REDUCTION,

DECONTAMINATION AND

DETOXIFICATION OF

SardjonoFaculty of Agricultural Technology

Gadjah Mada University

DETOXIFICATION OF

MYCOTOXINS IN FOOD

• Introduction

• Occurrence of mycotoxins in foods

• The role of food processing on reduction,

CONTENT :

• The role of food processing on reduction,

decontamination and detoxification of

mycotoxins

• Detoxification by indigenous strain

• Conclusion

INDONESIA

TROPICAL COUNTRY

population : 220 million

Self sufficiency in the production of basic food �agricultural crops (cereals, other carbohydrate source, beans)

High humidity ( RH > 78%)

Warm temperature (25 -32˚C)Ideal for fungal growth

Improperly / poor handling and storage condition

MYCOTOXICOSES

Mycotoxigenic fungi and occurrence of mycotoxins

STORAGE

PRODUCTION

RAW MATERIALS FOR

HARVEST AND POST

HARVEST HANDLING

PROCESSED FOODS

PROCESSING

RAW MATERIALS FOR

FOOD INDUSTRY

MYCOTOXINS

CONTAMINATION IN

FOOD CHAINSTORAGE

CONSUMPTION

Dry and heat

resistant fungi?

MYCOTOXINSMYCOTOXINS COMODITYCOMODITY PRODUCERPRODUCER HEALTH RISKHEALTH RISK

AflatoxinsAflatoxins

(B1, B2, G1, G2)(B1, B2, G1, G2)

Peanut, corn, grain and Peanut, corn, grain and their producttheir product

AspergillusAspergillus flavusflavus

AspergillusAspergillus parasiticusparasiticus

Carsinogenic, Carsinogenic, embryotoxicembryotoxic

Aflatoxin M1 Aflatoxin M1 MilkMilk AflatoxinsAflatoxins-- ContamintedContamintedfeed feed

CarsinogenicCarsinogenic

Fumonisins Fumonisins

( B1,B2 )( B1,B2 )

Corn, wheat and other Corn, wheat and other graingrain

FusariumFusarium moniliformemoniliforme CarsinogenicCarsinogenic

AccutAccut

THE FIVE FUNGAL MYCOTOXINS IN AGRICULTURAL PRODUCT

ZearalenoneZearalenone Corn, wheat, barleyCorn, wheat, barley

FusariumFusarium graminearumgraminearum

F. F. CulmorumCulmorum

F. F. crookwellensecrookwellense

CarsinogenicCarsinogenic

Reproduction Reproduction systemsystem

DeoxynivalenolDeoxynivalenol

NivalenolNivalenol Corn, wheat, barleyCorn, wheat, barley

FusariumFusarium graminearumgraminearum

F. F. CulmorumCulmorum

F. F. crookwellensecrookwellense

CarsinogenicCarsinogenic

Reproduction Reproduction systemsystem

Ochratoxin AOchratoxin A

(OTA)(OTA) Coffee, cocoa, wheatCoffee, cocoa, wheat

AspergillusAspergillus ochraceusochraceus

PenicilliumPenicillium vericosumvericosum carsinogeniccarsinogenic

Natural co-occurrence of aflatoxins and

Fusarium mycotoxins in corn

in corn from South East Asia (Yamashita, et al (1995))

Mean (range) concentration (ng/g) in positive sample

FMB1 FMB2 AFB1 AFB2

Yamashita, A., Yoshizawa T., Aiura Y., Sanchez P.C., Dizon E.I., Arim R.H.

and Sardjono. 1995. Fusarium mycotoxins (Fumonisins, Nivalenol andZearalenone ) and aflatoxins in corn from South East Asia.

Philippines

Thailand

Indonesia

419

(57-1820)

1580

(63-8,800)

843

(226-1780)

286

(58-1210)

251

(50-1400)

442

(231-556)

49

(1-430)

63

(1-606)

352

(1-3300)

14

(1-78)

14

(1-73)

90

(1-680)

Abbrevation : FMB1 and FMB1, Fumonisin B1 and B2 respectively; AFB1 and AFB2,

Aflatoxin B1 and B2, respectively

Central Java*)

Mycotoxins Concentration (ng/g)

Average Mycotoxins

Concentration (ng/g)

Average

AFB1

AFB2

AFG1

102

20

8

FMB1

FMB2

FMB3

788

182

108

Norhayati Ali, Sardjono, Yamashita, A., and Yoshizawa, T., 1998. Natural Co-

occurence of Aflatoxins and Fusarium Mycotoxins (fumonisins, deoxynevalenol, nivalenol dan zearalenone) in Corn From Indonesia.

AFG1 8 FMB3

DON

NIV

ZEA

108

27

109

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

Industrially-produced foods

Sample numberAnalyzed positive

24 14

Fumonisins (ppb)Range Average

22.8-104.6 50.1 foodsSmall industry-productsMaize flourMaize for foodsFormulated feed

17 164 -9 530 29

12.9-234.1 54.4- -

68.0-2471 127510.7-2257 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 category Zearalenone (ppb)

Range Average

Sample number

Analyzed positive

Industrially-produced foods

Small industry-products

Maize for foods

Formulated feed

5.5-18.6 9.1

21.2-526 199

6.1-6.3 6.2

6.3-526 25.5

21 4

17 5

13 2

21 18

* J. of Food Control 16(2005):65-71

HUMAN ORGAN TARGET

FO

OD

P

RO

CES

SIN

G

CONTAMINATED

RAW MATERIALS

SAFE PRODUCTS

DECONTAMINATION

DETOXIFICATION

HIGH LOAD!

FO

PR

SORTATION

PHYSICAL &

CHEMICAL

PRETREATMENT

•NOT FEASIBLE FOR LARGE SCALE

•SAFE ?

•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• 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 ?

BIOLOGICAL

BINDING

decontamination

BIOLOGICAL

METHOD ENZYMATIC

detoxification

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

BINDING

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, 1976Candida 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

DETOXIFICATIONDETOXIFICATION

Reduction

Degradation of lactone

Opening difuran ring

KOJI

( 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 spBGLactobacillus spPS

3 strains of yeast

MOROMI

AFB1

DEGRADATION

PRODUCT by

Lactobacillus spBG

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

Fusarium toxins

detoxification

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

MICROORGANISMS MYCOTOXIN MECHANISM REFFERENCE

Actinobacter calcoaceticus OTA Degradation Bata and Lasztity, 1999

Aspergillus fumigatus OTA Detoxification Varga et al., 2000

OTA and Patulin

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)

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)

HEATING

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

CEREALS

PROCESSINGCHEMICAL

PHYSICAL

BIOLOGICAL

CLEANING,

DEHUSKING

POLISHING

DRY MILLING

WET MILLING

BAKING

ROASTING

FRYINGNS

CO

NTA

MIN

AT

ED

MA

TE

RIA

LS

BIOLOGICALFRYING

COOKING

EXTRUSION

MALTING &

BREWING

PRODUCTS??

MY

CO

TO

XIN

RA

W M

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, 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 MILLINGMost 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 flourDON and ZEA : highest in bran and lowest in the flour

FumonisinFumonisin 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

Distribution of fumonisins in wet milling processKamimura, H.,1999

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)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 0.11G2 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

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%

COOKING AND CANNING

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

EXTRUSIONA 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 corn survive Martinez, 1989

dough

AFB1 Corn flour (+ 50µg/kg) 25% reduction Cazzaniga et al., 2001AFB1 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. 20%) 75% lost Camargo et al., 1989

OTA 50% moist; 150C partially decomposed Boudra et al.,1995)

DON quite stable Wolf-Hall et al., 1999

ZEA varried with screw configuartion 65-83% reduction Ryu et al.,1999

FUMONISIN extrusion of dry mill product 30-90% reduced Saunders et al.,2001

FMB1 & FMB2 cooking, extrusion, gelatinization 30-55% reduction Meuster, 2001

FMB1 & FMB2 corn flakes, extrusion and roasting 60-70% lost Degirolamo, et al.,2001

BREWING /

ALCOHOLIC

FERMENTATION DECOMPOSED /

CHANGE :

OTA

TRICHOTHECENETRICHOTHECENE

ISO TRICHOTHECENE

PATULIN

REFF : Scott et al,1992; Schwarz et al.,1995

DID NOT CHANGE � DON, FUMONISIN

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 :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 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 •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