REDUCTION, DECONTAMINATION AND DETOXIFICATION OF MYCOTOXINS IN FOOD Sardjono Faculty of Agricultural Technology Gadjah Mada University CONTENT : • Introduction • Occurrence of mycotoxins in foods • The role of food processing on reduction, decontamination and detoxification of mycotoxins • Detoxification by indigenous strain • Conclusion population : 220 million INDONESIA Self sufficiency in the production of basic food agricultural crops (cereals, other carbohydrate source, TROPICAL COUNTRY beans) High humidity ( RH > 78%) Warm temperature (25 -32˚C) Ideal for fungal growth Improperly / poor handling and storage condition Mycotoxigenic fungi and occurrence of mycotoxins MYCOTOXICOSES PRODUCTION HARVEST AND POST HARVEST HANDLING STORAGE RAW MATERIALS FOR FOOD INDUSTRY PROCESSING CONSUMPTION MYCOTOXINS PROCESSED FOODS CONTAMINATION IN STORAGE FOOD CHAIN Dry and heat resistant fungi? THE FIVE FUNGAL MYCOTOXINS IN AGRICULTURAL PRODUCT MYCOTOXINS COMODITY PRODUCER HEALTH RISK Aflatoxins Peanut, corn, grain and Aspergillus flavus Carsinogenic, (B1, B2, G1, G2) their product Aspergillus parasiticus embryotoxic Aflatoxin M1 Milk AflatoxinsAflatoxins--ContamintedContaminted Carsinogenic feed Fumonisins Corn, wheat and other Fusarium moniliforme Carsinogenic ( B1,B2 ) grain Accut Fusarium graminearum Carsinogenic Zearalenone Corn, wheat, barley F. Culmorum Reproduction F. crookwellense system Deoxynivalenol Fusarium graminearum Nivalenol Corn, wheat, barley F. Culmorum Carsinogenic F. crookwellense Reproduction system Ochratoxin A Aspergillus ochraceus (OTA) Coffee, cocoa, wheat Penicillium vericosum carsinogenic Natural co-occurrence of aflatoxins and Fusarium mycotoxins in corn Yamashita, A., Yoshizawa T., Aiura Y., Sanchez P.C., Dizon E.I., Arim R.H. and Sardjono. 1995. Fusarium mycotoxins (Fumonisins, Nivalenol and Zearalenone ) and aflatoxins in corn from South East Asia. in corn from South East Asia (Yamashita, et al (1995)) Mean (range) concentration (ng/g) in positive sample FMB1 FMB2 AFB1 AFB2 Philippines 419 286 49 14 (57-1820) (58-1210) (1-430) (1-78) Thailand 1580 251 63 14 (63-8,800) (50-1400) (1-606) (1-73) Indonesia 843 442 352 90 (226-1780) (231-556) (1-3300) (1-680) Abbrevation : FMB1 and FMB1, Fumonisin B1 and B2 respectively; AFB1 and AFB2, Aflatoxin B1 and B2, respectively Norhayati Ali, Sardjono, Yamashita, A., and Yoshizawa, T., 1998. Natural Co- occurence of Aflatoxins and Fusarium Mycotoxins (fumonisins, deoxynevalenol, Centralnivalenol Java*) dan zearalenone) in Corn From Indonesia. Concentration (ng/g) Concentration (ng/g) Mycotoxins Mycotoxins Average Average AFB1 102 FMB1 788 AFB2 20 FMB2 182 AFG1 8 FMB3 108 DON 27 NIV 109 ZEA 12 Important issue that corn from tropical country in the same growing conditions, can be simultaneously contaminated with aflatoxins, fumonisins, nivalenol, deoxynivalenol and zearalenone Level of Fumonisins contamination in maize-based foods and feed from Yogyakarta , Indonesia (Nuryono et al., 2004)* Sample category Sample number Fumonisins (ppb) Analyzed positive Range Average Industrially-produced 24 14 22.8-104.6 50.1 foods Small industry-products 17 16 12.9-234.1 54.4 Maize flour 4 - -- Maize for foods 9 5 68.0-2471 1275 Formulated feed 30 29 10.7-2257 968 * Mycotoxin Research 20(2004):2-9 Level of Zearalenone contamination in maize-based foods and feed from Yogyakarta , Indonesia (Nuryono et al., 2005)* Sample categorySample number Zearalenone (ppb) Analyzed positive Range Average Industrially-produced foods 21 4 5.5-18.6 9.1 Small industry-products 17 5 21.2-526 199 Maize for foods 13 2 6.1-6.3 6.2 Formulated feed 21 18 6.3-526 25.5 * J. of Food Control 16(2005):65-71 HUMAN ORGAN TARGET HIGH LOAD! CONTAMINATED RAW MATERIALS DECONTAMINATION DETOXIFICATION SAFE PRODUCTS FOODFO PROCESSINGPR SORTATION PHYSICAL & •NOT FEASIBLE FOR LARGE SCALE CHEMICAL •SAFE ? PRETREATMENT •PRODUCTION COST? Decontamination should : • Complete inactive, destroy, remove the toxins, reduce to acceptable levels • Not produce toxic residues in food • Preserve the nutritive value • Not alter acceptability or technological properties • Be integrated if possible, into regular food-processing and preparation step • Be cost effective • Not destroy or damage the equipment FOOD PROCESSING BIOLOGICAL PHYSICAL CHEMICAL REDUCTION, DECONTAMINATION, DETOXIFICATION ? BINDING decontamination BIOLOGICAL METHOD ENZYMATIC detoxification BINDING Organisms Action / mechanism Refference A few strains of LAB to bind AFB1 in food model El-Nazami et al., 1998 Pierides et al., 2000 Haskard et al., 2001 non covalently binding AFB1 Zhang and Ohta, 1993 El-nazami et al., 1998 Turbic et al.,2002 Lactobacillus ramnosus bind to viable and non viable cell El- Nazami et al., 1998 association to hydrophobic pocket Haskard et al., 2000 Staphylococcus aureus to bind AFB1 Oatley et al., 2000 and Escherichia coli Lactobacillus and to bind AFB1 El-Nazami et al., 2000 Propionibacterium L. rhamnosus GG and to bind Zearalenone and its dere- El-Nazami et al., 2002 L rhamnisus LC705 vates Aspergillus oryzaeKKB4 to bind AFB1 (reduce in SLF) Sardjono et al.,2004 Neurospora spp to bind AFB1 (reduce in SLF) Sigit Setyabudi et al., 2005 Aflatoxins detoxification ORGANISMS MYCOTOXINS MEDIUM REFFERENCES Flavobacterium auranotiacum Aflatoxin SLF Ciegler et al., 1966 Aflatoxin M1 milk Lillehoj et al., 1971 Aflatoxin peanut milk Hao and Brackett, 1995 Coryenebacterium rubrum Aflatoxin Mann and Rehm, 1976 Candida lipolytica Aflatoxin Mann and Rehm, 1976 Aspergillus niger Aflatoxin Mann and Rehm, 1976 Trichoderma viridae Aflatoxin Mann and Rehm, 1976 Mucor ambigous Aflatoxin Mann and Rehm, 1976 Aspergillus niger Aflatoxin Faraj et al., 1993 Rhizopus sp Aflatoxin Bol and Smith, 1989 Nout, 1989 Neurospora sp Aflatoxin Nout, 1989 Aflatoxin B1 SLF Sigit., FMC et al., 2005 Aspergillus oryzae KKB4 Aflatoxin B1 SLF Sardjono et al., 2004 Lactic acid bacteria (moromi) Aflatoxin B1 SLF Sardjono et al., 2005 RESEARCH ON ISOLATION OF INDIGENEOUS POTENTIAL STRAINS Aspergillus oryzae KKB4 Extracellular enzymes DEGRADATION DETOXIFICATION Degradation of lactone KOJI Reduction Opening difuran ring ( Sardjono et al., 2004) TOXICITY TEST THE GROWTH OF Bacillus megaterium IN ENZYMATIC DEGRADATION PRODUCT ( Sardjono, et al., 2004) A. Normal growth. B Growth on degradation product. C Growth on reaction product form heated enzymes and AFB1 DETOXIFICATION BY SALT TOLERANT LACTIC ACID BACTERIA ISOLATED FROM MOROMI ( Sardjono, et al., 2005) Lactobacillus spSS MOROMI Lactobacillus spPS Lactobacillus spBG 3 strains of yeast AFB1 DEGRADATION PRODUCT by Lactobacillus spBG Fusarium toxins detoxification MICROORGANISMS MYCOTOXINS MECHANISMS REFERRENCE Aspergillus niger DON; T-2 Acetylation, deacetylation El Sharkawy and Abbas, 1991 Mucor muceda DON; T-2 Acetylation, deacetylation El Sharkawy and Abbas, 1991 Agrobacterium rhizobium DON Oxidized to 3-keto-4-DON Shima et al., 1997 Fusarium nivale DON acetylation of hydroxyl Yoshizawa & Morooka, 1975 Fusarium graminearum T-2 Acetylation Yoshizawa et al., 1980 Colonetria nivalis T-2 Acetylation Yoshizawa et al., 1980 "corn enzymes" DON reduction Miller and Young, 1985 Arabidopsis thalia DON Hydroxyl group of DON Poppenberger et al., 2003 Fusarium moniliforme FMB1 Alberts et al., 1990 Exophiala spinifera FMB1 Hydrolyzed ester bound Duvick, 1994 Rhinoclodiella atrovirensa FMB1 Hydrolyzed ester bound Duvick, 1994 Exophilia spinifera FMB1 Oxidative deaminasi Blackwell et al., 1999 Thamnidium elegans ZEA Zea-4-O-ß-glucoside Kamimura, 1986 Mucor baenieri ZEA Zea-4-O-ß-glucoside El Sharkawy and Abul-Hajj,1987 Rhizopus spp ZEA Zea-4-O-ß-glucoside Kamimura, 1986 OTA and Patulin MICROORGANISMS MYCOTOXIN MECHANISM REFFERENCE Actinobacter calcoaceticus OTA Degradation Bata and Lasztity, 1999 Aspergillus fumigatus OTA Detoxification Varga et al., 2000 Aspergillus niger OTA Degradation Varga et al., 2000 Aspergillus from grape OTA Degradation Abrunhosa et al., 2002 Paecilomyces Patulin Degradation Anderson et al., 1979 yeast from alcoholic fermentation Patulin Reduction Harwig et al, 1979 Stinson et al., 1978 yeast Patulin Convert to acladiol E & Z Moss and Long, 2002 Lactobacillus plantarum Patulin Reduced by intra. Enzym R.Shapira (unpublished) HEATING TREATMENT MATERIALS MYCOTOXINS EFFECT REFFERENCE 200°C, 12 min cofee bean aflatoxin 79% reduction Scott, 1984 200°C, 15 min 94% reduction 100°C, 2 h cottonseed meal 30% MC aflatoxin 50% reduction Mann, et al., 1967 100°C, 2 h cottonseed meal 6,60% MC 50% reduction 175°C corn, dry condition citrinin decomposed Kitabatake et al, 1991 160°C corn, moist citrinin destroyed Roasting (190-227°C cofee bean OTA 69-96% reduction van der Stegen, 2001 for 5-20 min) 200-250°C dry or wet grain OTA complete destruc- Boudra et al.,1995 tion 150°C, 44 h corn Zearalenone not destryed Bennet et al.,1980 150°C- 200°C corn Fumonisin B1 87-100% destruct. 218°C, 15 min cornmeal with5 µg/g Fumonisin B1 complete loss Castelo et al.,1998 Baking DON 50% loss Tanaka et al.,1986 Extrusion cooking flour DON 95% reduction Cazzaniga et al.,2001 Extrusion cooking flour aflatoxin 10-25% reduction Elias-Orozko, 2001 Extrusion (0.3% lime; 1.5% H2O2 tortillas aflatoxin complete destroy 100°C, + activated apple juice patulin almost all Kadakal et al.,2002 charcoal Kadakal and Nas, 2003 CLEANING, DEHUSKING POLISHING CEREALS DRY MILLING PROCESSING WET MILLING CHEMICAL PHYSICAL BAKING BIOLOGICAL ROASTING MATERIALS NS CONTAMINATED NS CONTAMINATED FRYING RAW M RAW COOKING EXTRUSION MYCOTOXIN PRODUCTS?? MALTING & BREWING CLEANING, DEHUSKING,