Mycotoxins Aflatoxin in Distillers Grains Project Update Hu Shi Graduate Research Assistant Dissertation Advisors Klein Ileleji PhD Associate Professor amp Ext Engineer ID: 269417
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Slide1
Reduction of
Mycotoxins (Aflatoxin) in Distillers Grains – Project Update
Hu Shi, Graduate Research AssistantDissertation 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
)Slide2
Introduction
Aflatoxins are secondary metabolites produced by a fungus. Two agricultural important fungi species: Aspergillus
flavus and Aspergillus parasiticusAflatoxins are highly toxic, carcinogenic, immunosuppressiveAflatoxins are heat stable; chemical and biological methods tend to affect the quality of cornGrain Postharvest Quality Group
http://www.icrisat.org/aflatoxin/aflatoxin.aspSlide3
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.Biomass Logistics and Particle Technology GroupSlide4
Mycotoxins
reduction: A systematic approachBiomass Logistics and Particle Technology GroupSlide5
Sorting Results: by
Simsek Lab, NDSU
Light-2 Dark-2
Light-1 Dark-1
1 Pass
2 Passes
3 PassesSlide6
Equipment
Screen Cleaner – 13/64 and 17/64 in round hole screensGrain Postharvest Quality Group
Density segregation:
Gravity TableSlide7
Flow diagram
Biomass Logistics and Particle Technology GroupSlide8
Aflatoxin – quantification of levels
Grain Postharvest Quality Group
Based on ELISA method(enzyme-linked immunosorbent assay )Major Components:QuickScan Scanner
Test Strips
Slide9
Sample density and MC
Sample
Density(g/cm3)Moisture Content
(
Mean± STD, %
)
Original sample
1.212±0.065
a
15.11±0.04
a
Screen 13/64 overs
1.213±0.058
a
15.19±0.03
a
Screen 17/64
overs
1.221±0.063
a
15.24±0.01
a
Screen 17/64 passes
1.206±0.101
a
15.03±0.04
b
Gravity table heavy kernels
1.218±0.070
a
15.02±0.10
b
Gravity table light kernels
1.118±0.162
b
14.66±0.06
c
Two-tail t test, alpha level 0.05
Density: N ~100 (unequal number of kernels )
MC: N=6
Grain Postharvest Quality GroupSlide10
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
Grain Postharvest Quality Group
Aflatoxins
level of Original Sample: 185±28 (
Mean±Std
, ppb)Slide11
Conclusion
For at least some corn samples:There is significant difference in size, shape, and density between moldy and sound corn kernelsBased 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 kernelsGrain Postharvest Quality GroupSlide12
A
pproaches to reduce aflatoxins in coproductsChemical: 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+ozoneGrain Postharvest Quality GroupSlide13
Thermal and pH stability of AFB1 in DWG and CDS
Biomass Logistics and Particle Technology Group
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/pelletsSlide14
Temperature and pH effect on AFB1 in DWG
Biomass Logistics and Particle Technology Group
At α=0.05 significant levelBoth Temperature and pH had significant effectTemperature 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 Slide15
Temperature and pH effect on AFB1 in
CDSBiomass Logistics and Particle Technology Group
At α=0.05 significant levelBoth Temperature and pH had significant effectTemperature levels are significant different from each otherpH levels are significant Alkaline cooking at 90°C for 1h resulted greatest degradation (75%)Slide16
Food additives Selected
Food additives that were previously studied for aflatoxin detoxificationSodium 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
Biomass Logistics and Particle Technology GroupSlide17
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
Biomass Logistics and Particle Technology Group
Literature review: food additives treatmentSlide18
Food additives uses and regulations
Food additives
Effects, UseLimits and RestrictionsSodium BisulfiteNaHSO3
Chemical Preservative
GRAS, Not in meats or foods recognized as a source of Vitamin B1 (REG-
182.3739)
Sodium chlorite
NaClO
2
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
(C
6
H
8
O
7
)
Sequestrant
, buffer
GRAS/FS
Ammonium Persulfate(NH
4
)
2
S
2
O
8
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 practiceSlide19
Effects of 1% food additives on AFB1 in coproducts
Biomass Logistics and Particle Technology Group
Food additives
DWG
CDS
pH
AFB1 levels
Mean ±
Std
(ppb)
pH
AFB1 levels
Mean ±
Std
(ppb)
Control
4.42
56±5
a
4.28
67±3
a
bisulfite
4.24
55±2
a
4.22
60±9
ab
chlorite
4.45
42±4
b
4.42
40±3
d
citric acid
3.96
50±3
a
4.1
45±7
cd
ammonium persulfate
3.97
40±3
b
4.22
54±4
bcSlide20
Effects of Concentration (citric acid) on AFB1
Concentration
DWG
CDS
pH
AFB1 levels
Mean ±
Std
(ppb)
pH
AFB1 levels
Mean ±
Std
(ppb)
0 (control)
4.42
56±5
4.28
67±3
1%
4.24
50±3
4.10
45±7
2%
4.45
45±0
3.95
28±2
5%
3.96
25±4
3.44
16±1
Biomass Logistics and Particle Technology GroupSlide21
Effects of Concentration (citric acid) on AFB1
Biomass Logistics and Particle Technology GroupSlide22
Grain Postharvest Quality Group
AcknowledgementsNC-213 Grant support (Thank you!)Mr. Scott Brand (ABE shop manager)Rob and Curtis (Beck’s Hybrids employees) Slide23
Grain Postharvest Quality Group
Questions?Slide24
Reference
Burgos-Hernandez, A., R. L. Price, K. Jorgensen-Kornman, R. Lopez-Garcia, H. Njapau and D. L. Park (2002). "Decontamination of aflatoxin B-1-contaminated corn by ammonium
persulphate during fermentation." Journal of the Science of Food and Agriculture 82(5): 546-552.Doyle, M. P. and E. H. Marth (1978). "Bisulfite Degrades Aflatoxin - Effect of Temperature and Concentration of Bisulfite." Journal of Food Protection 41(10): 774-780.Fischbac.H and A. D. Campbell (1965). "Note on Detoxification of Aflatoxins." Journal of the Association of Official Agricultural Chemists 48(1): 28Hagler, W. M., J. E. Hutchins and P. B. Hamilton (1982). "Destruction of Aflatoxin in Corn with Sodium Bisulfite." Journal of Food Protection 45(14): 1287-1291.Trager, W. and L. Stoloff (1967). "Possible Reactions for Aflatoxin Detoxification." Journal of Agricultural and Food Chemistry 15(4):
679
Natarajan, K. R., K. C. Rhee, C. M. Cater and K. F.
Mattil
(1975). "Destruction of Aflatoxins in Peanut Protein Isolates by Sodium-Hypochlorite." Journal of the American Oil Chemists Society 52(5):
160-163Mendez-Albores, A., G. Arambula-Villa, M. G. F. Loarea-Pina, E.
Castano-Tostado and E. Moreno-Martinez (2005). "Safety and efficacy evaluation of aqueous citric acid to degrade B-aflatoxins in maize." Food and Chemical Toxicology 43(2): 233-238.
Mendez-
Albores
, A., J. C. Del Rio-Garcia and E. Moreno-Martinez (2007). "Decontamination of aflatoxin duckling feed with aqueous citric acid treatment." Animal Feed Science and Technology
135
(3-4): 249-262
.
Mendez-
Albores
, A., J. Veles-Medina, E. Urbina-Alvarez, F. Martinez-Bustos and E. Moreno-Martinez (2009). "Effect of citric acid on aflatoxin degradation and on functional and textural properties of extruded sorghum." Animal Feed Science and Technology
150
(3-4): 316-329
.
Moerck
, K. E., P.
Mcelfresh, A. Wohlman and B. W. Hilton (1980). "Aflatoxin Destruction in Corn Using Sodium Bisulfite, Sodium-Hydroxide and Aqueous Ammonia." Journal of Food Protection 43(7): 571-574.Mutungi, C., P. Lamuka, S. Arimi, J. Gathumbi and C. Onyango (2008). "The fate of aflatoxins during processing of maize into
muthokoi - A traditional Kenyan food." Food Control 19(7): 714-721.Safara, M., F. Zaini, S. J. Hashemi, M. Mahmoudi, A. R. Khosravi and F. Shojai-Aliabadi (2010). "Aflatoxin Detoxification in Rice using Citric Acid." Iranian Journal of Public Health 39(2): 24-29.
Biomass Logistics and Particle Technology GroupSlide25
Grain Postharvest Quality Group
Difference in physical properties between moldy and sound corn kernelsSlide26
Kernel Size
and ShapeGrain Postharvest Quality Group
Where the major, intermediate, and minor diameters are, respectively, 2a,2b,2cSlide27
Kernel Density
Grain Postharvest Quality Group
Micro Pycnometer
Height Reader
Plunger
Pointer
Gage Oil
Note:
Accuracy: within 1%
-
checked using precision ball bearings
Procedure:
T
he plate driver
i
s rotated to move the plunger up and down, the reference volume is set when the pointer touches the oil
T
he plunger is moved down, and the kernel is placed in the chamber; the plunger is moved up again so that the kernel is submerged in the gauge oil and the pointer again makes contact with the upper surface of the oil.
The
d
ifference in height reading from dial indicator determines the kernel volume.
Plate DriverSlide28
Physical difference
Corn
Sample
Size
Sphericity
Density
(g/cm
3
)
Major Diameter
(mm)
Intermediate Diameter
(mm)
Minor Diameter
(mm)
Good kernels
12.016±1.226
a
7.842±0.854
a
5.337±0.891
a
0.644±0.079
a
1.215±0.092
a
Moldy kernels
10.551±1.261
b
7.963±0.928
a
5.969±0.966
b
0.757±0.093
b
1.147±0.101
b
Grain Postharvest Quality Group
Two-tail t test at alpha level of 0.05, (subdivided sample)
Size and Sphericity
: tested
115
Good and
131
moldy kernels
Density:
tested 56 Good and 48 moldy kernels Slide29
Test at Corn Inbred Processing
FacilityGrain Postharvest Quality GroupSlide30
Density Distributions
Original Sample
Higher Density kernelsGrain Postharvest Quality GroupSlide31
density distribution
Density < 1.15g/cm^3 Fraction:
Higher density kernels:7.2%
Lower density kernels: 47.7%
Original sample:8.5%
Density <1g/cm^3 Fraction:
Higher density kernels:2.0%
Lower density kernels: 19.3%
Original sample:1.4%
Grain Postharvest Quality Group
Lower
Density kernels
Cumulative distribution comparisonSlide32
density distribution
Density < 1.15g/cm^3 Fraction:
Higher density kernels:7.2%
Lower density kernels: 47.7%
Original sample:8.5%
Density <1g/cm^3 Fraction:
Higher density kernels:2.0%
Lower density kernels: 19.3%
Original sample:1.4%
Grain Postharvest Quality Group
Lower
Density kernels
Cumulative distribution comparisonSlide33
Flow diagram
Biomass Logistics and Particle Technology GroupSlide34
Distributions of physical properties for healthy and moldy corn kernels
Biomass Logistics and Particle Technology GroupSlide35
Density distributions
Biomass Logistics and Particle Technology Group
Original sample
GT LD Fraction
GT HD Fraction
Cumulative distributionsSlide36
AF level measurement method in coproducts
No method available for aflatoxin measurement in co-products (DWG and CDS)Comparison of methods: TLC, HPLC, ELISATLC method is simple but has low sensitivity, HPLC is most accurate, cumbersome sample preparation.
ELISA has moderate sensitivity and quick sample preparation and analysisELISA test strips available for DDGS---Standardization of Aflatoxin Quantification in DWG and CDSBiomass Logistics and Particle Technology GroupSlide37
Standardization
of AFB1 level in DWGBiomass Logistics and Particle Technology Group
Good dose response (R2>0.98)Non-zero Constants: initial aflatoxin level in DWG sampleScaling factor (1.837)
Higher
extraction efficiency for spiked AFB1Slide38
Standardization of AFB1 level in
CDSBiomass Logistics and Particle Technology Group
Good dose response (R2>0.98)Zero Constants: aflatoxin free in CDS sampleScaling factor (1.837)
Higher
extraction efficiency for spiked AFB1Slide39
Color Sorting Results: % Rejected Kernels
Sample
# Passes1% Kernels Rejected
1
1
2.57 (
±
0.31)c
2
4.88 (
±
1.78)b
3
8.15 (
±
0.42)a
2
1
2.93 (
±
0.60)c
2
5.36 (
±
0.75)b
3
7.31 (
±
1.57)a
1
Number of passes through the color sorter
*Values in parenthesis represent standard deviation, values in the same column with the same letter are not significantly different (
α
=0.05)