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 Aflatoxins--Contaminted Aflatoxins Contaminted 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. F. crookwellense system Deoxynivalenol Fusarium graminearum Nivalenol Corn, wheat, barley F. Culmorum Carsinogenic F. 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 RAWMATERIALS CONTAMINATED PRETREATMENT CHEMICAL PHYSICAL & SORTATION HIGHLOAD!

FOODFO • • • PRODUCTION COST? SAFE ? NOT FEASIBLEFOR LARGE SCALE PROCESSINGPR DETOXIFICATION DECONTAMINATION SAFE PRODUCTS 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 modelzami El-Na 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, 1998 et al. association to hydrophobic pocket Haskard et00 al., 20 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-Nazami2002 et al., 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 MYCOTOXINSMEDIUM REFFERENCES Flavobacterium auranotiacumAflatoxin SLF Ciegler et al., 1966 Aflatoxin M1milk 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 MYCOTOXINSMECHANISMS REFERRENCE

Aspergillus niger DON; T-2 Acetylation, deacetylationEl Sharkawy and Abbas, 1991 Mucor muceda DON; T-2 Acetylation, deacetylationEl Sharkawy and Abbas, 1991 Agrobacterium rhizobium DON Oxidized to 3-keto-4-DONShima 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 MYCOTOXINMECHANISM REFFERENCE

Actinobacter calcoaceticusOTA 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 fermentationPatulin Reduction Harwig et al, 1979

Stinson et al., 1978 yeast Patulin Convert to acladiolMoss E & Zand Long, 2002

Lactobacillus plantarumPatulin Reduced by intra. R.ShapiraEnzym (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°Ccofee 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, POLISHING

Generate little heat ‰ no significant thermal breakdown Molds and mycotoxins are concentrated in dust, broken grains or seed coat Surface cleaning, washing and drying, steaming ‰ reduction of AFB1 were 35%, 47-85% and 90-93% (Kutit and Merko, 1991) Polishing ‰ remove NIV, DON and ZEA, all were found in bran fraction, except NIV also found in pearled barley In maize commercial mill ‰ AF and FMB reduced by 30-40%, no reduction on ZEA DRY MILLING Most mycotoxins tend to cencentrated in the bran fraction and outer layer of grain ‰ produce fraction with lower concentartion of mycotoxins

AFB1 : products (cornmeal or four contaminated only 7-10% f from original AFB1 OTA : lower in product, higher in bran and others fraction DON and ZEA : highest in bran and lowest in the flour Fumonisin Fumonisin concentration (ug/kg) Corn fraction B1 B2 B3 Whole corn 1540 716 152 Grits 135 39 10 "C" grade flour 358 122 46 Corn meal 148 52 28 Germ + bran 4210 2010 447 Broggi et al ., 2002 WET MILLING

PRODUCE STARCH AT EXTREEMELY LOW LEVEL OF MYCOTOXINS

AFB : were lost in steep liquor and concentrated in the ger ZEA,NIV, DON ; similar result (NIV and DON soluble in steep liquor)

B1 2.59 Zea mays B2 0.45 Feed (ppm) Cleaning Corn step B1 0.02 Steeping liquor

Milling Germ B1 0.15 B2 0.05 Fraction & Fiber B1 0.45 Separation B2 0.25 Gluten B1 0.72 B2 0.28 Dry Sacharification

B1 ND Corn Starch Convet into Corn oil B2 ND Sugar

Kamimura, H.,1999 Distribution of fumonisins in wet milling process BAKING, ROASTING, FRYING ( Scott 1983, Reiss 1978, Subirade 1996, Scudamore et al., 2003, Samar et al, 2001. Castelo et al., 2001

AFB1 : destroyed during dough fermentation lost during baking ( up to 25%) almost complete destruction in fried snack (pretreatment : 3% NaOH, autoclaving and frying 15 min at 196°C) OTA : Little lost during baking (cacao for flavor?) DON : The maximum reduced during dough fermnt. 46% FUMONISIN : Reduced about 28% (baking at 200°C for 20 min) No significance lost spiked corn fried at 170°C, degradation occurred started at 180°C Frying corn chips at 190°C for 15 min.‰ 67% (ppb) Raw peanut B1 0.24-2.38 Shelled peanut B2 Tr- 0.26 Steep tankes G1 ND-0.19 B1 0.034 G2 ND-Tr B2 0.012 Steep water Steep peanut B1 0.73 B1 0.66 B2 0.15 B2 0.09 Coat splitting G1 0.16 Seed coat Peanut kernel B1 0.50 G2 0.02 Sorting B2 0.11

B1 0.64 Damage kernel kernel (normal) ND B2 0.06 Frying Fry kernel B1 Tr B1 0.42 Frying oil B2 Tr Product ND

Distribution of aflatoxins in butter-peanut process COOKING AND CANNING

Nixtamalization (alkaline cooking and heating) Technology used for preparing traditional foods from corn (tortillas, for example), significantly reduced the concentration of aflatoxin. (Ulloa-Sosa and Shroeder, 1969; Price and Jogersen, 1985; Elias-Orozco et al., 2002) ‰ UP TO 79% REDUCED

NIV, DON : reduced 60-100% in aqueous bicarbonate at 10-50% by weight (Lauren and Smith, 2001) reduced 40% and 60% boiled noodle at 98C for 10 min ZEA : not reduced by 12 days at 110C heating after treatment with bicarbonate (Kamimura, 1980) T2 toxin: 30% reduced in the same heating (Kamimura,,1980) FUMONISIN : 11-15% reduced; 90% after alkaline steeping OTA : stable during cooking; citrinin ‰citrinin H2 EXTRUSION A system in which raw materials are passed through continuous processing machinery within which compressed and sheared at elevated temperature and pressure ‰ resident time is short

MYCOTOXINS PROCESS EFFECTS REFF

AFB1 & AFB2 Extrusion at 150C, spiked cornsurvive Martinez, 1989 dough AFB1 Corn flour (+ 50µg/kg) 25% reduction Cazzaniga et al., 2001 DON Corn four (+5 mg/kg) 95% reduction AFB1 & AFB2 Extrusion rice flour (140-200C; screw speed 130 rpm; moist. 75%20%) lost Camargo et al., 1989 OTA 50% moist; 150C partially decomposedBoudra et al.,1995) DON quite stable Wolf-Hall et al., 1999 ZEA varried with screw configuartion65-83% reduction Ryu et al.,1999 FUMONISIN extrusion of dry mill product 30-90% reduced Saunders et al.,2001 FMB1 & FMB2 cooking, extrusion, gelatinization30-55% reduction Meuster, 2001 FMB1 & FMB2 corn flakes, extrusion and roasting60-70% lost Degirolamo, et al.,2001 BREWING / ALCOHOLIC

FERMENTATION DECOMPOSED / CHANGE :

OTA TRICHOTHECENE ISO TRICHOTHECENE PATULIN

DID NOT CHANGE ‰ DON, FUMONISIN

REFF : Scott et al,1992; Schwarz et al.,1995 THE CONTRIBUTION OF SEVERAL FOOD PROCESSING ON REDUCTION OF MYCOTOXINS

1. EDIBLE OIL PROCESSING ‰ Aflatoxin were accumulated into “soap stock” during neutralization process . Peanut oil without purification are harmfull ‰ example : aflatoxin in “instant noodle”

2. WHEAT PROCESSING ‰ mycotoxins (espc.fumonisin) are accumulated in bran, germ and steeping water, small part stilll in final products

3. CORN STRACH PROCESSING‰ mycotoxins are accumulated in germ, bran and “corn steep liquor “‰ for feeding. In case of “marning” processing, reduction of aflatoxins occurred during boiling in lime solution

4. PEANUT BUTTER PROCESSING ‰ aflatoxin found in seed coat, steeping water, frying oil and trace are remain in final product

5. CIDER PROCESSING : safe for patulin 6. COFFEE PROCESSING : OTA remove by initial cleaning, reduction in roasting (16% from starting concentration )

AFB 93% lost during roasting

7. BEVERAGE : part of mycotoxins were removed by sorting the decayed fruit. Patulin 40% removed by clarification procedure NIXTAMALIZATION : Not effective for ZEA, FUMONISINS and OTA

NEUTRALIZATION : Effective for AFB1

ROASTING : reduce part of mycotoxins , almost removed AFB by lime cooking as pretreatment

BAKING : Reduce relatively high part of mycotoxins, except OTA

FERMENTATION : destroyed by binding, enzymatic detoxification, other reactions CONCLUSION

•Prevention of mycotoxins contamination is not always possible? •Procedure suitable for destruction of multiple mycotoxins still limited •Almost impossible to establish physical and chemical treatment would be useful for destruction mycotoxins simultaneously •Biological methods more promising for detoxification •Be possible to introduce modification to commercial process to get the reduction of mycotoxins in retail product