NATURAL INHIBITORS FOR INHIBITING THE PRODUCTION OF AFLATOXINS 1* Alim-Un-Nisa, 1 Naseem Zahra, 1 Sajila Hina, 1 Khalid Saeed, 2 Muhammad Sabir Hussain, 1 Lubna Liaquat, 1 Qurat-Ul-Ain Syed 1 Pakistan Council of Scientific and Industrial Research (PCSIR Laboratories Complex, Ferozepur road, Lahore-54600, Pakistan 2 Department of Chemistry, Government College of Science Wahdat road, Lahore, Punjab, Pakistan Correspondence author: nisaalim64@yahoo.com ABSTRACT Aflatoxins are the carcinogenic substances and their control is very important aspect related to health concerns. In present study, the inhibitory effects of the 8 different kinds of natural powdered spices were tested by introducing these materials into culture media for aflatoxin production. The results showed that black cumin + cinnamon oil, onion + cinnamon oil, Nutmeg, Mace + mustard oil, coriander + mustard oil completely inhibited the Production of aflatoxin, whereas turmeric completely inhibit (100%) at concentration 2.0% and at other concentrations (0.6, 1.0, 1.4%) it showed 84, 86, 90% inhibition. The black pepper and ginger significantly inhibit the aflatoxin production while garlic showed low inhibitory effect against production of aflatoxin. Key Words: Aflatoxin, Inhibitory effect, Thin Layer Chromatography INTRODUCTION Aflatoxins are toxic secondary metabolites produced by certain strains of A. flavus, A. parasiticus, A. nomius, A. tamarii, A. pseudotamarii and A. bombycis (Goto et al. 1997 and Peterson et al. 2001). Aflatoxins have received greater attention because of their carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans (Ghosh et al. 1997). Several analytical methods are available for the detection of aflatoxins, including enzyme-linked immunosorbent assays (ELISAs), Thin Layer Chromatography, and liquid chromatography, or a combination of immuno-affinity columns and TLC or liquid chromatography (WHO, 2002). It is estimated that human consumption of aflatoxins ranges from 0 to 30,000 ng/kg/day with an average of 10 to 200 ng/kg/day (Revankar, 2003). Eugenol and thymol extracted from clove and thyme respectively completely inhibit the growth of both Aspergillus flavus and Aspergillus versicolor at 0.4 mg/ml or less and 2 mg/ml, anethol extracted from star anise seeds inhibited the growth of all the strains (Hitokoto et al. 1980). The essential oil of white wood gave the highest inhibition followed by the essential oils of cinnamon and lavender, respectively. Also the inhibitory effects of these three essential oils at different concentrations were examined. It was found that the essential oil of whitewood at 1.5625% (v/v) and of cinnamon and lavender at 50% (v/v) were the concentrations for fungal growth inhibition (Dusanee et al. 2007). Similarly, 1% of caraway was sufficient to inhibit the growth completely for A. flavus. The results of colonies diameter measuring showed that garlic and origanum extracts have no significant suppressing ability on micellar growth of A. flavus, while garlic was more efficient in other test cultures (Dimić et al. 2007). The effect of ten powdered spice plants was evaluated at the concentration of 1, 2, 3 and 4% to observe the mycelial growth and sporulation of Aspergillus Niger and Eurotium repens. These powdered spices also tested against Aspergillus flavus in culture medium at the same concentration previously mentioned. It is observed that clove completely inhibit the mycelial growth. Cinnamon and anis totally inhibited the production of aflatoxin B 1 and B 2, while bay leaf and basil inhibited the synthesis of aflatoxin starting from the concentration of 2% (Sára et al. 2004). Several studies showed that different substances, such as essential oils, flavanoids, could inhibit the aflatoxin production and growth of Aspergillus (Lokman, 2010). The antifungal activity of some chemicals, herbal compounds/spices and plants at different concentrations were evaluated against the toxin producing Aspergillus flavus and Aspergillus parasiticus growth. The chemicals: Benzoic acid (0.1 0.5 %), propionic acid (0.1 0.5 %) and copper sulphate (0.2 0.5 %) showed complete inhibition of Aspergillus flavus Aspergillus parasiticus growth. In case of the herbal compounds/spices, clove (0.5 %) and clove oil (0.5 %), while among the plants garlic (0.5 %) and onion (0.5 %) showed complete inhibition in both cases (Arshad et al. 2012). The fungitoxic activities have been reported by several extracts of plants, while some of them i.e. cloves, cinnamon, Chinese cassia and thymes along with their mixture showed complete reduction in aflatoxins synthesis (El-Maraghy et al. 1995, Awuah, 1999 and Yin and Tsao 1999). MATERIALS AND METHODS All the samples were purchased from local food market of Lahore in February 2012. Samples were used in ungrind form. 37
Samples 1. Rice 2. Chilly 3. Corn Natural Inhibitors: 1. Garlic (Allium sativum) 2. Black pepper (Piper nigrum) 3. Ginger (Zingiber officinale) 4. Turmeric (Curcuma longa) 5. Black Cumin (Nigella sativa) 6. Cinnamon oil (Cinnamomum zeylanicum) 7. Onion Powder (Allium cepa) 8. Nutmeg, Mace (Myristica, fragrans) 9. Mustard oil (Brassica juncea) 10. Coriander Seeds (Coriandrum sativum) All the spices and oils were purchased from local market of Lahore in February 2012. Spices were ground by electric grinder. Preparation of sample: Three different samples were used in the study: rice, corn, chili for testing antifungal potential of natural inhibitors. 50g of each sample was soaked in 22-23% of distilled water in 250ml conical flask. Then they were sterilized at 121 o C for 15mins. Cultivation of Samples: Sterile samples with initial moisture content 22-23% were mixed with various concentrations of inhibitors and inoculated with Aspergillus flavus culture and incubated at 35 o C for 90 days. The samples without inhibitors were used as control. Aspergillus Flavus on peanut Aspergillus Flavus on corn Aflatoxin B 1 & B 2 Aflatoxin B 1, B 2, G 1, G 2 38
Analysis of Aflatoxins by TLC (Thin Layer Chromatography): The analysis of sample for aflatoxin production was done by Thin Layer Chromatography (TLC) according to Association of Official Analytical Chemist (AOAC) 2005, AOAC Official method (972.26). Method: In the 1 st experiment 50g sterilized rice sample was taken and mixed with garlic powder at concentration 0.30g, 0.50g, 0.70g, 1.0g and inoculated with 0.050g of Aspergillus flavus strain. The samples without inhibitors were used as control. All the samples were shaken to mix and incubated at 35 o C for 90 days. The samples were checked and analyzed for production of aflatoxin after every 15 days (table 1). In the 2 nd experiment 50g sterilized rice sample was taken and mixed with black pepper powder at concentration 0.30g, 0.50g, 0.70g, 1.0g and inoculated with 0.050g of Aspergillus flavus strain. The samples without inhibitors were used as control. All the samples were shaken to mix and incubated at 35 o C for 90 days. The samples were checked and analyzed for production of aflatoxin after every 15 days (table 2). In the 3 rd experiment 50g sterilized chili sample was taken and mixed with ginger powder at concentration 0.30g, 0.50g, 0.70g, 1.0g and inoculated with 0.050g of Aspergillus flavus strain. The samples without inhibitors were used as control. All the samples were shaken to mix and incubated at 35 o C for 90 days. The samples were checked and analyzed for production of aflatoxin after every 15 days (table 3). In the 4 th experiment 50g sterilized chili sample was taken and mixed with turmeric powder at concentration 0.30g, 0.50g, 0.70g, 1.0g and inoculated with 0.050g of Aspergillus flavus strain. The samples without inhibitors were used as control. All the samples were shaken to mix and incubated at 35 o C for 90 days. The samples were checked and analyzed for production of aflatoxin after every 15 days (table 4). In the 5 th experiment 50g sterilized corn sample was taken and mixed with black cumin powder at concentration 0.20g, 0.40g, 0.60g, 0.80g and cinnamon oil at concentration 0.024g, 0.030g, 0.036g, 0.048g respectively then inoculated with 0.050g of Aspergillus flavus strain. The samples without inhibitors were used as control. All the samples were shaken to mix and incubated at 35 o C for 90 days. The samples were checked and analyzed for production of aflatoxin after every 15 days (table 5). In the 6 th experiment 50g sterilized corn sample was taken and mixed with onion powder at concentration 0.20g, 0.40g, 0.60g, 0.80g and cinnamon oil at concentration 0.024g, 0.030g, 0.036g, 0.048g respectively then inoculated with 0.050g of Aspergillus flavus strain. The samples without inhibitors were used as control. All the samples were shaken to mix and incubated at 35 o C for 90 days. The samples were checked and analyzed for production of aflatoxin after every 15 days (table 6). In the 7 th experiment 50g sterilized rice sample was taken and mixed with nutmeg, mace powder at concentration 0.50g, 0.60g, 0.70g, 0.80g and mustard oil at concentration 0.024g, 0.030g, 0.036g, 0.048g respectively then inoculated with 0.050g of Aspergillus flavus strain. The samples without inhibitors were used as control. All the samples were shaken to mix and incubated at 35 o C for 90 days. The samples were checked and analyzed for production of aflatoxin after every 15 days (table 7). In the 8 th experiment 50g sterilized rice sample was taken and mixed with coriander powder at concentration 0.50g, 0.60g, 0.70g, 0.80g and mustard oil at concentration 0.024g, 0.030g, 0.036g, 0.048g respectively then inoculated with 0.050g of Aspergillus flavus strain. The samples without inhibitors were used as control. All the samples were shaken to mix and incubated at 35 o C for 90 days. The samples were checked and analyzed for production of aflatoxin after every 15 days (table 8). RESULTS AND DISCUSSION Aflatoxins have been studied extensively because of their high toxicity to certain domesticated animals as well as humans. Aflatoxins have received greater attention because of their carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans. Many Strategies, including natural control, biological control, control of insects pests, development of resistant cultivar, have been investigated to manage aflatoxins in crops. Among them, natural control appears to be most promising approach for control of Aflatoxin in post-harvested crops. 8 different spices have been studied including Garlic, Black pepper, Ginger, Turmeric, Black Cumin + Cinnamon oil, Onion Powder + Cinnamon oil, Nutmeg-Mace + Mustard oil and Coriander Seeds + Mustard Oil were examined for production of aflatoxin. Many of them completely inhibited the growth of A. flavus but some cannot inhibit the aflatoxin production successfully. The effect of different kinds of spices has been discussed and results have been shown in tables and graphs. The effect of garlic at various concentrations on aflatoxin 39
Table 1: Inhibitory effects of garlic powder in rice on the toxin production by toxigenic A. flavus Concentration of Garlic 0.6 1.0 1.4 2.0 15 28.51 2.38 2.02 1.52 1.01 30 34.14 3.11 2.90 2.09 1.26 45 40.03 5.90 4.17 3.02 2.97 60 42.03 7.86 6.19 4.76 3.20 75 42.03 8.01 6.97 5.28 4.18 90 42.03 9.41 7.24 6.06 5.12 Fig 1: Graph Showing inhibitory effects of garlic powder on aflatoxin production at various concentrations 40
Table 2: Inhibitory effects of black pepper powder in rice on the toxin production by toxigenic A. flavus Concentration of Black Pepper 0.6 1.0 1.4 2.0 15 26.69 15.84 14.09 13.21 11.27 30 32.63 17.54 16.65 15.31 13.86 45 45.40 20.07 18.99 17.46 15.93 60 45.92 25.65 24.06 23.97 18.54 75 45.93 26.08 25.86 24.65 19.96 90 45.93 27.55 26.34 25.94 21.43 Fig 2: Graph Showing inhibitory effects of black pepper powder on aflatoxin production at various concentrations 41
Table 3: Inhibitory effects of ginger powder in chili on the toxin production by toxigenic A. flavus Concentration of Ginger 0.6 1.0 1.4 2.0 15 25.34 13.20 11.99 8.94 5.04 30 33.36 13.86 12.63 10.44 7.10 45 43.13 14.71 13.02 11.19 9.69 60 43.39 18.10 17.30 13.65 11.41 75 43.87 21.05 19.11 16.31 14.99 90 43.87 27.04 25.69 22.38 20.05 Fig 3: Graph Showing inhibitory effects of ginger powder on aflatoxin production at various concentrations 42
Table 4: Inhibitory effects of turmeric powder in chili on the toxin production by toxigenic A. flavus Concentration of Turmeric 0.6 1.0 1.4 2.0 15 22.63 0.0 0.0 0.0 0.0 30 28.50 0.0 0.0 0.0 0.0 45 30.70 3.12 2.90 1.26 0.0 60 46.20 4.90 4.20 3.43 0.0 75 46.21 6.60 4.49 3.91 0.0 90 46.25 7.13 6.06 4.53 0.0 Fig 4: Graph Showing inhibitory effects of turmeric powder on aflatoxin production at various concentrations 43
Table 5: Inhibitory effects of black cumin powder + cinnamon oil in corn on the toxin production by toxigenic A. flavus Concentration of Black Cumin + Cinnamon oil 0.4 + 0.048 0.8 + 0.060 1.2 + 0.072 1.6 + 0.096 15 25.15 0.0 0.0 0.0 0.0 30 32.63 0.0 0.0 0.0 0.0 45 43.39 0.0 0.0 0.0 0.0 60 45.91 0.0 0.0 0.0 0.0 75 45.92 0.26 0.0 0.0 0.0 90 45.92 0.41 0.0 0.0 0.0 Fig 5: Graph Showing inhibitory effects of black cumin + cinnamon oil on aflatoxin production at various concentrations 44
Table 6: Inhibitory effects of onion powder + cinnamon oil in corn on the toxin production by toxigenic A. flavus Concentration of Onion Powder + Cinnamon oil 0.4 + 0.048 0.8 + 0.060 1.2 + 0.072 1.6 + 0.096 15 26.56 0.0 0.0 0.0 0.0 30 36.69 0.0 0.0 0.0 0.0 45 46.26 0.0 0.0 0.0 0.0 60 46.27 0.0 0.0 0.0 0.0 75 46.27 0.0 0.0 0.0 0.0 90 46.28 0.49 0.0 0.0 0.0 Fig 6: Graph Showing inhibitory effects of onion powder + cinnamon oil on aflatoxin production at various concentrations 45
Table 7: Inhibitory effects of Nutmeg, Mace powder + Mustard oil in rice on the toxin production by toxigenic A. flavus Concentration of Nutmeg, Mace + Mustard oil 1.0 + 0.048 1.2 + 0.060 1.4 + 0.072 1.6 + 0.096 15 26.77 0.0 0.0 0.0 0.0 30 36.69 0.0 0.0 0.0 0.0 45 46.20 0.0 0.0 0.0 0.0 60 46.20 0.0 0.0 0.0 0.0 75 46.21 1.26 1.01 0.0 0.0 90 46.23 1.69 1.23 0.23 0.0 Fig 7: Graph Showing inhibitory effects of nutmeg, mace + mustard oil on aflatoxin production at various concentrations 46
Table 8: Inhibitory effects of coriander powder + Mustard oil in rice on the toxin production by toxigenic A. flavus Concentration Coriander + Cinnamon oil 1.0 + 0.048 1.2 + 0.060 1.4 + 0.072 1.6 + 0.096 15 22.38 0.0 0.0 0.0 0.0 30 32.70 0.0 0.0 0.0 0.0 45 43.13 0.0 0.0 0.0 0.0 60 51.36 0.0 0.0 0.0 0.0 75 51.36 0.49 0.0 0.0 0.0 90 51.38 0.78 0.0 0.0 0.0 Fig 8: Graph Showing inhibitory effects of coriander + mustard oil on aflatoxin production at various concentrations 47
production has shown in table 1. It was found that at concentration 0.6, 1.0, 1.4 and 2.0% of garlic, the inhibition of aflatoxin production was 77, 82, 85, and 87% respectively on day 90 of storage. The results showed that garlic had significant inhibitory effect against aflatoxin production. The effect of black pepper at various concentrations on aflatoxin production has shown in table 2.It was found that at concentration 0.6, 1.0, 1.4 and 2.0% of black pepper, the inhibition of aflatoxin production was 40, 42, 43 and 53% respectively on day 90. The results showed the black pepper had low inhibitory effect against aflatoxin production. The effect of ginger at various concentrations on aflatoxin production has shown in table 3.It was found that at concentration 0.6, 1.0, 1.4 and 2.0% of ginger, the inhibition of aflatoxin production was 38, 41, 48 and 54% respectively on day 90. The results showed the ginger had low inhibitory effect against aflatoxin production. The effect of turmeric at various concentrations on aflatoxin production has shown in table 4. It was found that at concentration 0.6, 1.0, 1.4 and 2.0% of turmeric, the inhibition of aflatoxin production was 84, 86, 90 and 100% respectively on day 90. The results showed the turmeric had high inhibitory effect against aflatoxin production. The effect of black cumin + cinnamon oil at various concentrations on aflatoxin production has shown in table 5. It was found that at concentration 0.4+0.048% the inhibition of aflatoxin was 99% but at concentration 0.8+0.060, 1.2+0.072 and 1.6+0.096% of black cumin + cinnamon oil, the inhibition of aflatoxin production was 100% on day 90. The results showed that black cumin + cinnamon oil completely inhibit the aflatoxin production. The effect of onion powder + cinnamon oil at various concentrations on aflatoxin production has shown in table 6. It was found that at concentration 0.4+0.048% the inhibition of aflatoxin was 99% but at concentration 0.8+0.060, 1.2+0.072 and 1.6+0.096% of onion powder + cinnamon oil, the inhibition of aflatoxin production was 100% on day 90. The results showed that onion powder + cinnamon oil completely inhibit aflatoxin production. The effect of nutmeg, mace + mustard oil at various concentrations on aflatoxin production has shown in table 7. It was found that at concentration 1.0+0.048, 1.2+0,060, 1.4+0.072 and 1.6+0.096 % of nutmeg, mace + mustard oil, the inhibition of aflatoxin production was 96, 97, 99 and 100% respectively on day 90. The results showed the nutmeg, mace + mustard oil had strong inhibitory effect against aflatoxin production. The effect of coriander + mustard oil at various concentrations on aflatoxin production has shown in table 8. It was found that at concentration 1.0+0.048% the inhibition of aflatoxin was 98% but at concentration 1.2+0.060, 1.4+0.072 and 1.6+0.096% of coriander + mustard oil, the inhibition of aflatoxin production was 100% on day 90. The results showed that onion powder + cinnamon oil completely inhibit aflatoxin production. Conclusion The inhibitory effect varies widely, depending on the type of natural and chemical inhibitors. Black cumin + cinnamon oil, onion + cinnamon oil, Nutmeg, Mace + mustard oil, coriander + mustard oil completely inhibited the Production of Aflatoxin, whereas turmeric completely inhibit (100%) at concentration 2.0% and at other concentrations (0.6, 1.0, 1.4%) it showed 84, 86, 90% inhibition. The black pepper and ginger significantly inhibit the aflatoxin production while garlic showed low inhibitory effect against the production of aflatoxin. Thus some spices as preservatives may successfully replace synthetic pesticides and provide an alternative method to increase shelf life of food product and protect our staple food from the toxic fungal contamination. References 1. Goto T, Ito Y, Peterson SW, Wicklow DT, 1997. Mycotoxin Production Ability of Aspergillus tamari. Mycotoxins 44: 17-20. 2. Peterson SW, Ito Y, Horn BW, Goto T, 2001. Aspergillus bombycis, a New Aflatoxigenic Species and Genetic Variation in Its Sibling Species, A. nomius. Mycologia 93: 989-703. 3. Ghosh, SK, Desai MR, Pandya GL, 1997. Airborne aflatoxin in the grain processing industries in India. Am Ind Hyg Assoc J 58: 583 586. 4. WHO, 2002. Evaluation of Certain Mycotoxins in Food. WHO Technical Report Series 9061: 11. 5. Revankar SG, 2003. Clinical Implications of Mycotoxins and Stachybotrys. J Med Sci 325:262-264. 6. Hitokoto H, Morozumi S, Wauke T, Sakai S, Kurata H, 1980. Inhibitory effects of spices on growth and toxin production of toxigenic fungi. Applied and Environmental Microbiology 39:818 822.T. 7. Dusanee, S. Yaowapa, S. Prapaporn, S. Saowalak, P. Oraphan and C. Sittichai, 2007, KMITL Science and Technology Journal, 7, 1. 8. Dimić GR, Kocić-Tanackov SD., Karalić D., 2007. Growth inhibition of some Eurotium and Aspergillus species with spice extracts. Zbornik Matice srpske za prirodne nauke 113: 63-70. 9. Sára MC, Marcelo CP, Mario LVR, Caroline LA, Rozane AS, 2004. Effect of powdered spice treatments on mycelial growth, sporulation and production of aflatoxins by toxigenic fungi. Ciênc Agrotec Lavras 28: 856-862. 48
10. Lokman A, 2010. Inhibitory effect of essential oil on aflatoxin activities, African Journal of Biotechnology 9: 2474-2481. 11. Arshad H, Shafqatullah, Javed A, Zia-ur-Rehman, 2012. Inhibition of aflatoxin producing fungus growth using chemical, herbal compounds/spices and plants. Pure Appl Bio 1: 8-13. 12. El-Maraghy SSM, 1995. Effect of some spices as preservatives for storage of Lentil (Lens esculenta L.) Seeds. Foliage Microbiology 40: 490 492. 13. Awuah RT, 1999. Inhibition of fungal colonization of stored peanut kernels with products from some medicinal/culinary plants. Peanut Science 26: 13 17. 14. Yin M, Tsao S, 1999. Inhibitory effect of seven Allium plants upon three Aspergillus species. International Journal of Food Microbiology 49:254 263. 15. Association of Official Analytical Chemist (AOAC).2005 Official method 972.26. 49