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
Grain Postharvest Quality Group
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)
Biomass Logistics and Particle Technology Group
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)
Grain Postharvest Quality Group
Acknowledgements NC-213 Grant support (Thank you!) Mr. Scott Brand (ABE shop manager) Rob and Curtis (Beck’s Hybrids employees)
Grain Postharvest Quality Group
Questions?
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