Nuts are one of the commonly consumed snacks but poor handling and storage practices can make them prone to foodborne infections. The study aimed at assessing the microbiological quality and risk assessment for aflatoxins in groundnuts and cashew nuts consumed in selected locations in Nigeria. The moisture content, colony counts, incidence of pathogenic bacteria, aflatoxin contamination, and risk assessment for aflatoxins were evaluated using standard methods. The moisture content and total viable count ranged from 5.00–8.60% and 5.5–89 × 103 cfug−1, respectively, while the fungal count was between 4–24 × 103 and 1.0–4.5 × 102 cfug−1, respectively. Eleven fungal species belonging to 5 genera were isolated from the nuts, with
Nuts are delicacies generally accepted by almost everyone due to their high nutritive value and taste [
Nuts are susceptible to fungal attacks at different stages and time. They may invade the nuts while still on the trees and this usually occurs when the hard shells or pods of the nuts are split open and the seeds attacked by insects or pests which creates room for the fungal spores to access the developing seeds. Other sources of contamination include harvesting, sorting, and washing of the nuts before storage. If the nuts are not properly handled at this stage, it could lead to mould growth especially when seeds are not properly dried to the recommended moisture level before storage [
Both bacteria and fungi are responsible for microbial biodeterioration of food crops leading to food and economic losses, thereby reducing the export value of the food crops [
Mycotoxins are secondary metabolites produced by certain filamentous fungi [
Many countries have set maximum tolerable levels (MTLs) for mycotoxins of human health concern in foods, yet these regulations seem not to confer the required safety on the populace if large amount of foods with high susceptibility to aflatoxins such as maize, groundnuts, and melons are consumed by the population [
Aflatoxins in foods are converted to aflatoxin-8,9-epoxide metabolite in the liver which seems to be responsible for many of the toxic effects in the body [
Despite the availability of literatures on mycotoxin contamination of groundnuts from different parts of Nigeria [
Nuts are among the most common snacks consumed by almost everyone in Lagos State due to the busy nature of its inhabitants. Lagos is a cross metropolitan state with diverse kinds of migrants from within and outside the country who migrated into the state to earn a living. As a result of the busy schedule of the citizens, most people spend more time at work than at home and as a result eat less of homemade foods and more of snacks such as biscuits, nuts, and fried foods. Since nuts are perceived to be healthier and also have pleasant tastes, people have a great tendency to consume them instead of other snacks such as biscuits, chips, and popcorn. Due to the high population density of Lagos, new migrants now settle in Ogun state, a neighboring state to Lagos State, hence the choice of part of the state in the sampling process.
Twenty-seven samples of roasted cashew nuts and 15 samples of raw groundnuts were purchased from 9 and 5 locations in Lagos State and Obafemi Owode Local Government area, Ogun state, Nigeria. Samples were collected at random from three different traders in each location. Thus triplicate samples of roasted cashew nuts were purchased from the 5 zones that make up Lagos State, namely, “Eti-Osa” (“Ikota,” “Obalende,” and “Ajah”), “Badagry” (Market and “Topo”), “Ibeju-Lekki” (“Awoyaya and Lakowe”), “Mushin,” and “Ikeja,” while groundnut samples were purchased from the five major markets (“Ajebo,” “Fidiwo,” “Obafe,” “Ogunmakin,” and “Mowe”) in “Obafemi Owode” Local Government, Ogun State. Samples collected from the three different traders in each location were mixed together to form a composite sample/location. The composited samples were blended aseptically using Marlex Electronic Mixer Grinder (China) and 50 g portions kept in Ziplock envelopes. Samples that were not used immediately were kept at −20°C prior to analysis.
Moisture content was determined out according to AOAC (2000) method. Briefly, samples (5 g) were oven-dried in a preweighed dish at 105°C for 4 h and cooled in a desiccator continuously until a constant weight was obtained. The moisture content was calculated from reduction in weight and expressed as a percentage of the original weight as illustrated below:
The total viable count (TVC) and fungal counts were determined by the dilution plate technique [
The total viable and fungal counts were determined using the following formula:
This was determined using the method described by Adebajo and Diyaolu [
Identification of the fungal isolates was based on the examination of both the macroscopic characteristics (colony colour, morphology, and size) and microscopic characteristics (conidia morphology and size) features of the isolates on specific media and comparison with appropriate identification keys [
Isolates of
Isolates belonging to the
The data obtained from toxigenic screening of
This was determined as described by Atanda et al. [
Aliquots (0.1 ml) of decimally diluted samples were spread plated on Mannitol salt agar at 32°C for 72 h for presumptive staphylococcal count. Colonies surrounded by bright yellow zones were counted. Confirmation of
The presumptive coliforms were counted on MacConkey agar No. 3 at 37°C for 24 h [
Suspected aflatoxigenic fungi from both nuts were selected for aflatoxin analysis using the Enzyme Linked Immounosorbent Assay (ELISA) technique. Commercially available kit Agraquant®, total aflatoxin test kit (4–40 ng/kg), was used for the quantitative analysis of aflatoxins.
Five grams of the ground sample was weighed into a tightly sealed jar to which 25 ml methanol (70%) was added and allowed to settle for 5–10 min. The sample was then filtered using a No. 1 Whatman filter paper. Each of the fifty microlitres of the filtrate and aflatoxin standards in separate dilution wells was mixed with 100
This result was read at 450 nm, using ELISA plate reader. Optical densities of standards (0 ngg−1, 4 ngg−1, 10 ngg−1, 20 ngg−1, and 40 ngg−1) and those of samples were also recorded. A graph of the standard concentration versus optical densities was plotted and extrapolations were made to determine levels of total aflatoxins in the roasted cashew nuts samples.
The exposure levels of the nut consumers were determined as described by by Adetunji et al. [
Risk characterization for genotoxic and carcinogenic compounds such as aflatoxins is based on the margin of exposures (MOEs), which was calculated by dividing the Benchmark dose lower limit (BMDL) for aflatoxins- 170 ngkg−1bwday−1 [
In cases where MOEs were lower than 10,000, a public health concern is indicated which implied that aflatoxin exposures above 0.017 ngkg−1bwday−1 (as obtained by dividing 170 ngkg−1bwday−1 by 10,000) represented a risk of public health concern [
The estimated liver cancer risk for Nigerian adult consumers was calculated for aflatoxins because the ingestion of the toxin can be traced to the development of liver cancer [
The JECFA estimated potency values for AFB1 which corresponded to 0.3 cancersyear−1100,000−1population/ngkg−1bwday−1 (uncertainty range: 0.05–0.5) in HBsAg-positive individuals and 0.01 cancersyear−1100,000−1population/ngkg−1bwday−1 (uncertainty range: 0.002–0.03) in HBsAg-negative individuals [
The data obtained from the analysis were reported as means of three replicates and subjected to analysis of variance (ANOVA) at 95% confidence level to test for significance using SPSS 16.0 software (Windows version, SPSS, IL, USA). Colony counts of nuts were calculated as cfu/g. The relationship between aflatoxigenicity of
The average moisture content of the roasted cashew nuts from Lagos State ranged 5.0–8.6% and were not significantly different (
Roasted cashew nuts from “Ikeja” had the highest TVC count (64.5 × 103 cfug−1) while “Obalende” had the lowest count of 5.5 × 103. Furthermore, a mean TVC count of 29.67 × 103 cfug−1 was recorded for cashew nuts from Lagos State while the mean viable bacterial count for the groundnuts was 71.20 × 103 cfug−1 with “Mowe,” having the highest viable count of 89.00 × 103 cfug−1. The fungal counts also ranged 1.0 × 102 cfug−1–4.5 × 102 cfug−1 with a mean count of 2.30 × 102 cfug−1 for cashew nuts while groundnuts had counts that ranged from 4.00 × 103 cfug−1 for “Fidiwo” to 24.00 × 103 cfug−1 for “Mowe” (Table
Moisture content and colony counts of groundnuts and cashew nuts.
Groundnut | Cashew nut | ||||||
---|---|---|---|---|---|---|---|
Location | Moisture content | Total viable | Fungal count | Location | Moisture Content | Total viable count | Fungal count |
“Ajebo” | 5.1 | 70.0 | 5.0 | “Mushin” | 5. | 37. | 3. |
“Fidiwo” | 6.1 | 58.0 | 4.0 | “Ajah” | 5. | 25. | 4. |
“Obafe” | 5.6 | 78.0 | 24.0 | “Ikota” | 5. | 9. | 1. |
“Ogunmakin” | 6.5 | 61.0 | 4.0 | “Lakowe” | 6. | 13. | 1. |
“Mowe” | 7. | 89.0 | 10.0 | “Awoyaya” | 6. | 49. | 4. |
“Badagry” | 5. | 52. | 1. | ||||
“Topo” | 5. | 9. | 1. | ||||
“Obalende” | 8. | 5. | 1. | ||||
“Ikeja” | 6. | 64. | 2. | ||||
Mean | 6.12 | 71.2 | 9.40 | Mean | 6.04 | 29.67 | 2.30 |
Mean values with the same superscript along the same column are not significantly different (
The incidence of pathogenic bacteria in the roasted cashew nuts is shown in Table
Incidence of pathogenic bacteria in roasted cashew nuts from Lagos State, Nigeria.
Market | | Coliform |
---|---|---|
“Lakowe” | 0 | |
“Topo” | 0 | 1 |
“Obalende” | 0 | |
“Ikota” | 0 | 1 |
“Awoyaya” | | |
“Badagry” | | |
“Ajah” | 0 | - |
“Mushin” | 0 | |
“Ikeja” | 0 | |
Mean values with the different superscripts along the same column are significantly different
Nine fungal species belonging to 5 major genera were isolated from the roasted cashew nuts in Lagos State while 7 were isolated from the groundnut samples (Table
Percentage occurrence of fungal isolates in Nigerian nuts.
Sample type | Location | Fungal Isolate (%) | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
| | | | | | | | | | | ||
Cashew | “Mushin” | ND | 33. | ND | ND | ND | ND | ND | ND | ND | | ND |
“Topo” | ND | ND | ND | ND | ND | ND | 2 | ND | 5 | ND | 2 | |
“Awoyaya” | 2 | ND | ND | 2 | 2 | ND | ND | ND | ND | ND | 2 | |
“Lakowe” | 16. | ND | ND | 33. | ND | ND | 16. | ND | 16. | 16. | ND | |
“Obalende” | 2 | 2 | ND | ND | ND | ND | ND | 2 | 2 | ND | ND | |
“Badagry” | ND | 100 | ND | ND | ND | ND | ND | ND | ND | ND | ND | |
“Ikota” | 2 | 2 | ND | ND | ND | ND | ND | ND | 2 | 2 | ND | |
“Ajah” | ND | ND | ND | ND | ND | ND | ND | ND | ND | 10 | ND | |
| 50 | 37.5 | ND | 12.5 | 12.5 | ND | 25 | 12.5 | 50 | 50 | 25 | |
Groundnut | “Ajebo” | 28.57 | 42.85 | ND | ND | ND | ND | 14.28 | 14.28 | ND | ND | ND |
“Fidiwo” | 83.33 | ND | 16.67 | ND | ND | ND | ND | ND | ND | ND | ND | |
“Obafe” | 4.76 | 4.76 | ND | ND | 80.95 | ND | ND | 9.52 | ND | ND | ND | |
“Ogunmakin” | ND | 100.00 | ND | ND | ND | ND | ND | ND | ND | ND | ND | |
“Mowe” | ND | ND | ND | ND | 87.50 | 12.5 | ND | ND | ND | ND | ND | |
Overall | 60 | 60 | 20 | ND | 40 | 20 | 20 | 40 | ND | ND | ND |
ND: not detected; mean occurrence with same superscript along the same row is not significantly different (
The aflatoxigenicity test carried out on the
Aflatoxigenicity of fungal isolates from cashew nuts from Lagos State on Neutral Red Desiccated Coconut Agar (NRDCA).
Location | Aflatoxin producing potential |
| |
“Ikeja” | Negative |
“Obalende” | Positive |
“Mushin” | Negative |
“Badagry” Market | Positive |
“Ikota” | Positive |
“Mushin” | Positive |
“Lakowe” | Positive |
The aflatoxin concentration of the cashew nuts ranged from 0.1 ngg−1 to 6.8 ngg−1 with “Ikeja” and “Lakowe” having the least (0.1 ngg−1) and highest (6.8 ngg−1) concentrations while the aflatoxin concentration of the groundnuts ranged 29–33.78 ngg−1 (Table
Aflatoxin concentration of nuts.
Cashew nut | Groundnut | ||
---|---|---|---|
Location | Aflatoxin | Location | Aflatoxin |
“Lakowe” | 6.8 | “Ajebo” | 29.0 |
“Obalende” | 5.8 | “Fidiwo” | 33.0 |
“Ikota” | 5.4 | “Obafe” | 32.6 |
“Ikeja” | 0. | “Ogunmakin” | 32.8 |
“Badagry” | 6. | “Mowe” | 33.7 |
Mean concentrations with same superscript along the same column are not significantly different (
A significant (
Correlations analysis between aflatoxins concentration and aflatoxigenic potential of isolate.
Aflatoxin | Aflatoxin | |
---|---|---|
Aflatoxin | ||
Pearson correlation | 1 | −.98 |
Sig. (1-tailed) | .002 | |
| 5 | 5 |
Aflatoxin potential | ||
Pearson correlation | −.98 | 1 |
Sig. (1-tailed) | .002 | |
| 5 | 5 |
The range of exposures of consumers to aflatoxin contamination due to consumption of groundnuts in Obafemi Owode Local Government, Ogun State, and cashew nuts in Lagos State ranged 25.13–29.28 ngkg−1bwday−1 (Table
Risk Assessment due to consumption of groundnuts from Obafemi Owode Local Government Area, Ogun State, Nigeria.
Location | Mean aflatoxin | | | |
---|---|---|---|---|
“Ajebo” | 29.0 | 25.13 | 6.76 | 1.24 |
“Fidiwo” | 33.0 | 28.60 | 5.94 | 1.41 |
“Obafe” | 32.6 | 28.29 | 6.01 | 1.40 |
“Ogunmakin” | 32.8 | 28.50 | 5.97 | 1.41 |
“Mowe” | 33.7 | 29.28 | 5.81 | 1.45 |
Mean | 32.26 | 27.96 | 6.10 | 1.38 |
Values with different superscripts along the same column are significantly different at
Risk Assessment due to consumption of cashew nuts from Lagos State, Nigeria.
Location | Mean aflatoxin | | | |
---|---|---|---|---|
“Lakowe” | 6.8 | 0.24 | 708.33 | 0.01 |
“Obalende” | 5.8 | 0.20 | 850.00 | 0.01 |
“Ikota” | 5.4 | 0.19 | 894.73 | 0.01 |
“Ikeja” | 0. | 0.00 | 0.00 | 0.00 |
“Badagry” | 6. | 0.22 | 772.72 | 0.01 |
| 4.9 | 0.17 | 1000 | 0.01 |
Values with different superscripts along the same column are significantly different at
Furthermore, the HCC prevalence rate for groundnut consumers in Obafemi Owode Local Government, Ogun State, ranged 1.24–1.45 cases 100,000−1peopleyear−1 with a mean value of 1.38 cases 100,000−1peopleyear−1 while there is little or no risk of HCC (0.00–0.01 cases 100,000−1peopleyear−1) for the cashew nut consumers in Lagos State.
The moisture contents of the roasted cashew nuts were above the permissible recommended moisture limit of 5% [
The high moisture contents of the samples may also be as a result of improper packaging or lack of use of good packaging materials. Nuts are colloids materials (hygroscopic), which tend to absorb moisture from the surrounding atmosphere until it reaches equilibrium, Oladapo et al. [
The difference in the microbial load of some of the samples may be due to the varying climatic conditions in the different zones across the two states. “Ikota,” “Ajah,” and “Obalende” (“Eti-Osa” zone) are situated in the humid forest zones of Lagos State. The high humidity in this zone may be a contributing factor to the higher microbial counts of this zone and probably coupled with improper drying, poor handling, poor processing techniques, and storage methods of the nuts. Several authors had reported a dense population of fungi in groundnut samples [
The high incidence of
Although cashew nut is known to be an excellent substrate for microbial growth, outbreak of serious infections through its consumption had rarely been recorded. However, the probable presence of
Staphylococcal food poisoning is also one of the most common causes of reported foodborne diseases. Contaminated equipment and environmental surfaces also can lead to
This report further reemphasizes the suitability of NRDCA for screening of toxigenic fung in foods before further analyses with HPLC or other sophisticated methods. It is also readily more available as it can be easily composited as described by Atanda et al. [
Eighty percent (80%) of the tested samples had aflatoxin concentrations above the European Union (EU) permissible limit of 4
The high concentration of aflatoxins in the nuts especially groundnuts is reflected in the high PDI and MOE values of the nuts. Groundnut consumers in the local government state are therefore at high risk of exposures to aflatoxicosis and liver cancer and other forms of disabilities due to consumption of the contaminated nuts. The evaluation of risk assessments of aflatoxins by international bodies has been based on the carcinogenic potencies developed by JECFA, but, recently, the EC Scientific Panel on Contaminants in the Food Chain [
Although the risk of exposure through cashew nut consumption was low in Lagos State, there is still cause for concern because the smallest molecule of the aflatoxin metabolite could evoke changes in human DNA leading to mutations, selective cellular proliferation, and cancer [
Liver cancer is ranked as the second and third common cancer in men and women, respectively, and the leading cause of deaths in men and the third leading cause of cancer deaths in Africa women [
It was reported that the maximum tolerable limit for aflatoxins permitted in foods for liver cancer risk to be increased by 1 out of 100,000 and 10,000 population in Nigeria in a lifetime is 2 ppb and 24 ppb, respectively [
Several authors had reported the existence of a positive correlation between aflatoxin exposure and HCC in HBV positive individuals [
The inverse correlation between toxigenic isolates and aflatoxin concentrations confirms the fact that certain requirements (suitable temperature, water activity, relative humidity, and storage environment) must be met before the elaboration of aflatoxins in the food substances [
This study showed that the nuts were contaminated with pathogenic bacteria and toxigenic fungi, resulting in aflatoxin contamination of the products, thereby posing health risks to their consumers. There is need for improvement of sanitary practices, regular monitoring, and enforcement of standards for foods sold in open markets in order to reduce the menace of food borne diseases and mycotoxicoses among the populace. In addition, proper packaging materials and good storage practices should be enforced.
The authors disclose no conflicts of interest.
The authors are grateful to the technical staff of the Department of Biological Sciences, McPherson University, Nigeria, to the Department of Animal Health, Northwest University, Mafikeng Campus, South Africa, for their technical support, and also to National Research Foundation South Africa for the financial support towards this research work.