Animal Production and Health, Polytechnic School of Abomey-Calavi, University of Abomey-Calavi, Abomey-Calavi, 01 BP: 2009 Cotonou, Benin. World Journal of Advanced Research and Reviews, 2025, 28(01), 469-481 Publication history: Received on 26 August 2025; revised on 04 October 2025; accepted on 06 October 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.28.1.3439 Abstract The sanitary condition of consumers is comparable to the microbiological condition of fish. This study aimed at defining processing and preservation methods and determining their effect on the sanitary condition of fish in the Lokossa township. 27 processors were interviewed. 60 whole, pre-processed, smoked and fried samples of S. melanotheron and C. guineensis were taken. The identified processes were smoking (33%), frying (85.19%) and salting-drying (3.70%). All the processing operations began with trimming (scaling, gutting and cutting), drying and salting. Both species had similar microbial counts (p > 0.05) for total coliforms, faecal coliforms, S. aureus and C. perfringens, regardless of processing (whole, pre-processed, fried or smoked). FMAT counts and enterobacteria in S. melanotheron were significantly different between types of processing (p < 0.05). For C. guineensis, enterobacteria loads varied among pre-treated and whole fish, and smoked and fried fish. E. coli occurred in all samples, and 16.67% of the samples had Salmonella regardless of species. Minimizes the bacteriological load of the fish by smoking and frying under sanitary conditions. Keywords: Food safety; Microbiology; Tilapia; Coptodon guineensis; Sarotherodon melanotheron; Health; Food technology 1. Introduction Fish is an essential source of protein for humans and livestock. In 2022, fish production reached a record 223.2 million tonnes, with per capita consumption standing at 20.7 kg. This increase is linked to the growing global population and high protein requirements. Similarly, in Benin, the annual national production of fish is estimated at 88,677 tonnes, while the annual requirement is 202,000 tonnes. The deficit is made up by importing frozen fish (see references [2] and [3]). However, even if efforts are made to meet quantitative needs, a healthy diet and improved health are said to guarantee food security, according to [1]. In Benin, 1.1 million people are food insecure, and the phenomenon affects several departments. These include the Mono department, which has a food insecurity rate of 27% [4]. In order to overcome this issue, the target population in Mono needs to consume a varied, balanced and healthy diet. While this population has some access to cereals through agriculture, they have very limited access to protein-rich foods. Fish is the most widely consumed animal protein in the diet of the population in this department because it is available and accessible to low-income households. This is due to the department's many lakes, including Ahémé and Toho, and World Journal of Advanced Research and Reviews, 2025, 28(01), 469-481 470 its rivers, such as the Couffo and Mono, and their tributaries. Once caught, the fish are consumed either fresh or, more often, after processing, as they are highly perishable. Immediately after capture, the muscles are practically sterile, containing only bacteria on the skin, gills and in the viscera [5]. Most of this bacterial flora (with the exception of Clostridium botulinum, Vibrio para haemolyticus and Listeria monocytogenes) is banal in nature, and therefore harmless or only responsible for altering the marketability of products [6]. Fish can be contaminated by pathogens during handling, processing and marketing [7]. There are different processes to enhance the preservation of fish in the Mono department. In this perspective, there is a necessity to review these processes with the aim of enhancing the quality of the final product. Sensory and sanitary quality are the most required qualities by the consumers. There has been some work to assess the microbiological quality of fish in the coastal departments (cf. references [8, 9] in the Atlantic at Abomey Calavi [10, 11, 12, 13, 14, 15, 9], and in the Mono and Ouémé departments [9]. The studies by [9] focused only on fresh fish. To ensure a healthy food supply in Mono, we have decided to examine the impact of processing and preservation methods on the hygiene of fish in Lokossa, a town in south-west Benin. This study will characterise these processes and assess the bacteria present in fresh fish before and after processing. 2. Materials and methods 2.1. Study area Figure 1 Geographical location of the township of Lokossa Located in the north-western part of the Mono department, at an altitude of 31 meters, the township of Lokossa has a latitude of 6° 37' 60'' North and a longitude of 1° 43’ 0'' east. It extends over 1,605 km and is one of 6 districts in the World Journal of Advanced Research and Reviews, 2025, 28(01), 469-481 471 department. It covers an area of 260 km2, which represents 16% of the Mono's surface area and 0.23% of Benin's total surface area (112,622 km²) [16]. Bordered to the North by the township of Dogbo in the Couffo department, to the South by the the township of Athiémé and Houéyogbé, to the east by that of Bopa and to the west by Togolese territory, this township is divided into five districts: Lokossa, Agamè, Koudo, Houin and Ouèdèmè-Adja. These districts are subdivided into 8 town districts and thirty-seven (37) villages, making a total of forty-five (45) localities. The main town of the township (Lokossa) is located around 106 km from Cotonou (Benin's economic capital). It is also the capital of the Mono township [16]. Figure 1 shows the administrative map of Lokossa. 3. Methodology Data were collected in two stages: a survey and microbiological analyses. The survey focused on fish processing in the Lokossa township, while the analyses focused on germ research. 3.1. Survey of transformation processes/conservations It carried out a survey of fish processing methods in two two districts of the township, namely Houin and Lokossa Centre, which run through Lakes Toho and Doukon respectively. The questionnaire format solicited the following information: identification of respondent, activity carried out, dependents, kind of fish processed, processing units, cleanliness of processing unit, process and brief description of the same, and storage time. Twenty-seven women were interviewed during the survey: twenty-three at Houin district and four at central Lokossa. They were randomly chosen from among the most diligent and regular fish processors. 3.2. Fish sampling The Houin district, and more specifically the Duimè riverbank, has been chosen to collect fish samples for microbiological analysis. The site is packed with fishermen, fishmongers and processors. Two species of fish (Coptodon guineensis and Sarotherodon melanotheron), the most heavily fished in the township, were selected for data collection to assess microbiological quality. Samples of fresh, pre-treated, fried and smoked fish were acquired from fishmongers and processors in the arrondissements of Lokossa center and Houin (Township of Lokossa). Microbiological analyses were carried out at the Laboratory of Animal Biotechnology and Meat Technology of the Department of Animal Production and Health of the Polytechnic School of Abomey-Calavi of the University of Abomey-Calavi. Each sampled fish was aseptically placed in a Stomacher bag and then in a cooler fitted with carboglass. In total, the sanitary quality study was carried out on 30 fish of each species (Table 1). Table 1 Sampling plan Types of fish Fish species Number of fish sampled Whole fish C. guineensis 10 S.melanotheron 10 Pre-treated fresh fish C. guineensis 10 S.melanotheron 10 Fried fish C. guineensis 5 S.melanotheron 5 Smoked fish C. guineensis 5 S.melanotheron 5 Total 60 World Journal of Advanced Research and Reviews, 2025, 28(01), 469-481 472 3.3. Microbiological analyses 3.3.1. Microorganisms tested in samples The flora tested were : Total Aerobic Mesophilic Flora (TAMF), total and fecal coliforms (with a search for Escherichia coli), which provide information on the freshness of the flesh and the slaughtering conditions, respectively; Pseudomonas, which are psychotrophic germs that indicate fish spoilage and can develop in fish kept at ambient temperatures (25 to 40°C), as well as pathogenic germs such as Salmonella, Staphylococci and Sulfite-Reducing Anaerobes (SRA). Samples were analysed in accordance with ISO standards specific to each germ: • TAMF : [17]; • Salmonella : [18]; • Suspected pathogens staphylococci: [19]; • Sulfite-Reducing Anaerobes (SRA) : [19]; • Total coliforms :[20]; • Fecal coliforms : [20]; • E.coli : [21]; • Pseudomonas : [22]. 3.4. Test sample, preparation of dilutions and inoculum Dilutions are prepared for microbiological examination in accordance with standard NF V08-010. Twenty-five grams of flesh from the head, body, tail and gills of the fish were aseptically removed into a sterile Stomacher bag, then 225 ml of buffered peptone water was added and the whole homogenised in the Stomacher for 30 seconds to one minute. The crushed material was then left to stand for a maximum of 45 minutes at room temperature to revive the germs. This mixture thus constituted the mother suspension, which was used to prepare successive dilutions in geometric progression of reason 10. The various dilutions were carried out using this stock solution. One millilitre of the said solution (10-1), taken after homogenisation, was inoculated into 9 ml of sterile Tryptone salt or diluent (0.1%peptone + 0.85% NaCl) to obtain dilution 10-2. After homogenisation, 1 ml of this 10-2 dilution was also inoculated into 9 ml of sterile Tryptone Salt to obtain dilution 10-3. This was followed by dilutions 10-4 and 10-5. 3.5. Criteria for assessing the good bacteriological quality of fish For good fish quality, the microbiological criteria below were used to assess the analytical results obtained in our work. These are: • Absence of Sulfite-Reducing Anaerobes (SRA) in 1 g ; • Absence of pathogenic germs, in particular Salmonella, in 25 g ; • Absence of coliforms in 1 g ; • Total" aerobic flora < 25 germs/g for freshly obtained meat and <500 germs/g for cold-preserved meat. 3.6. Statistical analysis Survey data on the processing of fresh fish and fish frying and smoking processes in the township were analysed using the Proc Freq procedure in [23], and frequency significances were determined using the chi-squared test. The Generalised Linear Model procedure (Proc GLM) was used for analysis of variance. The factors used to explain variation in bacterial loads were treatment mode, fish species, and the interaction between treatment mode and species. The F-test was used to determine the significance of each effect, and the means were calculated and compared using the Student's t-test. Frequencies of Pseudomonas, E. coli and Salmonella were calculated using the Proc Frec procedure in SAS and compared using the chi-squared test and two-tailed Z-test. For each relative frequency, a 95% confidence interval (CI) was calculated. This was done using the following formula: World Journal of Advanced Research and Reviews, 2025, 28(01), 469-481 473 CI=1,96√[P(1−𝑃)] N P is the relative frequency. N is the sample size. 4. Results 4.1. Activities carried out by processors Several activities were carried out by processors in the township of Lokossa (table 2). All the respondents process and sell fish, and 11.11% of them also farm. Those who also fish and rear livestock represent only 3.70% of the women surveyed. In addition to farming, the women were involved in other activities such as selling porridge (15.38%) and other products (30.77%). Table 2 Activities carried out by processors in Lokossa Variables Numbers Percentage (%) CI Agricultural activities Agriculture 27 11.11b 11.85 Breeding 27 3.70b 7.12 Fishing 27 3.70b 7.12 Fish farm 27 0.00b 0.00 Fish processing and sales 27 100.00a 0.00 Other activities Sale of porridge 27 15.38ab 13.61 Retailer of various products 27 30.77a 17.41 Production of palm oil 27 7.69b 10.05 Production of palm oil, food sales 27 7.69b 10.05 Production and sale of “akassa” 27 7.69b 10.05 Processing and sale of “come” 27 7.69b 10.05 Clothes launderer 27 7.69b 10.05 Catering 27 7.69b 10.05 Main activities Agriculture 27 0.00b 0.00 Breeding 27 0.00b 0.00 Fishing 27 0.00b 0.00 Fish farm 27 0.00b 0.00 Fish processing and sales 27 77.78a 15.68 Other activities 27 11.11b 11.85 Percentages between the classes of the same column followed by different letters differ significantly at the threshold of 5% ; CI: Confiance Interval; % : percentage. 4.2. Processed fish species and processing methods Table 3 shows the different species of fish processed in the township of Lokossa and the techniques used. The fish species most commonly used for processing is tilapia (92.59%), whose proportion is higher (p<0.001) than that of the other species: dentex, sea bream and horse mackerel. The proportions of dentex, sea bream and processed horse mackerel were 11.11%, 7.41% and 7.41% respectively of all fish and did not differ significantly between them (p>0.05). The processing methods used in the township are: smoking, frying and salting-drying. World Journal of Advanced Research and Reviews, 2025, 28(01), 469-481 474 Of these processes, frying was the most used (85.19%), followed by smoking (33%) and salting-drying (3.70%). Finally, cold preservation, drying, smoking and frying were the preservation techniques used in the township. Preservation by drying was the most used, with 77.79% of processors surveyed using a preservation technique. Table 3 Processed fish species, processing and storage techniques Variables Township of Lokossa Numbers Percentage (%) CI Species of fish purchased for processing and preservation Tilapia 27 92.59a 9.88 Dentex 27 11.11b 11.85 Dorade 27 7.41b 9.88 Horse mackerel 27 7.41b 9.88 Processing methods used Smoking 27 33.33b 17.78 Frying 27 85.19a 13.40 Salting and drying 27 3.70c 7.12 Production of lanhouin 27 0.00c 0.00 Types of conservation technique Ice preservation 27 25.93b 16.53 Regular drying 27 77.79a 15.68 Regular smoking 27 3.70c 7.12 Other: regular heating in oil 27 3.70c 7.12 Percentages between the classes of the same column followed by different letters differ significantly at the threshold of 5% ; CI: Confiance Interval; % : percentage. 4.3. Description of fish processing and preservation processes Figure 2 describes the various fish processing and preservation processes in the township of Lokossa. All processing begins with trimming (scaling, gutting and cutting), and these trimming stages vary depending on the species. The fish were then washed or cleaned to remove any residues. The rest of the process depended on each processing method. For smoking, the cleaned fish is drained, salted (optional operation) soaked in oil and exposed to the smoke over a low fire. During smoking, the fish is sprayed with peanut oil. This process took around 4 hours for 50 kg of fish. As for frying, after trimming, the fish were cleaned and rinsed with water, drained, then salted (optional) before being fried in red oil or peanut oil. This process took around 3 and a half hours for 50 kg of fish. Salting-drying was compulsory after trimming and draining. The fish were then exposed to the sun. This process lasted between 48 and 72 hours, sometimes longer depending on the humidity in the air. Finally, to preserve the processed fish, processors used drying and smoking techniques. Each processing procedure corresponds to a processing diagram (Figure 2). World Journal of Advanced Research and Reviews, 2025, 28(01), 469-481 475 Figure 2 Diagram of fish processing in the township of Lokossa 4.4. Microbiological quality of fish 4.4.1. Effect of treatment method and species on the microbial load in fish Table 4 shows the effect of treatment and species on microbial load. For each of the fish species (S. melanotheron and C. guineensis), the microbial loads were similar for total Coliforms, fecal Coliforms, Staphylococcus aureus, Clostridium perfringens (p>0.05) whatever the treatment method (whole, pre-treated, fried and smoked). However, the highest Total Aerobic Mesophilic Flora (TAMF), load (p<0.05) was obtained on whole S. melanotheron samples. TAMF loads were identical on the other treated S. melanotheron samples. For Enterobacteria, the highest loads were obtained in whole and pretreated S. melanotheron and in whole and pretreated C. guineensis. No cases of Enterobacteria were obtained in the fried and smoked samples of either species. World Journal of Advanced Research and Reviews, 2025, 28(01), 469-481 476 Table 4 Effect of treatment method and species on the microbial load in fish Types of treatments Load of S. melanotheron Total Aerobic Mesophilic Flora (CFU/g) Total coliforms (CFU/g) Fecal coliforms (CFU/g) Staphylococcus aureus (CFU/g) Clostridium perfringens (CFU/g) Enterobacteri a (CFU/g) Mean SD Mean SD Mean SD Mean SD Mean SD Mean DS Whole fish 1330000.00 a 89566.8 5 925.00a 671.75 722.5 a 958.12 37.75a 31.46 26.75a 28.63 236.5 a 25.5 9 Pre-processed fish 642166.67b 113844. 19 0.00a 00.00 1400a 0.00 11.25a 3.88 33.00a 2.82 226.6 0a 0.28 Fried fish 620000.00b 0.00 0.00a 0.00 0.00a 0.00 0.00a 0.00 0.00a 0.00 0.00b 0.00 Smoked fish 320000.00b 0.00 0.00a 0.00 0.00a 0.00 17.50a 0.00 0.00a 0.00 0.00b 0.00 Significance test * NS NS NS NS * Load of C. guineensis Whole fish 737833.33a 714884. 95 925.00a 671.75 300.0 0a 212. 132 8.75a 9.54 45.00a 7.77 184.0 0a 23.2 0 Pre-processed fish 1037500.00 a 83 674 307 1250.00a 212.13 800.0 0a 848.52 18.75a 15.9 70.25a 45.6 269.0 0a 20.6 Fried fish 141333.33a 0.00 0.00a 0.00 0.00a 0.00 30.00a 0.00 0.00a 0.00 0.00b 0.00 Smoked fish 189333.33a 0.00 0.00a 0.00 0.00a 0.00 0.50a 0.00 0.00a 0.00 0.00b 0.00 Significance test NS NS NS NS NS * NS : p>0.05 ; * : p<0.05 ; DS : Standard Deviation ; Means between the classes of the same column followed by different letters differ significantly at the threshold of 5%; CFU/g: Colony Forming Unit per gram World Journal of Advanced Research and Reviews, 2025, 28(01), 469-481 477 4.5. Frequencies of Salmonella, Pseudomonas and Escherichia coli The frequency of Pseudomonas in S. melanotheron was higher (50%) than in C. guineensis (16.67%). Pseudomonas sp was found in whole fish and pretreated fish (50% in each case), while no cases were observed in fried and smoked fish (Table 5). The frequency of salmonella was 50% in the case of pre-treated fish, while no salmonella cases were found in the case of whole, fried and smoked fish. The frequency of the presence of salmonella in the samples analysed was 16.67% for each of the species (S. melanotheron and C. guineensis) (Table 6). Finally, Escherichia coli was present in all the fish samples analysed (Table 7). Table 5 Pseudomonas frequencies Source of variation Number Percentage of Pseudomonas sp (%) Presence CI Species S. melanotheron 12 50.00a 40.01 C. guineensis 12 16.67a 29.82 Treatments Whole fish 8 50.00a 49.00 Fried fish 5 0.00b 0.00 Smoked fish 5 0.00b 0.00 Pre-treated fish 8 50.00a 49.00 Percentages between the classes of the same column followed by different letters differ significantly at the threshold of 5% ; CI: Confiance Interval; % : percentage. Table 6 Salmonella frequencies Source of variation Percentage of salmonella sp (%) Number Presence CI Species S. melanotheron 12 16.67a 29.82 C. guineensis 12 16.67a 29.82 Treatments Whole fish 8 0.00b 0.00 Fried fish 5 0.00b 0.00 Smoked fish 5 0.00b 0.00 Pre-treated fish 8 50.00a 49.00 Percentages between the classes of the same column followed by different letters differ significantly at the threshold of 5% ; CI: Confiance Interval; % : percentage. Table 7 Frequencies of Escherichia Coli Source of variation Percentage of E. coli (%) Number Presence CI Species S. melanotheron 12 100.00 0.00 C. guineensis 12 100.00 0.00 Treatments Whole fish 8 100.00 0.00 Fried fish 5 100.00 0.00 Smoked fish 5 100.00 0.00 Pre-treated fish 8 100.00 0.00 I CI: Confiance Interval ; ; % : percentage.