Preliminary study on hazards and critical control points of kokoro, a Nigerian indigenous fermented maize snack

Description of production area

Imashayi, Joga and Iboro communities are places with a very small population in Yewa
North Local Government Area situated at 7°14?00?N; 3°02?00?E. It has an area of 2,087 km² (806 sq mi) and a population of 181,826 as at the 2006 census. Imashayi is located
at Latitude 7.0833333/7°4?59.9982?, Longitude 3.0833333/3°4?59.9988? while Joga is
positioned between Imashayi and Iboro which occupies Latitude 7.1/7°5?59.9994?, Longitude
3.1/3°6?0.0?. The three communities are agrarian with an expanse of fertile soil.
Ilaro is the host community to a Federal polytechnic; Ibese is the host community
to Dangote Cement Company (Figure 1). Other cities, towns and places close to these study communities include Joga-Orile
and Awaiye. The closest major cities include Abeokuta, Shagamu, Ikorodu and Lagos
(GoMapper 2013]).

Figure 1. Map of Yewa North showing study area. Source: Ministry of Lands, Surveys and Urban
Development, Ogun (2008).

Sample collection

Sixty samples of kokoro, twenty each from Ibadan (Oyo State), Sango-Ota (Ogun State)
and Iyana Ipaja/Oshodi (Lagos State) were purchased from ten different food vendors
selected at random in each State. Sampling was done during both rainy and dry seasons.
Samples were also collected from local producers at each stage of production and of
finished product. Samples purchased from food vendors were transported in cellophane
packs wraps as purchased and were kept in ice packs before reaching the laboratory
under 24 h. In the same vein, samples from each steps of production were transported
in sterile specimen containers held in ice packs. All samples were taken to the laboratory
within the same day of collection for microbiological analysis.

Traditional kokoro production

Preparation involves the use of maize (Zea mays). The grains are washed and boiled for about an hour (depending on the hardness/how
dry the grains are). The water used for boiling is decanted and grains steeped overnight
to ferment. Fermented grains are sieved from steep water and milled without the addition
of water. Salt (NaCl) and wet milled onion (Allium cepa) are added and mixed with the milled grains. The mixture is molded into small balls
about the size of a medium sized orange. Each ball/mold by experience is estimated
to produce a specific quantity of the final product. The small scale producers of
kokoro can buy the balls from large scale producers at determined price per dozen
to produce the final product at profit after sales. The balls are cut into small sizes
and kneaded to produce thin circular or straight snack about 24 cm long, these are
slightly fried in ground nut oil for 2–4 min to a light-brown semi-finished product.
They are covered in basket/basin overnight before a second (final) frying for 1–2 min
to make the product ready for consumption as a light-brown/dark-brown crispy snack.
The products are packaged in transparent cellophane or displayed openly for sale (Figures 2, 3).

Figure 2. Flow diagram of kokoro preparation and handling.

Figure 3. Kokoro after final processing.

Hazard analysis

The hazard analysis and critical control point’s evaluations were conducted on the
production protocol of three large scale kokoro producers, one from each of the three
communities. Selection was based on the interest of the producers to participate in
the exercise in which details of the procedures involved were explained by the researchers
during the preliminary visits to the communities. Observations were made of the raw
materials used, the personnel involved, the equipment and utensils, the environment,
kokoro production and packaging practices to identify sources of actual and potential
contaminants. Samples of raw materials and swabs of food contact surfaces were taken
at different stages of production (immediately after boiling, during steeping, milling,
kneading, after first and second frying and when on display for sales). The thermometer
was cleaned in 70% ethanol and dried before use to avoid being a source of contamination
to the samples. The analysis also included measuring the temperature–time exposure
period and determination of pH during steeping, before frying and after production.
The pH was measured using pH and temperature meter (ADWA-AD 1040).

A schematic diagram of kokoro production (Figure 2) was made based on the observations made and questions asked during processing. Previous
researchers (Creswell and Clark 2011]; Meysenburg et al. 2014]) used different types of charts and maps for monitoring the production processes
in some food products. Potential sources of contamination, critical control points
that needed to be monitored and the likelihood of microbial survival, multiplication
or destruction were noted as described by Oranusi et al. (2003]).

Isolation and enumeration of microorganisms

Ten gram of each sample were blended with sterile warring blender and homogenized
in 90 mL sterile peptone water. The raw grains where however not blended but soaked
for 10–20 min and washed out by vigorous agitation. The resultant homogenate was diluted
10^?2 to 10^?3 for the heat treated (boiled and fried) samples and 10^?4 to 10^?7 for the raw materials. From the appropriate dilutions, aliquot 0.1 mL was spread
plated in triplicate onto different media prepared based on the manufacturer’s instruction.
Plate count agar (PCA), eosin methylene blue (EMB) agar and (PDA) potato dextrose
agar (all from Biolab, India) were inoculated for total aerobic plate count (TAPC),
coliform count and fungal count, respectively. Bacillus cereus medium and mannitol salt agar (both from Oxoid, England) were inoculated for isolation
of B. cereus and S. aureus while Salmonella–Shigella agar (Fluka, Germany) were inoculated after 24 h pre-enrichment of sample homogenate
in Selenite F-broth, for isolation of Salmonellae. All inoculated plates were incubated
at 37°C for 24–48 h for colony formation and enumeration. Exception to this incubation
protocol was PDA plates that were incubated at 29 ± 2°C for 72–120 h and a plate of
EMB incubated at 44°C and 24–48 h for faecal coliform organisms. Colonies formed at
the expiration of incubation period were counted using digital colony counter (Gallenkamp,
England). Counts were expressed as cfu/g of sample. Samples of swabs of food contact
surfaces and water were cultured for the presence of coliform organisms, S. aureus, B. cereus and other organisms concerned with food safety. Characteristic discrete colonies
on the different media were isolated, and purified by repeated sub-culturing on Nutrient
agar (Oxoid). Pure cultures were stored on agar slants at 4°C for further characterization.
For the confirmation of coliform organisms, the method as described by Oranusi et
al. (2003]) was adopted. Colonies on EMB were inoculated into lactose broth in test tubes with
inverted Durham (bell) tubes. Incubation was done for 24–48 h at 37 and 44°C. Gas
production and/or color change of dye constituted a positive presumptive test. The
broth was inoculated onto EMB plates for 37°C incubation. Typical colonies on EMB
appearing bluish black with greenish metallic sheen characteristic of E. coli or brown mucoid colonies characteristic of E. aerogenes that are Gram negative and non-spore bearing confirmed the presence of coliform organisms.
Isolates were stored on agar slants at 4°C for further characterization.

Identification of isolates

Isolates on slants were purified by repeated sub-culture on nutrient agar. Pure cultures
were identified based on standard methods of Jolt et al. (1994]). Identification of characteristic bacteria isolates was based on colonial morphology,
microscopy and biochemical tests using Biomerieux^® Sa API biochemical test kits. Fungal isolates were identified based on their morphology,
microscopy and pigmentation on media with reference to standard identification keys
and atlas (Tsuneo 2010]).

Proximate analysis of samples

The chemical compositions of the samples were determined according to the procedure
outlined by the Association of Official Analytical Chemists (AOAC 1980]). The kokoro samples were analyzed for moisture content, carbohydrate, protein, lipid,
ash and fiber. Moisture content was obtained by drying samples in moisture dish in
an oven at 105°C until constant weights was obtained. For Ash content, pre-dried samples
obtained from moisture content analysis were ashed in furnace at 550°C overnight.
Crude protein value was obtained from nitrogen which was earlier determined by MicroKjedalh
method and by multiplying by 6.25 (conversion factor for nitrogen to protein). Crude
fat was obtained by exhaustively extracting 2.0 g of each sample in a Soxhlet apparatus
using petroleum ether (b.p. 40–60°C) as the extractant. Determination of crude fibre
was done by trichloroacetic acid method (Oladipo and Jadesimi 2012]) while carbohydrate content was obtained by difference from the combined percent
of moisture, protein, ash and fat from 100 (Nwanze et al. 2006]).

Statistical analyses

The statistical analysis of the data was done using the SPSS 20.0 software for windows
(SPSS 2011]). The values obtained were confirmed using one-way ANOVA at 0.05 level of significance.
Further test on those found to be significant was done using Duncan multiple range
tests (DMRT).

Significance and impact of study

Kokoro is an ancient snack valued especially within the South western and North central
regions of Nigeria comprising of over ten states. It is widely distributed as well.
However, this study has shown the various critical and hazard points towards ensuring
the safety of this product for consumers. The result of this study is significant
as it will be implemented in the bid to commercialize kokoro production in Nigeria
as well as ensuring safety and proper hygiene to safeguard public health. It will
further contribute to the development of food production database in Nigeria.