Natural occurrence of aflatoxin residues in fresh and sun-dried meat in Nigeria

Natural occurrence of aflatoxin residues in fresh and sun-dried meat in Nigeria

Olufunmilayo Gbonjubola Oyero^1,&, Akeeb Bola Oyefolu²

Department of Biology, The Polytechnic, Ibadan, Nigeria¹ Department of Microbiology, Lagos State University, Lagos, Nigeria²

^&Corresponding author

Olufunmilayo Gbonjubola Oyero, Department of Biology, the Polytechnic, Tel: +2348033265216, Ibadan, Nigeria

Meat refers to meat flesh, skeletal muscles, connective tissue or fat and others than meat flesh, including brain, heart, kidney, liver, pancreas, spleen, thymus, tongue and tripe that is used as food, excluding the bone and bone marrow and it contains high biological value protein and important micronutrients that are needed for good health throughout life [1]. However in Nigeria, meat consumption is correlated with the affluence of individuals, although man’s dependence on plant materials when consumed directly could support a substantially greater population than the animal products derived when fed to animals; this is true with respect to the total available calories, protein and other nutrients needed to sustain life.

Raw materials for animal feeds are exposed to microbial contamination at the time of their cultivation, processing, storage and distribution, while chemical composition of food, properties of the outside environmental and specific growth requirements determine the type of microorganisms and the course of physico-chemical reaction in the contaminated food [2].

In general, mycotoxin exposure is a critical problem in the hot and humid low income countries where poor methods of food handling and storage are common. In spite of occasional high profile incidents of acute poisoning outbreak, mycotoxins have not been widely prioritized from a public health perspective [3-4]. However attention has only been paid, largely to meet the stringent import regulations on mycotoxin contamination in the richer nations of the world. Humans become exposed directly by consuming contaminated commodities or indirectly by consuming products from animals that have ingested mycotoxin contaminated feeds [5]. While animal slaughter slabs, insufficient cleaning and disinfection of working areas, instruments and other equipment contribute greatly to meat contamination by toxigenic moulds, the conditions of the environment in the stores, refrigerators and shops are very suitable for the development of moulds inside the products, but more frequently on the surface of various sorts of meat and meat products [2,6]. Feeds and foods are often contaminated with various moulds and when the temperature and relative humidity are optimal after contamination, there is also a risk of mycotoxin production [7]. Relatively low water activity (a_w < 0.9) and low pH-values (pH < 6.0) are particularly favorable for mould development [8].

Although mycotoxin contamination of food commodities is a global problem, the developed world through the application of modern agricultural practices and legislatively regulated food processing and marketing system have greatly reduced exposure in these populations [9]. The contrary is the case in developing countries where populations live largely on commodities from local markets where regulations relating to mycotoxin control and consumer protection rarely exist; even when they are available they are not strictly enforced. As a result, mycotoxins presence in food represents a constant health risk for man.

Meat as a source of animal protein is consumed heavily in Nigeria and is also is recommended by nutritionists as a major source of protein for growing children, the convalescent, the expectant mothers and the aged. Therefore, the objective of this was to determine the various contaminating fungi on meat, assaying for mycotoxin residues and ascertaining the general safety of meat consumed in Northern and Eastern parts of Nigeria; in order to create awareness on the presence, concentrations and distributions of mycotoxins in meat. It could also form the scientific basis for promulgation of regulations important in the decision-making process to establish meaningful limits for mycotoxins in foods, most especially meat and meat products [9].

Sample preparation, isolation, enumeration and identification of mycoflora

Five major markets for bulk purchase and retail of foodstuffs and animal produce brought largely from the Northern and Eastern parts of Nigeria were selected for this study. The markets (aleshinloye, bodija, moniya, oja-oba and oje) distributed in 5 local government areas (Onireke, Ibadan- north, Akinyele, Ibadan- northwest, and Ibadan- northeast respectively) are known for wide patronage by inhabitants of the South-western states. Ten samples each of fresh and sun-dried pieces of cow beef and edible organs (liver, kidney and heart) were purchased randomly from these markets. They were placed in labeled sterile polythene bags and then transferred immediately to the laboratory for mycological and mycotoxin residue analysis. Ten grams of sample was diluted with 90mL of physiological saline and homogenization was achieved using mortar and pestle. Two hundred microlitres (200µl) of ten-fold serially diluted homogenized sample was inoculated onto potato dextrose agar (PDA) incorporated with antibiotics. Incubation was at 25 degrees Celsius for 3-5 days. Fungal enumeration was by plate count while mycoflora identification was by micro-morphological characteristics [10].

Extraction, detection and quantification of aflatoxins in meat samples

A thin layer chromatographic (TLC) method previously described by Oyero and Oyefolu, 2009 [9] was used and done in triplicates. Aflatoxin content of 5g of meat was extracted with 2.5ml of distilled water and 25ml of chloroform while shaking on a mechanical shaker. Ten milliliter (10ml) of the filtrate obtained was evaporated to dryness on water bath. A mixture of the reconstituted extract (0.2ml) and chloroform (1ml) spotted on pre-coated TLC plate was developed in chloroform/acetone (9:1) tank and detection was done alongside standards under the UV light at 360nm. For quantification, 0.8ml of extract was applied to a 0.5 µm preparative TLC plate as a band. Following plate development and UV examination, areas containing the toxin was scrapped and extracted in chloroform. The optical density of 3ml of the extract was then determined on a UV spectrometer at 360nm. Aflatoxin concentration was then calculated using the absorbance values for the sample and standard.

The mycoflora content of the samples ranged from 6.5 x 10⁵ to 2.2 x 10⁶ CFU and 1.25 x 10⁶ to 2.95 x 10⁶ CFU in the fresh and sun-dried samples respectively. On the overall, the contaminating mycoflora load was found to be higher in the sun-dried samples. Mycological analysis of the meat samples resulted in the isolation of 18 fungal species, belonging to 8 genera, namely, Aspergillus, Alternaria, Fusarium, Cladosporium, Penicillium, Neurospora, Rhizopus and Yeast. Three of these (Aspergillus, Fusarium and Penicillium) are potential mycotoxin producers (Table 1). Apergillus, the predominant genus was identified into 7 species, these included A. niger, A. tamari, A. fumigatus, A. terreus, A. flavus, A. citrinum, and A. sydowii. The genus Penicillium and Rhizopus yielded 2 species each i.e. P. Oxalicum, P. chrysogenum, R. nigrificans and R. oligosporus. Others however gave only one specie each. However it was observed that no Penicillium and Neurospora Species were isolated from the fresh sample while members of the genera Alternaria, Fusarium and Yeast were also not recovered from the sun-dried meat.

The potential health hazard that may be associated with the consumption of the various meat parts was assessed by the analysis for mycotoxin residues. The two types of aflatoxins (AFB, AFG) commonly found in animal tissues were found in varying concentrations. The fresh samples, with the exception of the total AFG (AFG₁, AFG₂) in kidney gave the highest mean concentrations for all the aflatoxins detected (Table 2). This ranged from 0.0217 to 0.0852µg/kg and 0.0267 to 0.0741 µg/kg for total AFB (AFB₁ and AFB₂) and AFG respectively. This was followed by the beef, liver, and heart with 0.0559, 0.0339, 0.0217 µg/kg for AFB and 0.0741, 0.0414, and 0.0267 µg/kg for AFG respectively. However the total aflatoxin contents of dried samples were lower, ranging from 0.0029 to 0.0758µg/kg and 0.0031 to 0.1413 µg/kg for AFB and AFG respectively. In addition an exceptionally high aflatoxin concentration was found in all the kidney samples.

The determination of the level of fungal contaminants in the samples analysed revealed a high microbial load in the sun- dried meat. This may not be unconnected with the fact that during the sun drying process, meats are spread openly in the sun and on bare concrete floors, this often allow contamination by dust. However, the mycoflora on the fresh meat may have been derived from contaminants in water, air, floors and surroundings of the slaughter house, as well as the instruments used during the slaughtering and processing into table meat for sale. Also the situation of the meat processing plants, the slaughter of animals, insufficient cleaning and disinfestations of working areas, instruments and other equipment contributes greatly to meat contamination by toxigenic moulds [12].

The mycoflora of the meat were identified into the Aspergillus ,Alternaria, Fusarium, Cladosporium, Rhizopus, Penicillium, Neurospora and Yeast genera. The incidence of the various genera has been documented by several workers [2,13-17]. The mycotoxin producing ability of many of the species have also been demonstrated in vitro [6,17-19]. Consequently the presence of moulds in meat and meat products could cause a decrease in their biological value due to the enzymatic degradation of meat components [2]. However their metabolic interactions with bacterial pathogens has played important role in the outbreak of food-borne illnesses [20]. The potential health hazards of the mycoflora need not be overemphasized. This is because the fungi following inhalation/ingestion are capable of surviving in the respiratory and intestinal tracts, where they may continue to grow and produce their toxins. These have been implicated in the pathogenesis of several mycotic and aflatoxin induced diseases in man. The Alternaria, Aspergillus, Cladosporium and Penicillium genera have been implicated as a cause of rhinitis and severe asthma [21] while the contributory role of Fusarium and Penicillium species in systemic respiratory disease as well as opportunistic infections involving various body sites by the Aspegillus and Rhizopus sp have been reported. The Aspergillus sp especially is particularly dangerous to the asthmatics and those with weakened immune systems [22-24]. The non-isolation of the Fusarium, Alternaria and Yeast species from the dried meat samples however may be due to the fact that they require a moderately high moisture content of 22-23 % [25]. This requirement however was only found or met in the fresh samples. 

Aflatoxins commonly associated with animal tissue (AFB1, AFB2, AFG1, and AFG2) were found in all the samples analysed. In nature, several mycotoxins co-occur in contaminated commodity [26] and so the occurrence of AFB and AFG was not surprising. On the overall, total aflatoxin content was higher in the fresh samples. This may have arisen from the fact that the animals were fed on aflatoxin contaminated food till the point of slaughter. It has been suggested that a withdrawal of animals from contaminated feeds onto mycotoxin free diets for 3-4 weeks could have allowed a sufficient withholding period to clear the muscles and organs from toxins [27]. Although the concentrations of the aflatoxins determined were insignificant when compared to the African maximum permissible limits of 5mg/kg and 20mg/kg for AFB1 and total aflatoxins in foods respectively [28]; the overall impact of mycotoxins on health however is dependent on the concentrations and duration of exposure, the toxicity of the compound, the body weight of the individual, the synergistic effects of mycotoxin, environmental factors and other effects [29-30].

The AFB1 and AFG1 detected in samples are proven human carcinogens and are thus classified as Group 1 carcinogens with AFB2 as Group 2B probable human carcinogens [31]. Documented evidence have shown that AFB1 exposure exacerbates protein calorie malnutrition, thereby suppressing growth as well as immunoglobulin (IgA) response to some vaccine challenges, among the African children [32-33]. Consequently the resultant effects of AFB1 exposure may include growth and immune function suppression.

This study confirmed that meat generally sold in Nigeria are grossly contaminated by various fungi species including the potentially mycotoxigenic ones. The determination of aflatoxins though in acceptable concentrations should urgently be addressed with a view of ensuring the safety of the consumers.

The authors declare no competing interests

Akeeb O. Bola Oyefolu was involved in study design, laboratory analysis of samples and wrote the initial draft of the manuscript; Olufunmilayo Gbonjubola Oyero was involved in study conception, design, sample collection, laboratory analysis and writing of the final manuscript.

Table 1: Distribution of mycoflora of 80 meat samples collected from 5 major markets distributed in 5 local government areas of Oyo State, Nigeria

Table 2: Aflatoxin content of 80 meat samples collected from 5 major markets distributed in 5 local government areas of Oyo State, Nigeria

1. Anon. Nutritional composition of red meat at http://www.thefreelibrary.com/Nutritional+composition+of+red+meat,-a0169311692. Accessed 24/7/2010

2. Laciakova A, Pipova M, Mate D, Laciak V. Microbial analysis of the meat products focused on the occurrence of microscopic filamentous fungi. MESO. 2004; VI (5):24-28

3. Azziz-Baumgartner E, Lindblade K, Gieseker K, et al. Case-control study of an acute aflatoxicosis outbreak, Kenya, 2004. Environ Health Perspect. 2005; 113:1779–1783

4. South African Medical Research Council - Programme on Mycotoxins and Experimental Carcinogenesis (PROMEC Unit). Aflatoxin in Peanut Butter - Policy Brief No. 3 May 2001. http://www.mrc.ac.za/policybriefs/polbrief3.htm. Accessed 13/6/2010

5. Hseih P. H. Metabolism and transmission of mycotoxins. Proc of the Int workshop on mycotoxins, Sept 1981

6. Jesenská, Z. Microscopic Filamentous Fungi in Food-and Feedstuffs (In Slovak). Alfa, Bratislava. 1987; 318:36-39

7. Štyriak L, Conkoval E, Laciakova A, Bohm J. Prevention of fumonosin production by microorganisms. Czech J Anim Sci. 1998; 43:449-452

8. Hanssen HP. Mould control in the meat processing industry. Fleischwirtschaft. 1995; 75:52

9. Milicevic DR, Skrinjar M, Baltic T. Real and perceived risks for mycotoxin contamination in foods and feeds: Challenges for food safety control. Toxins. 2010; 2:572-592

10. Domsch KH, Gams W, Anderson TH. Compedium of soil fungi Vol 1,2. London: Academic Press 1981.

11. Oyero OG and Oyefolu AOB. Fungal contamination of crude herbal remedies as a possible source of mycotoxin exposure in man. Asian Pacific J Trop Med. 2009; 5(2):38-43

12. Ismail MA, Abou-Elala AH, Nassar A, Michail DG. Fungal contamination of beef carcasses and the environment in a slaughterhouse. Food Microbiol. 1995; 12:441-445

13. Zaky ZM, MA Ismail and RS Refai. Aspergillus flavus and aflatoxins residues in luncheon meat. Assuit Vet Med J. 1995; 33(66):114-118

14. Negat AS. Incidence of mycotoxins and mycotoxin – producing moulds in some Egyptian dairy products 4th Sci Cong. Egyptian Society for cattle diseases. 1997:74-80

15. Mahmoud A-lE, Sayed AM and Abou Ei-Alla AA. Mycoflora and Natural occurrence of mycotoxins in some meat products and livers of poultry and imported bulls. Pakistan J of Biological Sciences. 2001; 4(5):611-613

16. Andersen SJ. Compositional changes in surface mycoflora during ripening of naturally fermented sausages. J Food Protect. 1995; 58: 426-429

17. Cvetnic Z, Pepelnjak S. Aflatoxin-producing potential of Aspergillus flavus and Aspergillus parasiticus isolated from samples of smoked-dried meat. Nahrung. 1995;39(4):302-7. This article on PubMed

18. WU MT, Ayres JC, Koehler PN. Toxigenic Aspergilli and Pennicillia isolated from aged, cured meats. Appl Microbiol. Appl Microbiol. 1974 Dec;28(6):1094-6. This article on PubMed

19. Berwal JS and Dinchev D. Study of the mycoflora during the natural commercial fermentation of raw dry sausages. MecoeMecni Prodykti. 1991; 1:34-37

20. Polster MD, Hartlova D, Kralikova I. Frequency of the occurrence of Aspergillus flavus aflatoxicogenic variants in the environment of food-processing establishments. Cs Hyg. 1985; 9:442-446

21. de Ana SG, Torres-Rodríguez JM, Ramírez EA, García SM, Belmonte-Soler J. Seasonal distribution of Alternaria, ASP Cladosporium and Penicillium species isolated in homes of fungal allergic patients. J of Investig Allergol Clin Immunol. 2006; 16(16):357-363. This article on PubMed

22. Anon-Fungal disease. http://www.Innvista.com/health/microbes/fungi/diseases.htm. Accessed 13/6/2010

23. Anon-mystery death of teacher, 21 after inhaling fungus spores. http://www.thefreelibrary.com/mystery+death+of+teacher+21+after+inhaling+fungus+spores-9019779974. Accessed 13/6/2010

24. Anon-E medicine. http://emedicine.medscape.com/article/300341-overview Accessed 13/6/2010

25. Alex Ciegler. Fungi that produce mycotoxins; Condition and occurrence. Mycopathologia. 1978; 65:5-11. This article on PubMed

26. Charmley LL,Trenholm HL. Mycotoxins - Canadian Food Inspection Agency. http://www.inspection.gc.ca/english/anima/feebet/pol/mycoe.shtml/. Accessed 13/6/2010

27. Krogh P, Elling F, Hald B, Larsen AE, Lillehoj EB, Madsen A, Mortensen HP. Time dependent disappearance of ochratoxin A residues in tissues of bacon pig. Toxicology. 1976 Aug-Sep;6(2):235-42. This article on PubMed

28. FAO Corporate Document Repository. Worldwide regulations for mycotoxins in food and feed. http://www.fao.org/docrep/007/y5499e/y5499e07.htm. Accessed 2/11/2010

29. Kuiper-Goodman T. Risk assessment to humans of mycotoxins in animal derived food products. Vet Hum Toxicol. 1991; 33:325-333. This article on PubMed

30. Bennett JW, Klich M. Mycotoxin. Clin Microbiol Rev. 2003; 16:497-516. This article on PubMed

31. IARC (1993a). Aflatoxins: naturally occurring aflatoxins (Group 1), aflatoxins M1 (Group 2B). Int Agency Res Cancer. 1993; 56:245

32. Gong YY, Hounsa A, Egal S, Turner PC, Sut cliffe AE, Hall AJ, Cardwell K, Wild CP. Postweaning exposure to aflatoxin results in impaired child growth: a longitudinal study in Benin, West Africa. Environ Health Perspect. 2004; 112:1334-1338. . This article on PubMed

33. Turner PC, Moore SE, Hall AJ, Prentice AM, Wild C. Modification of immune function through exposure to dietary aflatoxin in Gambian children. Environ Health Perspect. 2003; 111:217-20. This article on PubMed