Assessment of the Bacteriological Quality of Massa Sold in University of Abuja

Masa, a traditional Nigerian food made from fermented grains, is widely popular across various regions, particularly among students at the University of Abuja. Despite its popularity, the safety of Masa sold by street vendors is a significant public health concern due to poor hygienic practices during its preparation and handling, which can lead to contamination by pathogenic microorganisms. To assess the bacteriological quality of Masa within the University of Abuja, twelve samples were collected from four vendors on campus and analyzed using standard microbiological methods. The findings revealed a substantial bacterial presence, with total bacterial counts ranging from 2.0×10⁸ to 3.0×10⁸ CFU/g and total coliform counts between 1.5×10⁶ and 2.7×10⁶ CFU/g. The study identified three bacterial species: Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus, which are known to pose serious health risks. The presence of these pathogens underscores the potential danger of consuming Masa from these vendors, as the high bacterial load renders the samples unsafe for consumption. This research highlights the urgent need for improved sanitary measures in the preparation and vending processes of Masa to safeguard public health and ensure the safety of consumers.

Masa is a cereal-based, spontaneously fermented street food that is commonly prepared and sold by vendors in public places for direct consumption, often served roadside (Badau et al., 2018; Trafialek et al., 2017). This ready-to-eat cake has become increasingly popular in developing countries like Nigeria due to its affordability, convenience, and quick availability, particularly in urban areas (Badau et al., 2018). Masa is usually made from maize (Zea mays), millet (Pennisetum glaucum), or rice (Oryza sativa), with the choice of cereal depending on the producer's preference. It is widely consumed as an accompaniment to breakfast porridges, especially in Nigeria's northern and southern regions (Owuzu-Kwarteng and Akanbada, 2014). Consumers highly value Masa for its brown, crisp edges and mild sour taste, which are considered key quality attributes (Ayo et al., 2014). The physical characteristics of Masa, such as thickness, weight, volume, and sensory properties like flavor, aroma, texture, and sourness, can vary significantly based on the type of cereal grain used in its production (Igwe et al., 2013).

Street food vending, including the sale of Masa, is a significant source of employment in many developing countries and contributes notably to household incomes (Choudhury et al., 2011; Lucca and Torres, 2006; Biswas et al., 2010; Feglo and Sakyi, 2012). According to the United Nations Food and Agricultural Organization (2012), approximately 2.5 billion people consume street food every day, underscoring the importance of this sector in global food systems (Da Silva et al., 2014). However, ensuring the safety of street food is a major challenge for producers, consumers, and public health officials. Foods that are heavily contaminated with pathogenic or spoilage bacteria are not only unpalatable but also pose significant risks of foodborne illnesses (Hertanto et al., 2018). The presence of pathogenic bacteria in Masa, such as those that cause foodborne diseases, raises serious public health concerns (Nazni and Jaganathan, 2014).

Foodborne illnesses can result from contamination by bacteria, parasites, toxins, and viruses. For example, Salmonella and Escherichia coli, two common pathogens, are responsible for significant mortality, with 52,000 and 37,000 deaths annually, respectively (WHO, 2015). The high risk of contamination by pathogens like Escherichia coli, Salmonella, and Shigella in street foods such as Masa is particularly alarming because these bacteria can cause severe foodborne illnesses, posing serious health risks to consumers. Given the widespread consumption of Masa and its potential for contamination, ensuring the safety of this popular street food is a critical public health priority. The need for strict sanitary measures during the preparation and handling of Masa is evident to mitigate these risks and protect consumer health.

Study Area

The study will be carried out at the University of Abuja, situated in the Federal Capital Territory (FCT), Abuja, Nigeria. Geographically, the university lies between latitude 9.0765° N and longitude 7.3986° E, located on the plains of the Guinean savannah in central Nigeria (University of Abuja Postgraduate Handbook, 2015).

Sample size

Twelve (12) samples will be randomly collected from various Masa vendors at different locations within the University of Abuja.

Sample Collection

A total of twelve (12) Masa samples were collected from four (4) vendors at the University of Abuja, with three (3) samples taken from each of the four different vendors. These samples were purchased and collected in sterile containers, then transported to the University of Abuja's Microbiological Laboratory for analysis. 

Materials and reagents

Culture Media

Salmonella Shigella agar, Nutrient agar and Eosin-methylene blue agar.

Glass Wares

Microscopic Slides, Pipettes, Conical Flasks, Test tubes, Petri dishes

Reagents

Distilled water, Buffered peptone water and hydrogen peroxide (H2O2)

Serial dilution

Serial dilution was conducted using test tubes, each containing 9ml of distilled water. The syringes used were sterilized between uses by heating the needle over a Bunsen burner. One gram (1g) of finely crushed Masa sample will be added to the first test tube (10⁻¹), which was gently swirled to mix the sample with the water. One milliliter (1ml) of this mixture will be transferred to the second test tube (10⁻²), and the process was repeated, transferring 1ml from the second to the third test tube (10⁻³). This procedure was continued to produce a series of dilutions with progressively lower concentrations.

Preparation of media

Every Media were prepared following the manufacturer’s instructions, including Nutrient Agar, Eosin-Methylene Blue Agar, and Salmonella Shigella Agar. Each medium was autoclaved at 121°C for 15 minutes, then poured into Petri dishes and allowed to cool and solidify. These media were utilized for isolating, identifying, growing, and maintaining microorganisms.

Isolation and Identification of bacteria

The bacteria were isolated and enumerated by culturing them on both selective and non-selective media. Nutrient agar was used for total viable bacterial count (TVBC), while total coliform (TC) and fecal coliform (FC) counts were determined using nutrient broth. After 24 hours of incubation at 37°C, bacterial colonies were identified by their color on each agar plate. Pure cultures were then subjected to further characterization through biochemical tests, including Gram staining, catalase, citrate, and oxidase tests.

Biochemical/Confirmatory tests

Gram Staining

The procedure will be performed. A loopful of sterile distilled water was placed on a clean, grease-free slide using a sterile inoculating loop, and an isolate from the culture was mixed with the water. The smear was air-dried and then gently heat-fixed by quickly passing it over a Bunsen flame. The slide was first stained with crystal violet for 60 seconds, rinsed with water, and then treated with Lugol’s iodine for 30 seconds before rinsing again. It was decolorized with 70% alcohol for 10 seconds, counterstained with Safranin for 60 seconds, and allowed to dry. The slide was then examined under an oil immersion objective. Gram-positive cells appeared purple, while Gram-negative cells were red or pink.

Catalase test

The catalase test was performed on all bacterial isolates. A drop of hydrogen peroxide was placed on a glass slide, and colonies were picked with a sterile loop and added to the drop. Bubbles were observed, indicating the presence of catalase. Colonies that produce catalase break down hydrogen peroxide into water and oxygen, which is visible as air bubbles escaping from the solution.

Citrate test

Unique colonies were isolated using a sterilized wire loop and streaked onto citrate agar plates, taking care not to disrupt the agar. The plates were then incubated for 24 hours. Data was collected by observing any color changes that occurred on the agar.

Methyl Red Test

Five milliliters of glucose phosphate broth (comprising 1g glucose, 0.5% KH2PO4, 0.5% peptone, and 100mL distilled water) were dispensed into clean test tubes and sterilized. The tubes were then inoculated with the test organisms and incubated at 37°C for 48 hours. After incubation, a few drops of methyl red solution were added to each test tube, and the color change was observed. A red color indicates a positive reaction.

Oxidase Test

This test involved applying 2–5 drops of freshly prepared oxidase reagent (p-amino dimethylaniline) onto filter paper. The suspected organism was then picked up with a sterile wire loop and mixed with the oxidase reagent. A positive result was indicated by a color change to deep purple, while the absence of a color change indicated a negative result.

Coagulase Text

This test was conducted to distinguish Staphylococcus aureus from other Staphylococcus species. A clean, grease-free slide was used, and 2-3 drops of normal saline were added. The suspected organism was mixed with the saline, followed by the addition of 1-2 drops of plasma. The slide was then gently rocked. The presence of agglutination indicated a positive result, while the absence of agglutination indicated a negative result.

Indole Test

An isolate was inoculated in a test tube containing 3mL of sterile tryptone water and incubated at 37°C for 24 hours. After the incubation period, 0.5mL of Kovac’s reagent was added and the tube was gently shaken. This process was repeated for each isolate obtained from the samples. The development of a red color on the surface layer within 10 minutes indicated a positive result, while the absence of color change indicated a negative result.

Data analysis

Data were collected and presented in tables. The recorded parameters included:Frequency of occurrence of bacterial isolates, Prevalence of bacterial isolates, Bacterial load in each sample, Comparison of bacterial contamination across different vending locations, Identification of the most commonly isolated bacterial species.

Plate count

Table below presents the total bacterial count (TBC) and total coliform count (TTC) of bacterial isolates collected from four different vendors at Adelabu Market, located within the University of Abuja Main Campus. The TBC of bacterial isolates from the vendors ranged between 2.0 x 108 and 3.0 x 108 CFU/g, while the TTC varied from 1.5 x 106 to 2.7 x 106 CFU/g.

Table 1: Colony Forming Units of Bacterial Isolates (CFU/g)

KEYS: TBC = Total Bacterial Count, TCC = Total Coliform Count

Morphological characteristics of bacterial isolates

The morphological characteristics of the bacterial isolates are detailed in the Table below. All three isolates displayed an entire edge. Isolate A was cocci in shape, while Isolates B and C were rod-shaped. Regarding color, Isolate A appeared creamy, Isolate B was white, and Isolate C exhibited a blue hue on both general purpose and differential agar. In terms of elevation, Isolates B and C were flat, whereas Isolate A was raised. Optically, Isolate B was translucent, while Isolates A and C were opaque.

Table 2: Morphological characteristics of bacterial isolates

KEYS: I= Isolate, A= Isolate one, B= Isolate two, C= Isolate three

Biochemical characteristics

Two isolates, labeled A and B, were both Gram-positive and tested positive for citrate. Isolate C, on the other hand, tested negative for both indole and methyl red. Isolate A was positive for methyl red but negative for oxidase, while Isolates B and C were oxidase-positive, as summarized in the Table below.

Table 3: Biochemical Tests of Probable Organisms

Keys: I= Isolate, + = Positive, - = Negative,CA= Catalase test,GR= Gram reactionIN= Indole,MR= Methyl Red,CI= Citrate utilization test,OX= Oxidase test,CO= Coagulase test

Distributions of occurrence of microbial isolates

The frequency of occurrence for each identified isolate based on the microbiological examination is detailed in the Table below In Adelabu Market, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus were consistently found across all four Masa vendors, with a total frequency of 21 occurrences for each of these bacteria, as shown in the Table below.

Table 4: Distribution of occurrence of microbial isolates

Frequency of Occurrence of microbial isolates from Masa

The Table below shows that Bacillus subtilis exhibited the highest frequency of occurrence, appearing nine times, which corresponds to 42.9% of the total occurrences. In contrast, Pseudomonas aeruginosa had the lowest frequency, appearing four times, accounting for 19.0% of the total occurrences.

Table 5: Frequency of Occurrence of microbial isolates from Masa

Key: % = Percentage