Reduction of Mycotoxins (Aflatoxin) in Distillers Grains – Project Update

Hu Shi, Graduate Research Assistant

Dissertation Advisors:Klein Ileleji, PhD., Associate Professor & Ext. EngineerRichard Stroshine, PhD., ProfessorAgricultural and Biological Engineering Department

NC-213 2010 Team Award: Reduction of Mycotoxin Levels in Distillers Grains

Purdue (Ileleji & Stroshine) & NDSU (Simsek)

Grain Postharvest Quality Group

Introduction Aflatoxins are secondary

metabolites produced by a fungus. Two agricultural important fungi species: Aspergillus flavus and Aspergillus parasiticus

Aflatoxins are highly toxic, carcinogenic, immunosuppressive

Aflatoxins are heat stable; chemical and biological methods tend to affect the quality of corn

http://www.icrisat.org/aflatoxin/aflatoxin.asp

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Approach Investigate sorting out of infected kernels and

cleaning grain off immature and fines on mycotoxin reduction. Goal: reduce mycotoxin levels prior to bioprocessing

corn into ethanol.

Investigate mycotoxin reduction in distillers grains by food additives, microwave heating and ozone treatment during DDGS production. Goal: reduce mycotoxin in processed coproduct.

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Mycotoxins reduction: A systematic approach

Sorting Results: by Simsek Lab, NDSU

Light-2 Dark-2

Light-1 Dark-1

1 Pass

2 Passes

3 Passes

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Equipment

Screen Cleaner – 13/64 and 17/64 in round hole screens

Density segregation:

Gravity Table

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Flow diagram

17/64 overs

17/64 Passes

Screen Cleaner with 17/64 in Sieve

Run 2 timesHigher Density Kernel

Lower Density Kernel

Gravity Table

Run 2 times

Screen Cleaner with 13/64 in Sieve

13/64 overs

13/64 Passes (fines)

· Weight· Moisture content· Density· Aflatoxins level

Recombined

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Aflatoxin – quantification of levels

Based on ELISA method(enzyme-linked immunosorbent assay )

Major Components: QuickScan Scanner Test Strips

Results of cleaning and Sorting Test

Operation Percent removed(wt, %)

Percentreduction(%)

Aflatoxins level (Mean±Std, ppb)

Retained Removed

Screen 13/64 in 10.1 83.8 30 ±8 1404±143

Screen 17/64 in (run1) 3.3 1.8 27 ±1 246±27

Screen 17/64 in (run2) 2.8 9.4 13 ±2 197±32

Gravity Table (run1) 4.8 12.6 7±1 384±27

Gravity Table (run2) 5.4 16.4 <LOD 342±31

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Aflatoxins level of Original Sample: 185±28 (Mean±Std, ppb)

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ConclusionFor at least some corn samples: There is significant difference in size, shape, and

density between moldy and sound corn kernels Based on these differences, it is possible to

reduce the overall aflatoxin level in corn using cleaning and sorting

The Gravity table gave a significant reduction in addition to the 17/64 (in) screen cleaner for a sample having lower density, contaminated kernels

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Approaches to reduce aflatoxins in coproducts

Chemical: Sodium bisulphite (Concentration, Treatment time, temperature)

Ozone treatment using high voltage atmospheric plasma (Concentration, Treatment time)

Thermal: Microwave Heating (Heating Temperature, Treatment time)

Combination: Sodium bisulphite + Microwave heating, Sodium bisulphite+ozone

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Thermal and pH stability of AFB1 in DWG and CDS

Variables levels

Temperature 24, 60, 90 °C

pH 4.5(initial), 7, 10

3×3 Factorial design with triplicates

Experimental design Set up

Cooking time: 1.5 h Temperature was set in water bath and

monitored by thermocouple pH of samples were conditioned by

adding NaOH solutions/pellets

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Temperature and pH effect on AFB1 in DWG

24 60 900

20

40

60

80

100

pH 4.5pH 7pH 10

Temperature (°C)

AFB

1 le

vel (

ppb)

At α=0.05 significant level Both Temperature and pH

had significant effect Temperature level 90°C is

significant different from 24 and 60°C

pH level 10 is significant different from pH 4.5 and 7

Alkaline cooking at 90°C for 1h completely degraded AFB1 in DWG

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Temperature and pH effect on AFB1 in CDS

At α=0.05 significant level Both Temperature and pH

had significant effect Temperature levels are

significant different from each other

pH levels are significant Alkaline cooking at 90°C for

1h resulted greatest degradation (75%)24 60 90

0

20

40

60

80

pH 4.5pH 7pH 10

Tempearature (°C)

AFB

1 le

vel (

ppb)

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Food additives Selected Food additives that were previously studied for

aflatoxin detoxification Sodium bisulfite (Doyle and Marth 1978; Moerck, Mcelfresh et al. 1980; Hagler et al 1982)

Sodium chlorite (Trager and Stoloff 1967; Yang 1972; Natarajan, Rhee et al. 1975; Rhee, Natarajan et al. 1977)

Citric acid ( Mendez-Albores, Arambula-Villa et al. 2005; Mendez-Albores, Del Rio-Garcia et al. 2007)

Ammonium Persulfate(Tabata, Kamimura et al. 1994; Mutungi, Lamuka et al. 2008; Burgos-Hernandez et al. 2002)

Economic

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Food additives substrate Effect reference

Sodium bisulfite

None Bisulfite reacts with AFB1 and AFG1 reaction rate is first order with bisulfite concentration

Doyle and Marth 1978

Corn 2% sodium bisulfite for 24 h reduced aflatoxin from 235 to below 20 ppb Moerck, Mcelfresh et al. 1980

corn 8% sodium bisulfite for 14d for total degradation of aflatoxins Hagler et al 1982

Sodium chlorite

None 5% solution for a few second caused loss of aflatoxin fluorescence Fischbac.H and Campbell 1965

None 1.25% solution cause instantaneous reduction of aflatoxin Trager and Stoloff 1967

Peanut protein isolates

0.25% solution completely eliminated AFB1 during process of producing peanut protein

Natarajan, Rhee et al. 1975

Citric acid

Ground corn 1 N aqueous citric acid reduced 96.7% AFB1 (ratio 3ml/g) Mendez-Albores, Arambula-Villa et al. 2005

Duckling feed 1 N aqueous citric acid reduced 86% AFB1 (ratio 3ml/g) Mendez-Albores, Del Rio-Garcia et al. 2007

rice 1 N aqueous citric acid reduced 86% AFB1 (ratio 3ml/g) Safara, Zaini et al. 2010

Sorghum Addition of 1N citric acid degraded aflatoxin in sorghum during extrusion process from 17 to 92% depending on M.C. and temperature

Mendez-Albores, Veles-Medina et al. 2009

Ammonium Persulfate

  

None 1% ammonium persulfate solution, aflatoxin was completely destroyed in 16h at 40°C and 1 h at 100 °C

Tabata, Kamimura et al. 1994

None 64% reduction of aflatoxins in whole grains maize were degraded when soaked for 14h in 1% ammonium persulfate solutions

Mutungi, Lamuka et al. 2008

Corn grits Spiked aflatoxins were completely degraded at 60 C for 24 with 10 ml of 1% ammonium persulfate solution to 1 g corn grits

Tabata, Kamimura et al. 1994

corn Adding 2% ammonium persulfate in the liquefaction process reduced 87% of aflatoxin levels in the final products of ethanol productoin

Burgos-Hernandez et al. 2002

Literature review: food additives treatment

Food additives uses and regulationsFood additives Effects, Use Limits and Restrictions

Sodium BisulfiteNaHSO3

Chemical Preservative GRAS, Not in meats or foods recognized as a source of Vitamin B1 (REG-182.3739)

Sodium chloriteNaClO2

Microbial control agent GMP, used in food: 0.5-1.2g/L (CODEX STAN 192-195)

Modifier for food starch GMP, not to exceed 0.5 percent (REG-172.892)

Citric Acid(C6H8O7)

Sequestrant, buffer GRAS/FS

Ammonium Persulfate(NH4)2S2O8

Modifier for food starch not to exceed 0.075 percent (REG-172.892)

GRAS, generally recognized as safe

FS, permitted as ingredient in food

GMP, in accordance with good manufacturing practice

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Effects of 1% food additives on AFB1 in coproducts

Food additives

DWG CDS

pHAFB1 levels

Mean ± Std (ppb)pH

AFB1 levelsMean ± Std (ppb)

Control 4.42 56±5a 4.28 67±3a

bisulfite 4.24 55±2a 4.22 60±9ab

chlorite 4.45 42±4b 4.42 40±3d

citric acid 3.96 50±3a 4.1 45±7cd

ammonium persulfate

3.97 40±3b 4.22 54±4bc

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Effects of Concentration (citric acid) on AFB1

ConcentrationDWG   CDS

pHAFB1 levels

Mean ± Std (ppb) 

pHAFB1 levels

Mean ± Std (ppb)0 (control) 4.42 56±5   4.28 67±3

1% 4.24 50±3   4.10 45±72% 4.45 45±0   3.95 28±25% 3.96 25±4   3.44 16±1

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Effects of Concentration (citric acid) on AFB1

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 50

10

20

30

40

50

60

70

DWGCDS

Concentration of Citric Acid (%)

AFB

1 le

vel (

ppb)

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Acknowledgements NC-213 Grant support (Thank you!) Mr. Scott Brand (ABE shop manager) Rob and Curtis (Beck’s Hybrids employees)

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Questions?