Traditional beverages are drinks developed by the populations of a specific geographic region using local raw materials, whether cereal-based or not [1]. These beverages have an important place in global food systems due to their availability, unique sensory attributes, nutritional and socio-cultural value, affordability, and ease of production. They are consumed almost daily in Africa by both children and adults and can be fermented or not. Traditional fermented beverages generally result from spontaneous fermentation processes of carbohydrate-rich substrates (particularly cereals), driven by complex microbial communities composed mainly of lactic acid bacteria and yeasts [2]. Although these empirical fermentation practices significantly contribute to improving the organoleptic and nutritional characteristics of these beverages, they can also be a source of microbiological contamination when production conditions are not controlled [3]. Consequently, consuming these indigenous beverages could pose a risk of foodborne illness to consumers. It has been reported that more than two (2) million people worldwide, primarily children and the elderly, die each year from illnesses caused by the consumption of contaminated food and beverages [4].

In Sub-Saharan Africa, the production of traditional fermented beverages is often carried out in environments characterized by weak health oversight, limited adherence to good hygiene and manufacturing practices, and a lack of standardized processes. This is the case with Tchakpalo, a traditional fermented beverage made from corn and/or sorghum, produced in Benin and highly appreciated by the local population. As previously demonstrated by Dognon et al. [5], the production of Tchakpalo in the Grand Nokoué region relies on empirical and heterogeneous processes, involving in particular the use of rudimentary equipment, insufficiently controlled raw materials, and techniques likely to introduce or maintain undesirable microbial flora. Furthermore, Tchakpalo producers do not belong to any structured professional organization. This absence of institutional oversight and technical training results in production and marketing practices that fall far short of food industry standards. Consequently, questions arise regarding the health implications associated with these production and distribution conditions. Therefore, assessing the microbiological quality of Tchakpalo is an essential and indispensable step in evaluating the risks associated with its consumption.

It is within this context that the present study was conducted, with the aim of: (i) assessing the microbiological compliance of Tchakpalo samples sold in Grand Nokoué (Benin), and (ii) evaluating the adoption level of good hygiene practices byproducers during the production and sale of Tchakpalo.

The results obtained in this study highlight widespread microbiological contamination in the Tchakpalo samples analyzed in the study area. These results confirm that Tchakpalo sold in the Grand Nokoué exhibits significant microbiological contamination, consistent with several previous studies on traditional fermented beverages. In Benin, a recent study by Dognon et al. [5] highlighted the variability of Tchakpalo production processes and their empirical nature. This situation constitutes a major microbiological risk factor. Similarly, Konfo et al. [7] reported that cereal-based fermented beverages in Benin frequently exhibit high levels of hygiene indicator microorganisms due to poorly controlled processing conditions. [16] attributed this high contamination to inadequate hygiene conditions during the production, storage, and sale of fermented beverages. The consistent presence of Enterobacteria and the high prevalence of E. coli observed in this study corroborate the findings of [4]. These authors demonstrated that traditional fermented beverages in sub-Saharan Africa are often exposed to fecal contamination. Indeed, Enterobacteria serve as an indicator of fecal or environmental contamination, often linked to the use of contaminated water or inadequate hygiene practices [17][18]. The detection of E. coli in food is one of the most widely used hygiene criteria in the food industry [19]. E. coli is a key indicator of fecal contamination in food and beverages [20]. It has also been detected in traditional fermented beverages in Ghana, notably in Emuduro, a traditional beer characteristic of the Hausa people [21]. These risks are common in artisanal systems for the production of fermented beverages, particularly in West Africa, where health oversight remains limited [22].

In fact, in the municipalities of Grand Nokoué, Tchakpalo is produced in homes under unsanitary conditions that expose the production process (raw materials, equipment, water, intermediate products, finished products, etc.) to various types of contamination, particularly fecal and environmental contamination. Furthermore, the sales environment and the way Tchakpalo is sold pose a major risk of contamination. The sales environment (primarily along roadsides) exposes the beverage to contamination from air, dust, exhaust fumes, etc. Food placed close to the ground and exposed to dust has been linked to foodborne illnesses such as cholera and diarrheal diseases [23]. Several foodborne outbreaks have been linked to poor practices and poor hygiene among food handlers [24]. These observations are also consistent with recent studies by Paez-Lerma et al. [25] and [20] on traditional fermented beverages.

E. coli is a commensal bacterium found in the intestinal tract of mammals. Certain pathogenic strains of E. coli have been linked to several diseases in animals and humans [26]. The presence of E. coli in ready-to-eat food is also an indicator of the potential presence of enteric pathogens in that food [27]. Thus, the presence of E. coli in food makes it unsafe for human consumption. E. coli should not be detected in ready-to-eat food, even if a tolerance level is permitted. The U.S. Centers for Disease Control and Prevention (CDC) has estimated that E. coli O157:H7 infections cause 73,000 illnesses, 2,200 hospitalizations, and 60 deaths per year in the United States [28]. In addition to foodborne illnesses, E. coli strains can cause serious diseases such as diarrhea, dysentery, peritonitis, mastitis, sepsis, pneumonia, meningitis, hemorrhagic colitis, hemolytic uremic syndrome, and death [29]. In developed countries, E. coli is the most common pathogen associated with childhood diarrhea and is an emerging antimicrobial-resistant enteropathogen [30]. Based on their specific virulence properties, serotypes, and various epidemiological and clinical characteristics, seven distinct groups of diarrheagenic E. coli are recognized, namely: enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), diffuse-adhering E. coli (DAEC), and Shiga toxin-producing E. coli (STEC) [19].

With regard to S. aureus, its prevalence and abundance are closely linked to the rudimentary and manual nature of Tchakpalo production processes. Indeed, S. aureus is part of the normal flora of the skin and nasal passages in 20 to 50% of healthy individuals [31]. People working in the food sector therefore constitute a significant source of contamination. S. aureus is thus a good indicator of skin and mucosal contamination. This ecological specificity explains its constant presence in artisanal food products [18]. In Ethiopia, Erkihun et al. [32] showed that a significant proportion of food handlers (20%) were carriers of S. aureus. In Ivory Coast, Attien et al. [33] reported the frequent presence of S. aureus in street-sold beverages, including those sold hot, when hygiene conditions are inadequate. These results support the hypothesis that S. aureus contamination of Tchakpalo is primarily of human origin. Practices such as inadequate handwashing, direct handling of the product with bare hands, etc., may explain the high levels of S. aureus in Tchakpalo from Grand Nokoué. Furthermore, it should be noted that the presence of S. aureus in food poses a particular risk when the strains are enterotoxigenic. Indeed, staphylococcal foodborne illness (SFI) is primarily caused by a specific group of toxins known as enterotoxins [34]. Staphylococcal enterotoxins (SETs) are heat-stable and can cause food poisoning even after the product has been heat-treated. To date, more than 30 different SETs have been described, and SEA, SEB, SEC, SED, and SEE are the most commonly implicated in foodborne toxi-infections [31]. These heat-stable toxins can resist proteolytic enzymes (pepsin and trypsin). This property allows them to retain their biological activity even after cooking or passage through the gastrointestinal tract. According to the World Health Organization, STIs are among the leading causes of foodborne outbreaks worldwide. ASF is characterized by a sudden onset of symptoms (1 to 6 hours after ingestion), including vomiting, abdominal pain, and stomach cramps [35]. FDI can lead to hospitalizations, particularly among the YEPI (Young, Elderly, Pregnant women, and Immunocompromised Individuals) [36]. Thus, the prevalence and abundance of S. aureus observed in samples from Tchakpalo in the Grand Nokoué region suggest a potential public health risk, particularly if production and marketing conditions are not improved.

The fungal flora of interest in this study is molds. Although certain molds may be involved in the development of aromas in fermented food products, the majority of them are part of the spoilage flora, particularly in beverages. In Tchakpalo produced in the municipalities of Grand Nokoué, toxin-producing molds (Fusarium spp., Aspergillus spp., and Penicillium spp.) were present, with an average microbial load of 2.51 × 10² CFU/mL. The presence of these molds may be due to poor hygiene [37]. They may also originate from the grains used in Tchakpalo production. Indeed, [38] demonstrated that the grains used in traditional fermentations are frequently contaminated by potentially mycotoxin-producing fungal species, particularly those belonging to the genera Aspergillus and Penicillium. The high prevalence and large number of molds in Tchakpalo pose a serious public health problem, as these fungal genera are highly toxinogenic. Their pathogenicity is closely linked to the production of a group of toxins known as mycotoxins. Mycotoxins are secondary metabolites produced by various species of mold. Mycotoxin production is primarily associated with the genera Aspergillus, Penicillium, Fusarium, Claviceps, and Alternaria, and occurs under specific conditions [39]. There are more than 500 mycotoxins that are toxic and harmful to human, animal, and plant health. The most common and best-studied mycotoxins are those that pose the greatest risks to human health. These include aflatoxins, ochratoxins, fumonisins, zearalenone, trichothecenes, patulin, and citrinin [40]. Prolonged dietary exposure to these has been linked to cancers and diseases of the kidneys, liver, and immune system. Unfortunately, there is no single solution in the fight against mycotoxins. It is therefore important to take all necessary measures to reduce and control mycotoxin levels in food products. Molds can produce pectinases, which are degrading enzymes with a high potential to alter the taste, odor, and appearance of beverages. In light of the foregoing, it can be unequivocally concluded that the consumption of Tchakpalo in the municipalities of Grand Nokoué exposes consumers to serious risks of foodborne illness. However, these findings contrast with certain studies conducted on other African fermented beverages. For example, Chelule et al. [41] showed that in Mahewu (a South African fermented beverage), enterobacteria gradually disappear during fermentation due to the acidification of the medium. Similarly, Holzapfel [42] demonstrated that lactic fermentation, by lowering the pH and producing bacteriocins, constitutes an effective barrier against pathogens. The persistence of high microbial loads in Tchakpalo therefore suggests: insufficient acidification of the medium, post-fermentation recontamination, or limited dominance of lactic acid bacteria. From a public health perspective, the results confirm that traditional fermented beverages have a dual nature. As Tamang et al. [43] point out, they can have beneficial effects (nutritional value, improved digestibility, supply of functional microorganisms, etc.), but they can also serve as vectors for pathogens when hygiene conditions are insufficient.

The very low level of BPH adoption observed in this study is a key factor contributing to the high levels of microbiological contamination previously identified in Tchakpalo. The hygiene deficiencies noted largely account for the high prevalence of enterobacteria, E. coli, S. aureus, and molds in the Tchakpalo samples analyzed. Indeed, environmental conditions are often identified as major factors contributing to the deterioration of the sanitary quality of commercially available traditional fermented beverages, particularly in West Africa. Similar observations were reported by Tankoano et al. [44] regarding Dolo, a traditional fermented beverage from Burkina Faso. In that study, more than half of the Dolo production facilities visited exhibited major deficiencies regarding handler hygiene, water quality, and protection of products against environmental contamination. The authors established a significant relationship between these poor practices and the high levels of thermotolerant coliforms observed in Dolo. Irregularities in handwashing procedures and the lack of personal protective equipment constitute major hygiene failures. In fact, handwashing generally amounts to little more than rinsing the hands. Very few female producers wash their hands properly with soap before and during production or after handling a contaminated object. Similar results were obtained by Byakika et al. [45] during a study on the influence of food processors’ knowledge, attitudes, and practices on the microbiological quality of Obushera, a traditional fermented sorghum-based beverage in Uganda. These authors highlighted low levels of adherence to good hygiene practices among Obushera vendors, including a lack of systematic handwashing, failure to wear personal protective equipment, and constant exposure of Obushera to the external environment. These authors also reported high levels of contamination by coliforms and staphylococci, confirming the influence of hygiene practices on the product’s microbiological quality. In fact, the hands of food handlers are recognized as one of the main sources of food contamination. Poor handling practices facilitate the transfer of pathogenic microorganisms from the skin, mucous membranes, or the environment to ready-to-eat foods. Handling Tchakpalo simultaneously with banknotes, coins, cell phones, and other objects likely to carry contaminants during sales also constitutes a significant factor in cross-contamination.

Many studies have shown that frequently touched surfaces, particularly mobile phones and coins, can harbor a wide variety of potentially pathogenic microorganisms and contribute to their spread into food [46]. Research by Islam et al. [47] reported bacterial contamination in 99% of coins in circulation in Bangladesh. The authors isolated several potentially pathogenic bacteria, including E. coli, S. aureus, Salmonella typhimurium, and Klebsiella aerogenes, some of which exhibited multidrug-resistant profiles. Additionally, Appiah et al. [48] also demonstrated that banknotes and coins serve as significant reservoirs of pathogenic microorganisms (S. aureus, E. coli, Salmonella spp., and Klebsiella spp.). These findings confirm that coins and banknotes constitute a potential vector for cross-contamination capable of compromising the food safety of items handled simultaneously with them. The simultaneous handling of Tchakpalo with bare hands, along with coins and banknotes, observed among all female producers, constitutes an additional source of contamination for this beverage. The improper practices observed regarding the use of cell phones during Tchakpalo production and sales activities could contribute to cross-contamination of the product. Indeed, Almizwary et al. [49] reported that cell phones are significant reservoirs of potentially pathogenic microorganisms such as S. aureus, E. coli, Pseudomonas spp., and Klebsiella spp. According to these authors, frequent handling of these devices without proper hand hygiene increases the risk of transferring contaminants to ready-to-eat foods and beverages. The authors emphasize that poor hand hygiene after using a phone promotes cross-contamination. Khoothiam et al. [50] also demonstrated the presence of S. aureus on the mobile phones of food vendors in Thailand. The authors also identified strains carrying enterotoxin genes and exhibiting antibiotic resistance, thereby highlighting the potential role of mobile phones in the transmission of pathogenic microorganisms to food.

Although most producers use tap water, the fact that this water is not treated before being used in production and sales represents another potential source of contamination. In fact, during sales, producers add water directly to the Tchakpalo. It should be noted that even when water comes from a distribution network considered safe, its quality can deteriorate during transport, storage, or handling due to inadequate practices that promote microbial recontamination. In Ivory Coast, Seki et al. [51] demonstrated that bacterial contamination of water increased significantly after storage in households. These authors identified storage conditions, container cleanliness, and handling practices as key factors contributing to the deterioration of water’s microbiological quality. The risk is even greater for Tchakpalo producers, who rely on water from wells or boreholes. In fact, groundwater extracted from wells and boreholes can be contaminated by seepage from latrines, septic tanks, domestic wastewater, household waste, or agricultural activities located near the collection points [52]. Olalemi et al. [53] reported the presence of enteric bacteria indicative of fecal contamination in several groundwater sources used for human consumption in Nigeria. In Benin, Behanzin et al. [54] reported the presence of E. coli in 45.83% of the well and borehole water samples analyzed in Sèmè-Podji. More recently, Adanlokonon et al. [55] demonstrated that all wells studied in the municipality of Agbangnizoun were contaminated with bacteria indicative of fecal pollution. These results confirm that untreated groundwater can be a significant source of microbiological contamination of food and beverages, particularly when used as a raw material in the production of Tchakpalo. These findings therefore underscore the need to strengthen quality control of the water used by producers and to promote appropriate treatment before its incorporation into the production process, particularly during the sale of the product.

The observed storage conditions also contribute to an increased risk of microbiological contamination. Storing raw materials and Tchakpalo together on the floor at home, as well as at room temperature for long periods, are major factors contributing to the microbial contamination of Tchakpalo. Mishra et al. [56] recommend separating raw materials and finished products in storage areas to limit the risk of recontamination. Indeed, in artisanal production systems, prolonged exposure of ready-to-eat foods to room temperature can promote the proliferation of certain undesirable microorganisms and accelerate their microbiological spoilage. Sikombe et al. [57] demonstrated that storing Mabisi, a traditional fermented beverage from Zambia, at room temperature led to significant changes in the microbial flora and reduced the product’s microbiological stability, whereas refrigeration significantly improved its shelf life. The joint storage of raw materials and Tchakpalo on the floor at home, combined with prolonged storage at room temperature, also constitutes a potential source of contamination that may further compromise the microbiological quality of this beverage. These observations highlight the importance of improving storage practices to enhance the food safety of Tchakpalo.

In environmental terms, Tchakpalo is exposed to external contaminants. The lack of adequate protection against dust, insects, rodents, exhaust fumes, etc., combined with the proximity to latrines, garbage dumps, wastewater, or domestic animals, creates conditions conducive to cross-contamination. Indeed, food and beverages sold in open environments are particularly vulnerable to contamination from ambient air, dust, and various environmental vectors [58]. Insects and rodents are also recognized as mechanical vectors for many pathogens. Their presence near food can facilitate the transfer of fecal bacteria from waste, excrement, or contaminated water to products intended for human consumption. The complete lack of food safety training, medical supervision, and health inspections reflects a significant lack of institutional oversight in the Tchakpalo sector in Benin. Several studies have shown that food handlers who have not received any food safety training generally exhibit inadequate levels of hygiene knowledge and practices. Indeed, training in food safety is one of the key determinants of improved hygiene practices and reduced risks of food contamination [59].

Furthermore, the lack of regular medical monitoring represents an additional risk factor. Food handlers may be asymptomatic carriers of pathogenic microorganisms that can be transmitted to food during preparation or distribution. In a study conducted among butchers in Almada (Portugal), Oliveira et al. [60] assert that health status is an essential component of GHPs designed to prevent foodborne illnesses. Fekadu et al. [59] demonstrated that establishments subject to regular health inspections generally exhibit a higher level of compliance with food safety requirements than those not subject to any supervision. In our study, the low levels of hygiene observed among Tchakpalo producers. Fekadu et al. [59] showed that establishments with regular health inspections generally exhibit better compliance with food safety requirements than those without any supervision. In our study, the low hygiene levels observed among Tchakpalo producers are one of the main factors explaining the poor microbiological quality of this beverage in Grand Nokoué.

Thus, the low adoption of Good Hygiene Practices (GHP) observed in this study appears to be a major determinant of the degraded microbiological quality of Tchakpalo. This confirms that, in artisanal production systems, improving food safety necessarily involves strengthening the capacities of producers, standardizing processes, and establishing a minimum level of health oversight. It is therefore necessary to implement training, awareness, and health inspection programs to improve the safety of this traditional beer and reduce the risk of foodborne illness among consumers.

This study highlights widespread microbiological contamination of Tchakpalo sold in the municipalities of Grand Nokoué, characterized by a high prevalence and loads of microorganisms indicative of poor hygiene and health risks. The results also show a very low adoption level of good hygiene practices by producers, with major deficiencies concerning personal hygiene, water quality, storage conditions, equipment maintenance, the production environment, and health oversight of the sector. These deficiencies are likely the main factors explaining the poor microbiological quality observed. Beyond the mere presence of potentially pathogenic microorganisms, these results raise the question of the actual pathogenicity of the strains circulating in the Tchakpalo. In this regard, it appears essential to further investigate in order to characterize the virulence factors and antibiotic resistance profiles of the microbial strains, particularly those of S. aureus and E. coli isolated. Such an approach will allow for a more precise assessment of the risks to consumers and a better understanding of the potential role of this traditional beverage in the spread of pathogenic and multidrug-resistant bacteria. In a context marked by the global emergence of antibiotic resistance, these investigations are fully aligned with the One Health approach, which recognizes the close interactions between human health, animal health, and the environment.

The authors thank the Tchakpalo producers involved in this study.

This study was funded by the authors.

d’Avila DOGNON: Conceptualization, Formal analysis, Data curation, Funding acquisition, Methodology, Writing – original draft.

Agossou Damien Pacôme NOUMAVO: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing.

Sènan VODOUHE: Conceptualization, Methodology, Funding acquisition, Supervision, Validation, Supervision, Writing – review & editing.

Lucile ADJOVI: Methodology, Formal analysis, Investigation, Writing - original draft, Funding acquisition.

Balbine GBAGUIDI: Methodology, Formal analysis, Writing - original draft.

Ricardos AGUEGUE: Conceptualization, Writing – review & editing.

Kouakou Romaric KOUAKOU: Conceptualization, Writing – review & editing

Philippe SESSOU: Conceptualization, Writing - review & editing.

Farid BABA-MOUSSA: Supervision