The present overview was intended to evaluate the degree of total aflatoxins and ochratoxin A contamination in different samples of bitter, dark, milk, and white chocolates marketed in Pakistan. For that exploration, two hundred (
Chocolates have become an indispensable part of most people’s daily routine, eaten anywhere and anytime, whatever the occasion is, as a delicious dessert for over thousands of years worldwide especially among young generation [
The high intake of chocolates with high cocoa content has been accelerated as it offers important beneficial health effects but, on the other hand, these products tend to have the highest amount of aflatoxins and ochratoxin A [
Mycotoxins, a diverse and pervasive group of compounds produced by certain species of fungus [
In spite of various research studies on mycotoxins in chocolate and related products from many countries, no information is accessible on mycotoxin contamination in chocolates marketed in Pakistan. Owing to the considerable health hazards linked with them and to ensure a healthy food supply [
The outcomes will give some important references to highlight the danger appraisal and scrutinize the quality of chocolates with respect to mycotoxin contamination with the intention of preventing their accumulation in these products.
Two hundred samples (
HPLC grade acetonitrile, methanol, toluene, phosphate buffered saline (PBS), and glacial acetic acid were purchased from Merck (Germany). Double distilled water was used for the preparation of solutions. Analytical standards of aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), and ochratoxin A were purchased from Sigma-Aldrich. The laboratory glass wares used were kept at 10% (v/v) nitric acid (Merck, Germany) overnight and rinsed several times with ultrapure water before use.
The method for the analysis of mycotoxins was reported in literature [
25 grams of the homogenized chocolate sample and 1 g of NaCl were extracted in 100 mL of methanol : water solution (80 : 20, v/v). The mixture was shaken for 30 min by using Ultra-Turrax Homogenizer (Polytron, Switzerland). Homogenized solutions were filtered through Whatman Number 1 filter paper. 9 mL of the filtrate was added in phosphate buffered saline (24 mL) and applied to an Aflatest WB immunoaffinity column (Vicam, USA) at a flow rate of 1 mL/min. The column was washed with distilled water (30 mL). Aflatoxins were eluted with methanol (2 mL) into the amber vials and evaporated to dryness at 40°C under a stream of N2. The dry residue was redissolved in methanol : water (2 : 3, v/v; 1 mL) and filtered with the help of Millex PTFE 0.45 mm (Millipore, USA) for aflatoxin analysis.
All the measurements of the branded and local samples of chocolates were repeated three times and the data was statistically analyzed by using the software IBM SPSS Statistics, version 20. Regression analyses were applied to find out the coefficient of determination (
Chocolates are the most consumed sustenance in the world owing to their specific flavor and exceptional health benefits, but mycotoxin contamination results in prompt deterioration in their quality. During this research, a total 200 samples of chocolates were analyzed to evaluate the contamination level of total aflatoxins and ochratoxin A by HPLC. The mean recovery % and RSD values at different spike levels are calculated and summarized in Table
Recoveries of mycotoxins from spiked chocolates.
Spike level ( |
Aflatoxin B1 | Aflatoxin B2 | Aflatoxin G1 | Aflatoxin G2 | Ochratoxin A | |||||
---|---|---|---|---|---|---|---|---|---|---|
Mean recovery (%) | RSD (%) | Mean recovery (%) | RSD (%) | Mean recovery (%) | RSD (%) | Mean recovery (%) | RSD (%) | Mean recovery (%) | RSD (%) | |
2 | 91.5 | 4.34 | 84.7 | 3.25 | 77.8 | 4.65 | 79.8 | 2.82 | 92.8 | 1.65 |
4 | 55.6 | 2.72 | 60.9 | 5.12 | 58.6 | 1.40 | 59.2 | 2.54 | 63.7 | 3.48 |
6 | 88.9 | 1.70 | 70.5 | 2.02 | 91.9 | 1.17 | 92.9 | 1.83 | 91.9 | 2.16 |
The studied compounds were recognized by using retention time that matches against those of the calibration standards while the quantification was achieved by means of peak area that match the standards. The HPLC chromatograms of AFs and OTA standards were displayed in Figures
HPLC chromatogram of aflatoxin Standards.
HPLC chromatogram of OTA standard.
Occurrence of total aflatoxins and OTA was noticed in almost all the examined branded chocolate samples. The quantitative analysis of mycotoxin (aflatoxins and OTA) in chocolates showed that, in the branded samples, dark chocolates contained the highest concentration of aflatoxin (2.27
Incidence of total aflatoxins in branded and local chocolate samples.
Branded samples | Local chocolates | |||||||
---|---|---|---|---|---|---|---|---|
Number of branded samples ( |
Contaminated samples | Total AFs |
Concentration range ( |
Number of local samples ( |
Contaminated samples | Total AFs |
Concentration range ( | |
Bitter chocolate | 25 | 19 |
|
0.15–0.98 | 25 | 22 |
|
1.15–2.95 |
Dark chocolate | 25 | 24 |
|
1.02–2.56 | 25 | 25 |
|
1.32–2.62 |
Milk chocolate | 25 | 20 |
|
1.05–2.25 | 25 | 23 |
|
2.02–3.05 |
White chocolate | 25 | 20 |
|
0.08–0.2 | 25 | 21 |
|
2.08–3.85 |
Incidence of OTA in branded and local chocolate samples.
Branded samples | Local chocolates | |||||||
---|---|---|---|---|---|---|---|---|
Number of branded samples ( |
Contaminated samples | OTA |
Concentration range ( |
Number of local samples ( |
Contaminated samples | OTA |
Concentration range ( | |
Bitter chocolate | 25 | 21 |
|
0.15–1.28 | 25 | 25 |
|
0.05–1.3 |
Dark chocolate | 25 | 25 |
|
0.02–1.05 | 25 | 25 |
|
2.05–3.87 |
Milk chocolate | 25 | 23 |
|
0.05–2.05 | 25 | 23 |
|
1.89–2.67 |
White chocolate | 25 | 21 |
|
2.08–4.12 | 25 | 24 |
|
1.32–4.15 |
HPLC chromatogram of branded chocolate sample.
HPLC chromatogram of local sample is presented in Figure
HPLC chromatogram of local chocolate sample.
HPLC chromatogram of chocolate sample.
A comparison between local and branded samples of chocolates disclosed that the local samples contained total aflatoxins and OTA in greater concentration as compared to the branded samples and is displayed in Figures
Concentration of total AFs in branded and local samples of chocolates.
Concentration of OTA in branded and local samples of chocolates.
The incidence of ochratoxin A and aflatoxins in the chocolate has been reported in the last era throughout the world and is an attention-seeking topic of concern for researchers. The cooccurrence of aflatoxins and ochratoxin A in the chocolate samples was reported in studies carried out in Brazil. The authors observed aflatoxins in 80% of the examined samples. A strong relationship was observed between the occurrence of ochratoxigenic fungi and the ochratoxin A content in the chocolate samples [
The results of current research clearly exhibited that the level of total aflatoxins and ochratoxin A contamination in chocolates is quite high in Pakistan and comparable with the literature reported [
The high level of mycotoxin contamination in chocolate is a serious health hazard and the influenced authorities should consider this issue of contamination urgently. For consumer safety, necessary measures must be reinforced in all processing phases to take out the level of mold defilement. Moreover, maximum limits of mycotoxins for all food products should be specifically established in the country. Publications are accessible with the mycotoxins contamination of chocolates in different realms [
The additional researches must be required in order to collect enough records on the existence of mycotoxins and with the purpose of reducing the amount of these in different chocolate bars that are the most consumed in the community.
The present study provides the first description of the mycotoxin contamination in chocolates from Pakistan. Taking all the information into consideration, it is concluded that the defilement of chocolates with mycotoxins is quite high in local samples in contrast with branded chocolates. So it is necessary to create awareness among people about the contamination caused by mycotoxins. To guarantee the safety of consumers, the concerned administrative authorities are recommended to check this adverse issue of food contamination and quality control method of food ought to be practiced.
The authors declare that there are no conflicts of interest regarding the publication of this paper. It is confirmed that Department of Toxicology, Quality Operations Laboratory, University of Veterinary and Animal Sciences, did not provide any funding and did not lead to any conflicts of interest regarding the publication of this manuscript. There are no any other possible conflicts of interest in the manuscript.
The authors acknowledge the Department of Toxicology, Quality Operations Laboratory, University of Veterinary and Animal Sciences, for providing Laboratory facilities for the analysis of mycotoxins.