Optimisation of aflatoxin B1 reduction in pistachio nuts by kefir grains using statistical experimental methods

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F. Ansari
K. Rezaei
F. Khodaiyan
A. Rahmani



The presence of aflatoxin B1 (AFB1) in pistachio nuts has a serious impact on public health. Kefir grains (KGs) contain several probiotic microorganisms well known for their aflatoxin decontamination effects. In this study, the capacity of KG to remove AFB1 from pistachio nuts was investigated and the detoxification method was optimised using certain statistical approaches. Five variables (toxin concentration, KG level, contact time, incubation temperature and pre-treatment heat level) were considered in the study. Pistachio samples were spiked with 5, 10, 15, 20 and 25 ng/g AFB1 stock solutions and then treated with 5, 10, 15, 20 and 25% (w/w, based on the amount of pistachio paste) KG already heated at 70 and 110 °C. AFB1 concentrations in the samples were determined using high-performance liquid chromatography. Results of the study indicated that: (1) KG caused significant decrease in AFB1; (2) cell viability of KG was not necessary for its decontamination effect; (3) the amount of AFB1 reduction depended on toxin concentration and contact time; and (4) the interactions between toxin concentration and KG level, toxin concentration and contact time as well as incubation temperature and contact time had significant effects on AFB1 reduction. The optimum conditions for the highest AFB1 reduction (96%, compared to the control sample) included a 20 ng/g toxin concentration, 20% KG level, 6 h contact time and 30 °C incubation temperature. To verify the findings of this study, the efficiency of KG needs to be confirmed in vivo.

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Abdulkadar, A.H.W., Al-Ali, A. and Al-Jedah, J., 2000. Aflatoxin contamination in edible nuts imported in Qatar. Food Control 11: 157-160.
Association of Official Analytical Chemists (AOAC), 2000. Aflatoxin B1 and total aflatoxins in peanut butter, pistachio paste, fig paste, and paprika powder – immunoaffinity column LC with post-column derivatization. Official Methods of Analysis 999.07. (17th Ed.), Chapter 49. AOAC International, Gaithersburg, MD, USA.
Bueno, D.J., Casale, C.H., Pizzolitto, R.P., Salvano, M.A. and Oliver, G., 2007. Physical adsorption of aflatoxin B1 by lactic acid bacteria and Saccharomyces cerevisiae: a theoretical model. Journal of Food Protection 70: 2148-2154.
Devegowda, G., Arvind, B.I.R. and Morton, M.G., 1996. Saccharomyces cerevisiae and mannan oligosaccharides to counteract aflatoxicosis in broilers. In: Proceedings of Australian Poultry, Science Symposium, Sydney, Australia, pp. 103-106.
Dini, A., Khazaeli, P., Roohbakhsh, A., Madadlou, A., Pourenamdari, M., Setoodeh, L., Askarian, A., Nader Doraki, Farrokhi, H., Moradi, H. and Khodadadi, E., 2013. Aflatoxin contamination level in Iran’s pistachio nut during years 2009-2011. Food Control 30: 540-544.
El-Nezami, H., Kankaanpaa, P., Salminen, S. and Ahokas, J., 1998. Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1. Food and Chemical Toxicology 36: 321-326.
European Commission (EC), 1993. Commission regulation (EU) No. 315/1993, as regards laying down community procedures for contaminants in food. Official Journal of the European Union L037: 1-3.
European Commission (EC), 2010. Commission regulation (EU) No. 178/2010, amending regulation (EC) No. 401/ 2006 as regards groundnuts (peanuts), other oilseeds, tree nuts, apricot kernels, liquorice and vegetable oil. Official Journal of the European Union L52: 32-43.
Fajgelj, A. and Ambrus, A., 2000. Guidelines for single-laboratory validation of analytical methods for trace-level concentrations of organic chemicals. In: Fajgelj, A. and Ambrus, A. (eds.) Principles and practices of method validation. The Royal Society of Chemistry, London, UK, pp. 179-252.
Food and Agriculture Organization (FAO), 2012. Top production-pistachio, 2010. FAO, Rome, Italy.
Gratz, S., Wu, Q.K., El-Nezami, H., Juvonen, R.O., Mykkänen, H. and Turner, P.C., 2007. Lactobacillus rhamnosus strain GG reduces aflatoxin B1 transport, metabolism, and toxicity in Caco-2 cells. Applied and Environmental Microbiology 73: 3958-3964.
Haskard, C.A., El-Nezami, H.S., Kankaanpää, P.E., Salminen, S. and Ahokas, J.T., 2001. Surface binding of aflatoxin B1 by lactic acid bacteria. Applied and Environmental Microbiology 67: 3086-3091.
Haskard, C.A., El-Nezami, H.S., Peltonen, K.D., Salminen, S. and Ahokas, J.T., 1998. Sequestration of aflatoxin B1 by probiotic strains: binding capacity and localisation. Revue de Medecine Veterinaire 149: 571.
Hathout, A.S., Mohamed, S.R., El-Nekeety, A.A., Hassan, N.S., Aly, S.E. and Abdel-Wahhab, M.A., 2011. Ability of Lactobacillus caseiand Lactobacillus reuteri to protect against oxidative stress in rats fed aflatoxins-contaminated diet. Toxicon 58: 179-186.
Hernandez-Mendoza, A., Garcia, H.S. and Steele, J.L., 2009. Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1. Food and Chemical Toxicology 47: 1064-1068.
Institute of Standards and Industrial Research of Iran (ISIRI), 2010. Food and feed – mycotoxins-maximum tolerated level. National Standard 5925. ISIRI, Teheran, Iran.
Institute of Standards and Industrial Research of Iran (ISIRI), 2011. Food and feed – determination of aflatoxins B and G by HPLC method and clean up by imminoaffinity column-test method. National Standard 6872. ISIRI, Teheran, Iran,
International Agency for Research on Cancer (IARC), 1993. IARC Monographs on the evaluation of carcinogenic risks to humans, Volume 59. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines, and mycotoxins. IARC Press, Lyon, France, pp. 245-395.
Ismaiel, A.A., Ghaly, M.F. and El-Naggar, A.K., 2011. Milk kefir: ultrastructure, antimicrobial activity and efficacy on aflatoxin B1 production by Aspergillus flavus. Current Microbiology 62: 1602-1609.
Joint FAO/WHO Expert Committee on Food Additives (JECFA), 1998. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series 40: 897-913.
Kakisu, E., Bolla, P., Abraham, A.G., de Urraza, P. and De Antoni, G.L., 2013. Lactobacillus plantarum isolated from kefir: protection of cultured Hep-2 cells against Shigella invasion. International Dairy Journal 33: 22-26.
Lee, Y.K., El-Nezami, H., Haskard, C.A., Gratz, S., Puong, K.Y., Salminen, S. and Mykkänen, H., 2003. Kinetics of adsorption and desorption of aflatoxin B1 by viable and nonviable bacteria. Journal of Food Protection 66: 426-430.
Leite, A.M.O., Mayo, B., Rachid, C.T.C.C., Peixoto, R.S., Silva, J.T., Paschoalin, V.M.F. and Delgado, S., 2012. Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis. Food Microbiology 312: 215-221.
Li, F.Q., Yoshizawa, T., Kawamura, O., Luo, X.Y. and Li, Y.W., 2001. Aflatoxins and fumonisins in corn from the high-incidence area for human hepatocellular carcinoma in Guangxi, China. Journal of Agricultural and Food Chemistry 49: 4122-4126.
Liu, J.R. and Lin, C.W., 2000. Production of kefir from soymilk with or without added glucose, lactose, or sucrose. Journal of Food Science 65: 716-719.
Madrigal-Santillán, E., Madrigal-Bujaidar, E., Márquez-Márquez, R. and Reyes, A., 2006. Antigenotoxic effect of Saccharomyces cerevisiae on the damage produced in mice fed with aflatoxin B1 contaminated corn. Food and Chemical Toxicology 44: 2058-2063.
McKenzie, K.S., Sarr, A.B., Mayura, K., Bailey, R.H., Miller, D.R., Rogers, T.D., Norred, W.P., Voss, K.A., Plattner, R.D., Kubena, L.F. and Phillips, T.D., 1997. Oxidative degradation and detoxification of mycotoxins using a novel source of ozone. Food and Chemical Toxicology 35: 807-820.
Mukendi, N., Rollmann, B. and De Meester, C., 1991. Detoxification of aflatoxin B1 by different chemical methods and evaluation of the effectiveness of the treatments applied. Journal de Pharmacie de Belgique 46: 182-188.
Myers, R.H. and Montgomery, R.C., 2002. Response surface methodology: process and product optimization using designed experiments. Wiley, New York, NY, USA.
Oatley, J.T., Rarick, M.D., Ji, G.E. and Linz, J.E., 2000. Binding of aflatoxin B1 to bifidobacteria in vitro. Journal of Food Protection 63: 1133-1136.
Patel, U.D., Govindarajan, P.R.I.Y.A. and Dave, P.J., 1989. Inactivation of aflatoxin B1 by using the synergistic effect of hydrogen peroxide and gamma radiation. Applied and Environmental Microbiology 55: 465-467.
Peltonen, K., El-Nezami, H., Haskard, C., Ahokas, J. and Salminen, S., 2001. Aflatoxin B1 binding by dairy strains of lactic acid bacteria and bifidobacteria. Journal of Dairy Science 84: 2152-2156.
Pereyra, C.M., Cavaglieri, L.R., Chiacchiera, S.M. and Dalcero, A.M., 2010. Fungi and mycotoxins in feed intended for sows at different reproductive stages in Argentina. Veterinary Medicine International 1-7.
Phillips, T.D., Clement, B.V. and Park, D.L., 1994. Approaches to reduction of aflatoxins in foods and feeds. In: Eaton, D.L. and Groopman, J.D. (eds.) The toxicology of aflatoxins: human health, veterinary and agricultural significance. Academic Press, London, UK, pp. 383-4066.
Pizzolitto, R.P., Bueno, D.J., Armando, M.R., Cavaglieri, L., Dalcero, A.M. and Salvano, M.A., 2011. Binding of aflatoxin B1 to lactic acid bacteria and Saccharomyces cerevisiae in vitro: a useful model to determine the most efficient microorganism. In: Guevara-Gonzalez, R.G. (ed.) Aflatoxins – biochemistry and molecular biology. Intech, Rijeka, Croatia, pp. 323-346.
Pizzolitto, R.P., Salvano, M.A. and Dalcero, A.M., 2012. Analysis of fumonisin B1 removal by microorganisms in co-occurrence with aflatoxin B1 and the nature of the binding process. International Journal of Food Microbiology 156: 214-221.
Rahaie, S., Razavi, S.H. and Emam-djomeh, Z., 2010. The ability of Saccharomyces cerevisiae strain in aflatoxin reduction in pistachio nuts. Iranian Journal of Food Science and Technology 7: 81-88.
Raju, M.V.L.N. and Devegowda, G., 2000. Influence of esterified-glucomannan on performance and organ morphology, serum biochemistry and haematology in broilers exposed to individual and combined mycotoxicosis (aflatoxin, ochratoxin and T-2 toxin). British Poultry Science 41: 640-650.
Samarajeewa, U., Sen, A.C., Cohen, M.D. and Wei, C.I., 1990. Detoxification of aflatoxins in foods and feeds by physical and chemical methods. Journal of Food Protection 53: 489-501.
Set, E. and Erkmen, O., 2010. The aflatoxin contamination of ground red pepper and pistachio nuts sold in Turkey. Food and Chemical Toxicology 48: 2532-2537.
Shetty, P.H., Hald, B. and Jespersen, L., 2007. Surface binding of aflatoxin B1 by Saccharomyces cerevisiae strains with potential decontaminating abilities in indigenous fermented foods. International Journal of Food Microbiology 113: 41-46.
Slizewska, K., Nowak, A., Libudzisz, Z. and Blasiak, J., 2010. Probiotic preparation reduces the faecal water genotoxicity in chickens fed with aflatoxin B1 contaminated fodder. Research in Veterinary Science 89: 391-395.
Suzuki, T., Noro, T., Kawamura, Y., Fukunaga, K., Watanabe, M., Ohta, M., Sugiue, H., Sato, Y., Kohno, M. and Hotta, K., 2002. Decontamination of aflatoxin-forming fungus and elimination of aflatoxin mutagenicity with electrolyzed NaCl anode solution. Journal of Agricultural and Food Chemistry 50: 633-641.
Topcu, A., Bulat, T., Wishah, R. and Boyac?, I.H., 2010. Detoxification of aflatoxin B1 and patulin by Enterococcus faecium strains. International Journal of Food Microbiology 139: 202-205.
Tramšek, M. and Goršek, A., 2007. A novel approach for assuring and following inoculum activity during kefir grains growth studies – application of dynamic pH profiles. Acta Chimica Slovenica 54: 693-699.
Wild, C.P. and Turner, P.C., 2002. The toxicology of aflatoxins as a basis for public health decisions. Mutagenesis 17: 471-481.
Wilson, D.M. and Ryne, G.A., 1993. Factors affecting Aspergillus flavusgroup infection and aflatoxin contamination of crops. In: Eaton, D.L. and Groopman, J.D. (eds.) The toxicology of aflatoxins. Academic Press Inc., San Diego, CA, USA, pp. 309-325.
Zhang, Q., Xiong, K., Tatsumi, E. and Liu, H.J., 2012. Elimination of aflatoxin B1 in peanuts by acidic electrolyzed oxidizing water. Food Control 27: 16-20.
Zheng, Z., 2011. World production and trade of pistachios: the role of the US and factors affecting the export demand of US pistachios. University of Kentucky, Lexington, KY, USA, 123 pp.
Zhou, J., Liu, X., Jiang, H. and Dong, M., 2009. Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis. Food Microbiology 26: 770-775.
Zuo, R.Y., Chang, J., Yin, Q.Q., Wang, P., Yang, Y.R., Wang, X., Wang, G.Q. and Zheng, Q.H., 2013. Effect of the combined probiotics with aflatoxin B1-degrading enzyme on aflatoxin detoxification, broiler production performance and hepatic enzyme gene expression. Food and Chemical Toxicology 59: 470-475.