A survey of the incidence and level of aflatoxin contamination in a range of locally and imported processed foods on Malawian retail market

Food Control 39 (2014) 87e91

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A survey of the incidence and level of aflatoxin contamination in a range of locally and imported processed foods on Malawian retail market Limbikani Matumba a, b, *, Maurice Monjerezi a, Timothy Biswick a, Jonas Mwatseteza a, Wilkson Makumba b, David Kamangira c, Alfred Mtukuso c a b c

University of Malawi, Department of Chemistry, Chancellor College, P.O. Box 280, Zomba, Malawi Chitedze Agricultural Research Station, P.O Box 158, Lilongwe, Malawi Department of Agricultural Research Services, P.O. Box 30 779, Lilongwe 3, Malawi

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 May 2013 Received in revised form 12 September 2013 Accepted 17 September 2013

Samples of locally (Malawian) processed and imported maize- and groundnut-based food products (peanut butters, roasted groundnuts, peanut based therapeutic foods, instant baby cereals, maize puffs and de-hulled maize flour) were collected from popular markets of Lilongwe City, Malawi. The samples were analysed in order to determine the frequency and extent of aflatoxin contamination, using immuno-affinity column and reversed-phase liquid chromatography with post-column photochemical derivatization and fluorescence detection. No aflatoxins were detected in all samples of imported baby cereal and locally processed de-hulled maize flour. However, all locally processed maize based baby foods had aflatoxins above EU maximum tolerable level of 0.1 mg/kg. In 75% of locally processed maize puffs, aflatoxins were detected at levels of up to 2 mg/kg. Peanut based therapeutic foods had aflatoxin level between 1.6 and 2.9 mg/kg, exceeding the EU tolerable maximum level (0.1 mg/kg) set for food for health purposes. Locally processed peanut butters had aflatoxins levels in the range of 34.2e115.6 mg/kg, which was significantly higher than their imported counterparts (<0.2e4.3 mg/kg). Samples of locally processed skinned and de-skinned roasted groundnuts had aflatoxins in range of 0.5e2.5 mg/kg and 0.6e36.9 mg/kg, respectively. These results highlight the need for rigorous monitoring of aflatoxins in commercially available processed products in order to reduce likely health risks associated with dietary aflatoxin intake. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Aflatoxins Contamination Processed Maize Groundnuts HPLC

1. Introduction Several maize- and groundnut-based ready-to-eat food products are commercially available, some of which are promoted as infant/baby and therapeutic foods. However, maize and groundnuts are prone to pre-harvest and post-harvest contamination with aflatoxins. Aflatoxin contamination has been reported in samples of maize and groundnuts from Malawi (Matumba, Monjerezi, Chirwa, Lakudzala, & Mumba, 2009; Monyo et al., 2012) and across Africa (Bankole, Schollenberger, & Drochner, 2006; Sibanda, Marovatsanga, & Pestka, 1997; Shephard, 2003, 2008). There is potential that the contaminated raw materials pass on aflatoxins to the final product (Bullerman & Bianchini, 2007). * Corresponding author. University of Malawi, Department of Chemistry, Chancellor College, P.O. Box 280, Zomba, Malawi. Tel.: þ265 999682549. E-mail address: [email protected] (L. Matumba). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.09.068

Aflatoxins have been shown in many studies to be immunosuppressive, teratogenic, mutagenic, carcinogenic, genotoxic and hepatotoxic (Fung & Clark, 2004; Hendrickse, 1997; IARC, 1993; Peraica, Radic, Lucic, & Pavlovic, 1999; Preisler, Caspary, Hoppe, Hagen, & Stopper, 2000; Wangikar, Dwivedi, Sinha, Sharma, & Telang, 2005; WHO, 1998) to humans and animals, depending on the duration and level of exposure. Maize- and groundnut-based ready-to-eat food products may constitute aflatoxin exposure risk, particularly because they are consumed by infants/babies, malnourished children and people living with HIV/AIDS (Manary, Ndkeha, Ashorn, Maleta, & Briend, 2004; Ndekha, Manary, Ashorn, & Briend, 2005; Sandige, Ndekha, Briend, Ashorn, & Manary, 2004). It has been postulated that a synergy exists between HIV and AFB1 in AIDS development (Jiang et al., 2008; Jolly et al., 2013). In addition, aflatoxins cause decreased transport of soluble nutrients (Fink-Gremmels, 2008), disrupt protein, carbohydrate and lipid metabolism (Cheeke & Shull, 1985), alter growth

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diluent was passed through AflatestÒ affinity column as described earlier. For both maize- and groundnut-based foods, the columns were then washed with 23 mL of water/methanol (85/15, v/v) to remove maize intrinsic compounds and finally the aflatoxins were selectively eluted with 1 mL of 100% methanol followed by 1 mL of 100% HPLC water. The total volume of the eluent (2 mL) was mixed using a vortex mixer for 30 s after which the sub-sample was ready for HPLC analysis. In case the total aflatoxins exceeded 25 mg/kg, a sample was re-analysed ensuring that only 0.1e0.2 g sample equivalent was passed through the affinity column.

factor expression and impair child growth (Gong, Turner, Hall, & Wild, 2008; Khlangwiset, Shephard, & Wu, 2011). There is however, limited knowledge about the frequency and levels of aflatoxins in processed products in Malawi. In this context, this study reports, for the first time, on the occurrence of aflatoxins in industrial processed food products marketed in Malawi. The data presented in this study may be useful in facilitating improved food regulation and dietary risk management in Malawi. 2. Materials and methods

2.2.3. Aflatoxins determination using HPLC-FLD Determination of aflatoxins was done using Agilent 1200 Series HPLC System (Agilent, Waldbronn, Germany) consisting of G1322A degasser, G129A autosampler, G1330B thermostat, CY1311A quaternary pump, G1316A temperature controller and G1321A fluorescence detector (FLD). Chromatographic separation was achieved using ZORBAX EclipseÒ XDB-C18 column (150 mm  4.6 mm I.D., 5 mm particle size), protected by C18 security guard cartridge (4  3 mm i.d.) (both supplied by Agilent Technologies). An isocratic mobile phase consisting of water/methanol/acetonitrile (55/35/10, v/v/v) was used at a flow rate of 1 mL per min. The column oven temperature was maintained at 30  C and the injection volume was 40 mL for both standards and samples. Post-column derivatization (PCD) was achieved using a photochemical reactor (LCTech UVE, Dorfen, Germany). Fluorescence excitation and emission wavelengths were set at 365 and 440 nm, respectively. Retention times of AFG2, AFG1, AFB2 and AFB1 were 5.5, 6.4, 7.6 and 9.0 min respectively. Data acquisition and processing was achieved using chromatographic software (ChemStationÒ). Aflatoxin determination in samples was based on a five point external standard calibration curve, using a mixture of aflatoxin standards (AFB1 and AFG1, each ranging from 0.5 to 15 ng/mL, and AFB2 and AFG2, ranging from 0.125 to 3.755 ng/mL). Calibration curves, with strong regression (R2  0.995) were classified as valid. Quality control in the aflatoxin analysis was achieved using naturally contaminated reference materials (Product #: TR-A100, Batch #: A-C-268, R-Biopharm AG, Darmstadt, Germany). Five samples of each product type, spiked with 12.5 mg/kg total aflatoxins, were used to assess recovery and recoveries between 70 and 110% were classed as valid. The results were corrected by mean recovery rates obtained from the recovery experiments (Table 1). Limits of detection (LODs) and quantification (LOQs) were determined at a signal-to-noise (S/N) ratio of 3/1 and 10/1, respectively, for each food category separately. For data evaluation, half the values of LOD or LOQ of the respective category were assigned to values below the LOD and between the LOD and LOQ, respectively. Since aflatoxin concentration in the samples was not normally distributed, data were log transformed before statistical analysis. The statistical analysis was performed on SPSS version 16 (SPSS inc., Chicago, IL, USA). P values < 0.05 were considered statistically significant.

2.1. Food samples A total of 125 samples of local and imported food products were purchased from the market in Lilongwe City, Malawi, in December 2012. The local products were: 14 cans of peanut butters; 15 packs of de-skinned roasted groundnuts; 9 packs of un-skinned roasted groundnuts; 6 cans of peanut based therapeutic foods, 36 packs of instant baby cereals, 12 packs of maize puffs; 15 packs of de-hulled maize flour samples. Imported products included 7 packs of instant baby cereals and 11 cans of peanut butters. 2.2. Aflatoxin analysis by HPLC-FLD method 2.2.1. Chemical and reagents Acetonitrile, methanol and HPLC-grade water were supplied by Merck (Darmstadt, Germany). 5.0 mg/mL total aflatoxins (aflatoxin B1 (AFB1)/aflatoxin B2 (AFB2)/aflatoxin G1 (AFG1)/aflatoxin G2 (AFG2) (4/1/4/1, v/v/v/v)) were purchased from Trilogy Analytical Laboratory (Lot # 120316-090, Washington, MO, USA). After reconstitution in 10 mL acetonitrile, the standard solution was kept securely at 15  C, wrapped in aluminium foil to avoid photodegradation and held for 6 months. Working aflatoxins standard solutions were made by diluting the stock solution in methanol/ water (50/50, v/v). 2.2.2. Extraction and clean-up Modified AflatestÒ immuno-affinity procedures for extraction and clean-up of aflatoxins in cereals and nuts were used (VICAM, 1999). For maize-based samples, sub-samples (30 g) of finely ground products (to pass sieve #20) were added to 3 g of NaCl and extracted with 60 mL of methanol/water (80:20, v/v) and blended at high speed for 2 min. The extract (10 mL) was diluted four folds with HPLC grade water and filtered twice (firstly through a coarse fluted filter, and secondly through a glass filter) before passing a 20 mL (2 g sample equivalent) of the diluent through AflatestÒ affinity column (VICAM, Watertown, MA, USA). For all groundnut-based products, sub-samples (15 g) were added to 3 g of NaCl and extracted with 75 mL of methanol/water (70:30, v/v), blended at high speed for 2 min, the filtrate diluted two folds with water and re-filtered through a glass-fibre filter. A 30 mL (2 g sample equivalent) of the

Table 1 Recovery percentages of the aflatoxins and limit of quantifications (LOQs) for tested products. Aflatoxin

Extruded maize-soybeans mixture Recovery % b

AFB1 AFB2 AFG1 AFG2 a

x

RSD

86 83 85 82

3 4 5 4

LOQa (mg/kg) 0.5 0.2 0.6 0.3

Maize flour Recovery % b

x

RSD

92 88 83 74

5 4 4 3

Maize puffs LOQa (mg/kg) 0.7 0.3 0.7 0.3

Recovery % b

x

RSD

78 71 75 70

4 3 5 4

Peanut butter LOQa (mg/kg) 1.0 0.3 0.7 0.5

Recovery % b

x

RSD

96 93 84 76

2 3 4 3

LOQa (mg/kg) 0.5 0.2 0.6 0.3

Limit of quantifications (LOQs) determined at a signal-to-noise (S/N) 10/1. Mean Recovery rates were determined from five (5) analyses of spiked blank food (each product type) with AFB1 and AFG1, each at 5 mg/kg and AFB2 and AFG2, each at 1.25 mg/kg. b

L. Matumba et al. / Food Control 39 (2014) 87e91

3. Results and discussion 3.1. HPLC-FLD method performance Mean recoveries of AFB1 and AFG1 in spiked samples (each at 5 mg/kg) and AFB2 and AFG2 (each at 1.25 mg/kg) are provided in Table 1. The relative standard deviations (RSD) of the recoveries were generally low (5.0) for all types of aflatoxins which demonstrated the methods were well under control during the analytical sessions. The LODs for AFB1, AFB2, AFG1 and AFG2 in all tested products were 0.2, 0.08, 0.2, 0.08 mg/kg, respectively. The limits of quantifications (LOQs) of AFB1, AFB2, AFG1 and AFG2 in all tested products were 0.7, 0.3, 0.7 and 0.3 mg/kg respectively. 3.2. Occurrence of aflatoxins in analysed samples Data on the incidence and level of individual aflatoxins (AFB1, AFB2, AFG1 and AFG2) and total aflatoxins (AFB1þAFB2þ AFG1þAFG2) in the food products is provided in Table 2 and Fig. 1, respectively. The results were corrected by mean recovery rates (Table 1). The majority of the tested samples were baby cereals and peanut butters, as they were the most widely available on the market (Table 2). Aflatoxins were detected in 71% of the 125 food samples. Total aflatoxins ranged from LOD up to 115.6 mg/kg. AFB1 was detected most often, although AFB2, AFG1 and AFG2 were widely detected in the products (Table 1). The highest concentration of AFB2, AFG1 and AFG2 was found in samples of peanut butters (Table 1). 3.2.1. Maize-based products Exclusive breastfeeding is gradually replaced by complementary foods from the sixth month of life onwards (WHO, 2001) and cereals are among the most common weaning foods. In this study, a total of 36 samples of locally instant baby cereals, which are processed by extruding maize and soybeans, were analysed. All samples contained aflatoxins above 0.2 mg/kg, which is higher than the EU maximum tolerable level set for baby/infant foods (EC, 2010). Furthermore, in 33% of the baby cereal samples, total aflatoxins exceeded 3 mg/kg, a universal maximum tolerable level for all maize products for human consumption set by the Government of Malawi (MBS, 1998) (Fig. 1). The incidence of aflatoxins in the baby food is of concern considering that infants and children are more susceptible to toxins than adults, because of their lower body weight, higher metabolic rate and lower ability to detoxify toxins (Sherif, Salama, & Abdel-Wahhab, 2009). Soybeans are generally considered resistant to Aspergilli colonization and aflatoxins

89

contamination (Gupta & Venkitasubramanian, 1975; Njobeh et al., 2009). The aflatoxin contamination of the locally processed baby foods may therefore be attributable to carry over from the raw maize. If reduction of aflatoxin content through extrusion (Castells, Marín, Sanchis, & Ramos, 2005, 2006) and dilution (by addition of soya bean) is considered, the present results suggest the use of highly contaminated raw maize. On the other hand, all seven samples of imported instant baby cereals had no detected aflatoxins (Fig. 1). Maize puffs are commonly produced by extruding maize grit and are a popular snack among children. In the present study, aflatoxins were detected in 9 out of 12 samples (75%), with contamination levels ranging from LOD to 2.0 mg/kg (mean 1.0 mg/ kg) (Fig. 1). Although all the 9 samples were acceptable according to the universal aflatoxins regulation for all maize products set by Malawian Government, these samples were not fit as baby/infant food according the EU standards (EC, 2010; MBS, 1998). The concentrations of aflatoxins in the maize puffs were comparatively lower than in instant baby cereals (Fig. 1), although this difference was not statistically significant (p > 0.05). This difference could be attributed to the fact that the maize puffs are usually made from dehulled maize which contain reduced aflatoxin levels (Matumba et al., 2009; Njapau, Muzungaile, & Changa, 1998; Siwela, Siwela, Matindi, Dube, & Nsiramazanga, 2005). These findings are also supported by the fact that all (15) of the tested de-hulled maize flour samples had no detectable aflatoxins. 3.2.2. Groundnut-based products Peanut butter production involves dry-roasting of raw shelled groundnuts at 140e160  C, blanching, de-skinning, grinding and addition of other ingredients such as sugar, salt and stabilizers. Cumulatively, such processes have been reported to reduce aflatoxins concentration by as much as 89% (Siwela, Mukaro, & Nziramasanga, 2011). However, in present study, aflatoxins concentration in locally processed peanut butter ranged from 34.2 to 115.6 mg/kg and therefore not fit for human consumption according to any existing regulation globally (FAO, 2004). Considering the level of aflatoxin reduction during processing as earlier stated the present results indicate that peanut butter processors in Malawi use raw material with very high aflatoxins contamination. It is also interesting to note that despite the fact that de-skinning of roasted groundnuts significantly reduces aflatoxins concentration (Siwela et al., 2011) aflatoxin concentrations in de-skinned groundnuts were comparatively higher than in un-skinned roasted groundnuts (Fig. 1). These results may be attributed to grading of the raw

Table 2 Aflatoxin contamination in maize- and groundnut-based foods marketed in Lilongwe City, Malawi specifying the number analysed samples, percentage of positive samples (% pos), median, minimum (min) and maximum (max) concentrations concentration in mg/kg. Product

Aflatoxin AFB1

AFB2

AFG1

AFG2

Total aflatoxins

% Pos Median Minemax % Pos Median Minemax % Pos Median Minemax % Pos Median Minemax % Pos Median Minemax (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) Maize puffs (N ¼ 12) 75 Instant baby cereal (local) 100 (N ¼ 36) Peanut based therapeutic 100 food (N ¼ 6) Un-skinned roasted 22 groundnuts (N ¼ 9) De-skinned roasted 73 groundnuts (N ¼ 15) Peanut butter (local) 100 (N ¼ 14) Peanut butter (imported) 73 (N ¼ 11)

0.5 0.7

0.1e1.4 0.5e5.2

58 97

0.1 0.2

0.1e0.3 0.1e1.1

67 94

0.5 1.5

0.1e0.5 0.5e4.9

17 61

0.1 0.2

0.0e0.1 0.2e0.6

75 100

1.1 2.5

0.3e2.0 0.5e10.4

0.7

0.5e1.2

100

0.1

0.1e0.2

100

1.1

0.9e1.4

100

0.2

0.1e0.2

100

2.1

1.6e2.9

0.5

0.5e0.5

22

0.1

0.2e0.2

56

0.5

0.5e1.6

11

0.2

0.2e0.2

22

0.5

0.5e2.5

2.5

0.1e12.3

67

0.8

0.2e1.8

73

4.0

0.5e25.1

60

0.8

0.4e2.5

73

8

0.6e36.9

13.2e40.6 100

4.3

1.7e7.2

100

37.5

14.8e65.0 100

3.7

1.7e6.4

100

72

34.3e115.6

73

0.2

0.2e0.4

64

1.1

0.2

0.2e0.7

73

26.4 1.3

0.5e1.4

0.5e1.8

55

0.7

2.7e4.3

90

L. Matumba et al. / Food Control 39 (2014) 87e91

technical assistance provided by L. Singano, T. Mhango, H. Mbalame, C. Gadaga, D. Kalima, D. Bwanamiri, M. Kalitsiro and C. Tchuwa is highly appreciated. References

Fig. 1. Distribution of total aflatoxins (AFs) (AFB1 þ AFB2 þ FG1 þ AFG2) in maize- and groundnuts-based products marketed in Lilongwe City. Figures in parenthesis indicate fraction of aflatoxin positive samples. Reference lines (dotted) indicate the following maximum tolerable levels set for: AFB1 in baby/infant food and dietary food for medical purpose set by EU (0.1 mg/kg); AFs in maize products set by Malawi Government (3 mg/kg); AFs in ready-to-eat groundnuts and maize set by EU (4 mg/kg), AFs in ready-to-eat groundnuts and maize set by Codex Alimentarius Commission (10 mg/kg); and AFs in food guided by WHO (20 mg/kg).

groundnuts as raw material for different products: The aflatoxin incidence suggest that heavily affected nuts are processed into peanut butter, the intermediate into de-skinned nuts and the better quality nuts sold whole without de-skinning. In this case, the consumer would not visually detect mouldy nuts in the latter two products. On the other hand, 8 of the 11 analysed imported peanut butter samples had significantly lower total aflatoxins content (maximum, 4.3 mg/kg; mean, 2.7 mg/kg). In Malawi, peanut-based ready-to-use therapeutic foods are fed to malnourished children and AIDS patients (Manary et al., 2004; Ndekha et al., 2005; Sandige et al., 2004). In the present study, all 6 tested peanut based therapeutic foods had aflatoxin levels between 1.6 and 2.9 mg/kg, exceeding the EU maximum tolerable level for dietary food for health purpose (EC, 2010). The incidence of aflatoxins in the therapeutic foods is of concern considering that aflatoxins could further compromise health of the patients by depressing immunity and affecting nutrient absorption and utilization (Cheeke & Shull, 1985; Fink-Gremmels, 2008; Gong et al., 2008; Jiang et al., 2008; Jolly et al., 2013). The incidence of aflatoxins in baby food may exacerbate the incidence of stunting among children estimated at 50% in Malawi (ORC Macro, 2006). 4. Conclusion This is the first report on the incidence of aflatoxins in industrial processed food products marketed in Malawi. The results presented in this study, suggest that the presence of aflatoxins in locally produced baby foods and peanut butters may constitute a public health problem. This study highlights need for regular monitoring of processed products in the country, and the region. Having functional authorities (facilities) in the country is a great advantage in this respect in order to adequately safeguard local consumers and meet high standards of importing countries. Acknowledgements This research has been supported by Government of Malawi; World Bank; the European Union and Norwegian Embassy through Agricultural Sector Wide Approach-Support Project (ASWAp-SP). Their support is gratefully acknowledged. The authors also acknowledge donation of HPLC system by EU, through a SADC-Food Safety Capacity Building on Residues Control Project. Invaluable

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