A Comparison of Two Milling Strategies To Reduce the Mycotoxin

Article pubs.acs.org/JAFC

A Comparison of Two Milling Strategies To Reduce the Mycotoxin Deoxynivalenol in Barley Piyum A. Khatibi,† Greg Berger,‡ Jhanel Wilson,§ Wynse S. Brooks,∥ Nicole McMaster,† Carl A. Griffey,∥ Kevin B. Hicks,‡ Nhuan P. Nghiem,‡ and David G. Schmale, III*,† †

Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, Virginia 24061, United States Department of Crop, Soil, and Environmental Sciences, Rice Research and Extension Center, University of Arkansas, Stuttgart, Arkansas 72160, United States § Sustainable Biofuels and Co-Products Research Unit, USDA, ARS, Eastern Regional Research Center, Wyndmoor, Pennsylvania 19038, United States ∥ Department of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, Virginia 24061, United States ‡

S Supporting Information *

ABSTRACT: Winter barley (Hordeum vulgare L.), a potential feedstock for fuel ethanol production, may be contaminated with the trichothecene mycotoxin deoxynivalenol (DON). DON is a threat to feed and food safety in the United States and may become concentrated during the production of distillers dried grains with solubles (DDGS). DDGS is a coproduct of fuel ethanol production and is increasingly being used as feed for domestic animals. Therefore, new strategies to reduce the threat of DON in DDGS need to be developed and implemented for grain destined for fuel ethanol production. It is known that large concentrations of DON accumulate in the hulls of wheat and barley. Consequently, improved methods are needed to carefully remove the hull from the grain and preserve the starchy endosperm. Whole kernels from five Virginia winter barley genotypes were used to evaluate the abilities of two different milling strategies (roller milling and precision milling (FitzMill)) for their ability to remove the hull-enriched tissue from the kernel while maintaining starch levels and reducing DON levels in the endosperm-enriched tissue. After whole kernels were milled, DON and starch levels were quantified in the hull-enriched fractions and endosperm-enriched fractions. Initial milling experiments demonstrated that the precision mill system (6 min run time) is able to reduce more DON than the roller mill but with higher starch losses. The average percent DON removed from the kernel with the roller mill was 36.7% ± 5.5 and the average percent DON removed from the dehulled kernel with the precision mill was 85.1% ± 9.0. Endosperm-enriched fractions collected from the roller mill and precision mill contained starch levels ranging from 49.0% ± 12.1 to 59.1% ± 0.5 and 58.5% ± 1.6 to 65.3% ± 3.9, respectively. On average, the precision mill removed a mass of 23.1% ± 6.8 and resulted in starch losses of 9.6% ± 6.3, but produced an endosperm-enriched fraction with relatively very little average DON (5.5 ± 2.7 μg g−1). In contrast, on average, the roller mill removed a mass of 12.2% ± 1.6 and resulted in starch losses of 2.1% ± 0.5, but produced an endosperm-enriched fraction with high average DON (20.7 ± 13.5 μg g−1). In a time course precision milling experiment, we tested barley genotypes Nomini, Atlantic, and VA96-44-304 and attempted to reduce the starch loss seen in the first experiment while maintaining low DON concentrations. Decreasing the run time of the precision mill from 5 to 2 min, reduced starch loss at the expense of higher DON concentrations. Aspirated fractions revealed that the precision milled hull-enriched fraction contained endosperm-enriched components that were highly contaminated with DON. This work has important implications for the reduction of mycotoxins such as DON in barley fuel ethanol coproducts and barley enriched animal feeds and human foods. KEYWORDS: barley, hull, dehulling, milling, mycotoxin reduction, deoxynivalenol, starch retention, fermentation, distillers dried grains with solubles (DDGS)

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domestic animals. When grain, such as winter barley,6 containing DON is used as feedstock to produce fuel ethanol, DON becomes concentrated in the DDGS.7,8 The fuel ethanol industry relies on the sale of DDGS as feed for domestic animals for generating profits9 and DDGS contaminated with mycotoxins such as DON are unmarketable. Current methods to reduce or alleviate mycotoxin contamination in grain include blending clean grain with

INTRODUCTION Fusarium head blight (FHB), caused by the fungal plant pathogen Fusarium graminearum Schwabe (teleomorph Gibberella zeae), can be a devastating disease of grains such as wheat and barley.1 During infection, the fungus produces a mycotoxin called deoxynivalenol (DON).2 DON is a protein synthesis inhibitor4 and ingestion of DON may lead to vomiting, feed refusal, and a compromised immune system.5 Grain contaminated with DON may be rendered unusable for feed and food products.3 Distillers dried grains with solubles (DDGS), a coproduct of fuel ethanol production, is increasingly being used as feed for © 2014 American Chemical Society

Received: December 10, 2013 Accepted: April 15, 2014 Published: April 15, 2014 4204

dx.doi.org/10.1021/jf501208x | J. Agric. Food Chem. 2014, 62, 4204−4213

Journal of Agricultural and Food Chemistry

Article

reducing DON levels in the endosperm-enriched tissue. The samples were first cleaned using a dockage tester (Carter Day International, Minneapolis, MN). For each barley genotype, two 500 g samples were roller milled, and two 500 g samples were precision milled. The roller mill flow consisted of one pass through 16 corrugations per 25.4 mm (mm) rolls with a 0.127 mm gap. After roller milling, the material was sifted with a SWECO sifter (SWECO, Florence, KY) containing a 24SSBC sieve. The milled kernel fraction (endosperm-enriched fraction) passed through the sieve while the hull-enriched fraction remained on top of the sieve screen. The precision milling was carried out similarly to that described by Hicks et al.28 using a Fitzpatrick Comminuting Machine (Model D, W.J. Fitzpatrick Company, Chicago IL) also referred to as a FitzMill. Each 500 g sample was milled for 6 min using a 2AA sieve (1.9 mm) with the sharp blades rotating forward at 1750 rpm. Tests were run batch-wise in duplicate. Samples were loaded in the FitzMill chamber and during milling, the hullenriched fractions were collected beneath the mill and the dehulled kernel fractions (endosperm-enriched fraction) were retained in the milling chamber. A comparison of hull removal efficiency was determined by measuring the mass fraction (% dry weight basis, (% d.w.b)) of the barley hull and dehulled kernel remaining after processing through the roller mill or precision mill. Time Course Precision Milling. Three hulled barley genotypes (Nomini, Atlantic, and VA96-44-304) were chosen to determine the optimum run time for the FitzMill necessary to efficiently remove tissue enriched in hull from the kernel while minimizing starch loss. The grains used for these experiments were harvested in 2011 from barley genotypes that were planted in 2010 in a randomized complete block in mist-irrigated nurseries at Mt. Holly, VA and treated with Fusarium graminearum as described above. For each barley genotype, whole kernel subsamples of 500 g were dehulled using the FitzMill as described above except that samples were milled for only 2, 3, 4, or 5 min rather than the 6 min in the original experiment. A logarithmic trend line was applied to % mass fraction data derived from grain processed in the time course precision milling experiment.28 Additionally, some of the individual hull and kernel fractions from these experiments were aspirated using a Grainman model 63-11560VS Grain Cleaner Laboratory Aspirator (Grain Machinery Mfg. Corp., Miami, Florida, U.S.A.). The speed setting was 7, the feeder was set at 5, and the air control was set at 0.2. Two fractions were collected: the heavy fraction that was composed primarily of starchenriched fractions (endosperm) and the light fraction composed primarily of fibrous hull. Extraction of Deoxynivalenol. Samples containing approximately 10 g of whole kernels, hulls, and dehulled kernel fraction were ground in a coffee grinder (Hamilton Beach, model 80365, Southern Pines, NC) positioned at the expresso and 12 cup setting (Figure S1 in the Supporting Information [SI]). Mycotoxin extractions were performed following standard protocols on 1 g subsamples of the ground whole kernel, ground hull, and ground kernel (Figure S1).29−31 Each subsample was combined with 10 mL extraction solvent (86% (v/v) acetonitrile in DI water) in a capped polypropylene tube. The solvent−sample mixture was placed on a shaker at 200 rpm for 1 h at room temperature and then passed through a cleanup column composed of a 1 g mixture of Bakerbond C18 (VWR, Radnor, PA) and aluminum oxide (active, neutral) (Sigma-Aldrich, St. Louis, MO) at a 1:3 ratio. All samples were extracted for DON so the final concentration in isooctane was diluted ×20. If the sample was quantified to have a DON concentration of 0.5 μg g−1 or less on the GC−MS, then the sample extract was quantified at a smaller dilution of ×2.5. This was done to ensure that DON levels fell within the standard curve. For 1:20 dilutions, a 1 mL aliquot of eluent was transferred to a glass test tube and evaporated to dryness using a nitrogen evaporator set at 55 °C. Dried samples were then silylated with a 100 μL mixture of N-trimethylsilylimidazole (TMSI) and trimethylchlorosilane (TMCS) at a 100:1 ratio. Samples were then resuspended in 2 mL of isooctane containing 0.5 μg g−1 mirex, followed by 2 mL of water to quench the reaction. Samples were vortexed for 10 s, and 100 μL of the isooctane−mirex supernatant was removed and transferred to

contaminated grain (prohibited in Europe and not approved by the U.S Federal Drug Administration (FDA)), washing grain with water, and using adsorbents to bind mycotoxins.10,11 Strategies to prevent DON production during storage include electron beam irradiation12 and fungistats to prevent fungal growth.13 These strategies may be effective, but are likely to require high equipment and labor costs. New cost-effective and commercially viable methods need to be developed and implemented to reduce DON contamination in barley destined for food and feed products. Planting resistant barley cultivars can be one of the most effective ways to reduce FHB and DON accumulation; however, resistance conferring complete immunity to FHB has not been identified to date. In Virginia, new hulled and hulless barley cultivars that are resistant to FHB and DON accumulation are being developed.14,15 With hulled barley, the hull is tightly fixed to the pericarp epidermis, while with hulless barley the hull loosely covers the caryopses.16 Previous work has demonstrated that mycotoxins such as DON accumulate in the hull and outer regions of the kernel.17−20 Thus, an alternative to hulled barley would be to plant hulless genotypes whose hull can be easily removed and that have higher starch content.6 However, higher yields and higher revenues with hulled barley (bushels per acre) do not make hulless barley an attractive alternative for growers.6 Past attempts at milling or pearling hulled grain to reduce mycotoxin levels were proven effective and showed reductions that ranged from 30% to 66%.19−25 Inevitably, grain mass losses (i.e., starch, protein) occur and have ranged from 10% to 32%.22,25−27 Recent advances in hull removal have led to a more effective and simpler dehulling strategy that may be more cost-effective and may minimize losses in grain mass.28 The Fitzpatrick Comminuting Machine (precision mill) was shown to dehull kernels and produce fractions with much higher starch content than that of the original hulled kernel (71% vs 60% starch, respectively). Moreover, the process minimized starch loss to less than 1.5%.28 In this study, we evaluated the abilities of a FitzMill precision mill and a roller mill to remove the hulls and reduce DON in Virginia hulled barley samples without causing major starch losses. We hypothesized that the precision mill (compared to the roller mill) would more effectively remove hull-enriched tissue from hulled barley, thereby resulting in larger reductions of DON, higher starch concentrations, and smaller starch loss. Our work has important implications for the reduction of mycotoxins in barley fuel ethanol coproducts and barley enriched animal feeds and human foods.

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MATERIALS AND METHODS

Barley Genotypes and Infection. Five Virginia winter hulled barley genotypes (Callao, Nomini, Atlantic, VA06B-32, VA96-44-304) were planted in 2009 in a randomized complete block with two replications in mist-irrigated nurseries at Mt. Holly, VA.14 Research plots had dimensions of 1.5 m × 13.4 m and Fusarium graminearum colonized corn (Zea mays) kernels were applied to the plots at the boot stage to encourage infection and DON contamination in the harvested grain.7,15 Grain was harvested in summer 2010 using a small plot research combine. Roller Milling and Precision Milling. Ten hulled barley samples (five genotypes) from Virginia winter barley genotypes Callao, Nomini, Atlantic, VA06B-32, and VA96-44-304 were used to evaluate the abilities of two different milling strategies (roller milling and precision milling (FitzMill)) for their ability to remove the hullenriched tissue from the kernel while maintaining starch levels and 4205



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chromatography vials for gas chromatography−mass spectrometry (GC−MS) analysis. For 1:2.5 dilutions, the same method was followed, except that a 2 mL aliquot was dried down and resuspended in 0.5 mL of isooctane−mirex mixture. GC−MS Analysis for Deoxynivalenol Quantification. GC−MS analysis was conducted using an Agilent 6890/5975 system operating in selected ion monitoring (SIM) mode. A 1 μL volume of each sample or known standard was injected by an autosampler in splitless mode onto an Agilent HP-5MS column, 0.25 mm inner-diameter by 0.25 μm film thickness by 30 m length. The inlet temperature was set to 250 °C with a constant column flow rate of 1.2 mL/min. The initial column temperature was held at 150 °C for 1 min, increased to 280 °C at a rate of 30 °C/min, and held constant for 3.5 min. A postrun method ramped the oven temperature to 325 °C and was held for 2.5 min. Mirex (Sigma-Aldrich, St. Louis, MO) was used as an internal control at 0.5 μg g−1. SIM mode detected DON target ions at a mass:charge ratio of 512, with reference ions at 422 and 497.32 SIM mode detected mirex target ions at mass:charge ratio of 272 with reference ions at 276 and 237. DON was quantified using a standard curve of ratios of DON area to mirex area versus DON standard concentration and fit to a linear regression model. DON standards (Romer Laboratories, Austria) were prepared in a DON-free wheat matrix29 at concentrations of 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10, and 15 μg g−1. The limit of quantitation (LOQ) for this method was determined to be 0.1 μg g−1. Sample results were initially quantified using a standard curve with the first five standards (0.05 to 1 μg g−1). If raw DON values were quantified as greater than 1 μg g−1, calculations were performed using a standard curve containing all points. For quality assurance, samples of barley and wheat containing known levels of DON were concurrently prepared and interspersed among test samples for each GC−MS run. Quality assurance samples were considered acceptable if the quantified DON concentration did not deviate more than 20% of the expected concentration. DON recovery for barley using the DON extraction method described above has been reported to range between 100% and 105%.30 The percentage of DON removed by milling an individual kernel was determined by adjusting the DON values of each fraction by multiplying by the appropriate mass fraction (% d.w.b) (Figure 1 or Figure 2) and then calculating the proportion of DON in the hull-enriched tissue that was removed relative to the total DON (the DON concentration in the kernel’s hullenriched tissue plus the DON concentration in the kernel’s endosperm-enriched tissue). A logarithmic trend line was determined to be the best fit and was applied to the % DON removed from

dehulling whole kernels processed in the time course precision milling experiment. Statistical Analyses. A two-way analysis of variance (ANOVA) was performed to test for significant differences among genotypes, mill type, and time points during precision milling using PROC GLIMMIX in SAS (version 9.2; SAS Institute, Cary, NC). A Tukey−Kramer’s honestly significant difference (HSD) posthoc test was performed for variables that differed at P ≤ 0.05. A correlation analysis was performed between DON levels in the whole kernels and % hull mass fraction using JMP (version 9.0.0; SAS Institute Inc., Cary, NC). For the time course precision mill experiment, an analysis was performed to determine if % DON reduction and % hull mass fraction were correlated and if % starch loss and % hull mass fraction were correlated using JMP (version 9.0.0; SAS Institute Inc., Cary, NC). Starch Analysis. Starch content was determined through chemical analysis of the hull-enriched and endosperm-enriched fractions remaining after precision milling and roller milling. A logarithmic trend line was applied to starch content data derived from grain processed in the time course precision milling experiment.28 Fractions with high starch content were considered to be endosperm-enriched fractions, and fractions with low starch were considered hull-enriched fractions. Starch content of hull and kernel samples was determined by analyzing approximately 100 mg of sample using a starch determination kit (Megazyme International Ireland Ltd., Bray Business Park, Bray, Co. Wicklow, Ireland.33 Enzyme digestions were conducted following the Megazyme protocol,and the hydrolyzed starch solutions were diluted to 50 mL with water. This method was modified by use of an YSI 2700 Analyzer (YSI Incorporated, Yellow Springs, Ohio) fitted with an YSI 2710 turntable for automated glucose determination. The percentage of starch lost by milling an individual kernel was determined by adjusting the starch compositional values of each fraction by multiplying by the appropriate mass fraction (% d.w.b.) (Figure 1 or Figure 2) and then calculating the proportion of starch in the hull-enriched tissue that was removed relative to the total starch composition (the starch in the kernel’s hull-enriched tissue plus the starch in the kernel’s endosperm-enriched tissue).

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RESULTS Mass Fractions from the Roller Mill and Precision Mill. After milling whole kernels in the roller mill and precision mill, components were separated into endosperm-enriched fractions and hulled-enriched fractions. Under the conditions tested, the precision mill system resulted in larger hull-enriched mass fractions and smaller endosperm-enriched mass fractions than the roller mill system. An ANOVA confirmed an effect of milling strategy on hull-enriched and endosperm-enriched mass fractions (P ≤ 0.05) (Table S1). The roller mill resulted in endosperm-enriched percent mass fractions that ranged from 86.6% ± 1.3 to 89.6% ± 0.5 and hull-enriched percent mass fractions that ranged from 10.5% ± 0.5 to 13.4% ± 1.3 (Figure 1). The precision mill resulted in endosperm-enriched percent mass fractions that ranged from 70.8% ± 10.9 to 84.1% ± 0.4 and hull-enriched percent mass fractions that ranged from 16.0% ± 0.4 to 29.2% ± 10.9 (Figure 1). On average, the roller mill yielded endosperm-enriched and hull-enriched fractions of 87.8% ± 1.5 and 12.2% ± 1.6, while the precision mill yielded an average percent mass fraction of endosperm-enriched and hull-enriched fractions of 76.9% ± 6.8 and 23.1% ± 6.8, respectively (Figure 1). Average mass recovery was 97.3% and 99.8% for the roller mill and precision mill, respectively (not shown). Deoxynivalenol Levels in Barley Fractions. DON levels in whole kernels ranged from 14.3 ± 4.6 μg g−1 (Nomini) to 38.8 ± 14.6 μg g−1 (Atlantic) (Table 1). Whole kernels that were processed in the roller mill resulted in endospermenriched fractions containing DON concentrations that ranged

Figure 1. Percent mass fractions (% d.w.b.) of endosperm-enriched fractions and hull-enriched fractions from whole kernels processed in the roller mill (R) and precision mill (P). Five barley genotypes were used and run in duplicate and values given are mean ± standard deviation. 4206



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Figure 2. Time course precision mill percent mass fractions (% d.w.b.) of (A) hull-enriched fractions from whole kernels of barley genotypes Nomini (triangle, y = −8.85 ln(x) + 98.02, R2 = 0.99), Atlantic (circle, y = −10.37 ln(x) + 94.49, R2 = 0.98), and VA96-44-304 (square, y = −14.00 ln(x) + 91.88, R2 = 0.99) and (B) endosperm-enriched fractions from whole kernels of barley genotypes Nomini (triangle, y = 8.85 ln(x) + 1.98, R2 = 0.99), Atlantic (circle, y = 10.37 ln(x) + 5.51, R2 = 0.98), and VA96-44-304 (square, y = 14.00 ln(x) + 8.12, R2 = 0.99). Each time point for a given genotype was run in duplicate, and values given are mean ± standard deviation.

Table 1. Concentration of DON (μg g−1) in the Endosperm-Enriched Fractions and Hull-Enriched Fractions from Whole Kernels Processed in the Roller Mill and the Precision Mill; Five Barley Genotypes Were Used and Run in Duplicate roller milla genotype

whole kernela

Callao Nomini Atlantic VA06B-32 VA96-44304 grand mean

18.3 14.3 38.8 16.2 31.7

± ± ± ± ±

endosperm-enriched fractions

2.3 4.6 14.6 3.4 2.8

12.9 9.4 35.5 12.1 33.6

23.9 ± 11.9

± ± ± ± ±

precision milla

hull-enriched fractions

3.0 3.8 18.1 2.0 5.8

71.3 46.1 109.0 51.6 102.6

20.7 ± 13.7

± ± ± ± ±

% DON removedb

11.2 11.1 4.5 8.8 8.6

41.4 42.5 34.0 33.3 32.3

76.1 ± 28.0

± ± ± ± ±

endosperm-enriched fractions

3.8 0.0 8.4 1.0 0.3

3.0 5.6 8.9 3.4 6.4

36.7 ± 5.5

± ± ± ± ±

2.1 1.8 2.6 1.1 1.3

hull-enriched fractions 107.3 69.4 179.8 82.2 155.0

5.5 ± 2.7

± ± ± ± ±

8.2 26.7 14.4 3.3 2.8

118.7 ± 45.8

% DON removedb 91.1 69.8 87.6 85.9 90.9

± ± ± ± ±

5.3 1.0 9.0 2.5 0.2

85.1 ± 9.1

Values given are mean ± standard deviation. bThe percentage of DON removed by milling an individual kernel was determined by adjusting the DON values of each fraction by multiplying by the appropriate mass fraction (Figure 1) and then calculating the proportion of DON in the hullenriched tissue that was removed relative to the total DON (the kernel’s hull-enriched tissue plus the kernel’s endosperm-enriched tissue).

a

Table 2. Percent Starch (% d.w.b.) Analysis of Endosperm-Enriched Fractions and Hull-Enriched Fractions from the Roller Mill and the Precision Mill Derived from Five Hulled Barley Genotypes; Five Barley Genotypes Were Used and Run in Duplicate roller milla barley genotype Callao Nomini Atlantic VA06B-32 VA96-44-304 grand mean

endosperm-enriched fractions 57.4 57.4 59.1 59.0 49.0 56.4

± ± ± ± ± ±

3.7 1.3 0.5 0.1 12.1 5.8

precision milla

hull-enriched fractions 11.1 7.2 7.5 8.5 7.7 8.4

± ± ± ± ± ±

% starch loss

0.1 0.8 1.1 1.5 1.3 1.7

2.4 1.8 1.9 1.7 2.5 2.1

± ± ± ± ± ±

0.1 0.5 0.1 0.4 0.8 0.5

b

endosperm-enriched fractions 59.9 58.5 65.3 61.5 60.8 61.2

± ± ± ± ± ±

3.5 1.6 3.9 0.6 0.2 3.0

hull-enriched fractions 19.5 13.5 30.0 13.5 23.5 20.0

± ± ± ± ± ±

0.0 3.2 9.5 0.2 0.9 7.4

% starch lossb 8.2 4.2 16.7 5.0 13.7 9.6

± ± ± ± ± ±

0.5 1.0 10.5 0.4 1.7 6.3

Values given are mean ± standard deviation. bThe percentage of starch lost by milling an individual kernel was determined by adjusting the starch compositional values of each fraction by multiplying by the appropriate mass fraction (Figure 1) and then calculating the proportion of starch in the hull-enriched tissue that was removed relative to the total starch percentage (the kernel’s hull-enriched tissue plus the kernel’s endosperm-enriched tissue).

a

from 9.4 ± 3.8 μg g−1 to 35.5 ± 18.1 μg g−1 and DON levels in the hull-enriched fractions that ranged from 46.1 ± 11.1 μg g−1 to 109.0 ± 4.5 μg g−1 (Table 1). Conversely, whole kernels processed in the precision mill resulted in endosperm-enriched fractions containing DON concentrations that ranged from 3.0 ± 2.1 μg g−1 to 8.9 ± 2.6 μg g−1 and DON levels in the hullenriched fractions that ranged from 69.4 ± 26.7 μg g−1 to 179.8 ± 14.4 μg g−1 (Table 1). For the five barley genotypes analyzed, the grand mean DON concentration of the endosperm-enriched fractions and hull-enriched fractions from the roller mill was 20.7 ± 13.7 μg g−1 and 76.1 ± 28.0

μg g−1, while the grand mean DON concentration from the precision mill fractions yielded 5.5 ± 2.7 μg g−1 and 118.7 ± 44.9 μg g−1, respectively (Table 1). An ANOVA demonstrated a significant (P ≤ 0.05) effect of genotype and milling strategy on DON levels in the endosperm-enriched and hull-enriched fractions (Table S1). DON removed from the milled kernel using the precision mill ranged from 69.8% ± 1.0 to 91.1% ± 5.3 versus 32.3% ± 0.3 to 42.5% ± 0.0 using the roller mill (Table 1). Significant differences (P ≤ 0.05) in the % DON removed was observed between the mill used and genotype ∗ mill interactions (Table S1 in the SI). 4207



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Table 3. Precision Milled Aspirated Hull-Enriched Fraction and Endosperm-Enriched Fraction Analysis Showing % Mass Fraction, DON Concentration (μg g−1), and % Starch That Resulted in Clarified Hull-Enriched Fractions (H) and EndospermEnriched Fractions (E) precision milled aspirated samplesa aspirated hull-enriched fractions genotype Nomini

time (min)

% mass fraction

DON (μg g−1)

aspirated endosperm-enriched fractions

2 3 4 5

Atlantic

H E

81.5 18.5

4.8 98.6

3

VA96-44-304

H E

75.8 24.2

5.4 131.0

3

5 a

H E H E

72.4 27.7 69.9 30.1

% starch

H E H E

7.6 92.4 10.0 90.0

4.5 2.8 4.1 2.1

0.9 60.2 0.8 61.8

H E H E

4.9 95.1 4.0 96.0

3.8 3.5 2.6 1.8

3.2 62.2 1.3 66.1

H E H E

5.3 94.7 3.3 96.7

7.7 6.5 5.6 4.0

1.6 55.6 1.4 59.9

11.2 65.6

2

4

DON (μg g−1)

4.7 52.0

2

4 5

% mass fraction

% starch

9.1 131.4 9.2 109.4

8.6 59.3 9.9 60.9

Aspirated samples were single runs.

Starch Composition of Endosperm-Enriched and HullEnriched Fractions. Whole kernels processed in the roller mill resulted in starch levels in the endosperm-enriched fractions that ranged from 49.0% ± 12.1 to 59.1% ± 0.5 and starch levels in the hull-enriched fractions that ranged from and 7.2% ± 0.8 to 11.1% ± 0.1 (Table 2). Conversely, the precision mill resulted in starch composition in the endosperm-enriched fractions that ranged from 58.5% ± 1.6 to 65.3% ± 3.9 and starch levels in the hull-enriched fractions that ranged from 13.5% ± 3.2 to 30.0% ± 9.5 (Table 2). For the 5 barley genotypes analyzed, the grand mean starch concentration of the endosperm-enriched fractions and hull-enriched fractions from the roller mill was 56.4% ± 5.8 and 8.4% ± 1.7, while the average for the precision mill fractions was 61.2% ± 3.0 and 20.0% ± 7.4, respectively (Table 2). Starch lost from the milled kernel using the roller mill and precision mill ranged from 1.8% ± 0.5 to 2.5% ± 0.8 and 4.2% ± 1.0 to 16.7% ± 10.5, respectively (Table 2). Average starch loss from the roller mill was 2.1% ± 0.5 and average starch loss from the precision mill was 9.6% ± 6.3 (Table 2). An ANOVA demonstrated a significant (P ≤ 0.05) effect of milling strategy on starch levels in the endosperm-enriched fractions and an effect of genotype, milling strategy, and genotype ∗ mill interactions on starch levels in the hull-enriched fractions (Table S1). Time Course Precision Milling-Percent Mass Fraction. Three of the barley genotypes that were analyzed in the first analysis (Nomini, Atlantic, and VA96-44-304) were subject to time course precision milling to locate a milling time that resulted in lower starch loss. Longer precision milling times resulted in larger amounts of hull-enriched fractions and smaller amounts of endosperm-enriched fractions (Figure 2). Time

course precision milling generated hull-enriched mass fractions ranging from 8.2% ± 0.8 (2 min, Nomini) to 30.2% ± 2.0 (5 min, VA96-44-304) and endosperm-enriched mass fractions ranging from 69.8% ± 2.0 (5 min, VA96-44-304) to 91.8% ± 0.8 (2 min, Nomini) (Figure 2). Significant differences (P ≤ 0.05) for genotype, time, and genotype ∗ time interactions were observed for endosperm-enriched and hull-enriched % mass fractions in the time course precision mill experiment (Table S2). Aspirated samples revealed that hull-enriched fractions were contaminated with small amounts of components enriched in endosperm (which were separated by aspiration) and endosperm-enriched fractions that were contaminated with small amounts of components enriched in hulls (which were also separated by aspiration) (Table 3). Endosperm-enriched fractions that were contaminating the hull-enriched fraction ranged from 18.5% (Nomini, 5 min) to 30.1% (VA96-44-304, 5 min) and hull-enriched fractions that were contaminating the endosperm-enriched fraction ranged from 3.3% (VA96-44-304, 3 min) to 10% (Nomini, 3 min) (Table 3). Time Course Precision Milling-DON and Starch Levels. From 2 to 5 min, barley genotypes Nomini, Atlantic, and VA9644-304 had increases in the proportion of DON that was removed from the dehulled kernel, with percentages ranging from 45.0% ± 7.1 to 68.8% ± 8.5, 68.9% ± 7.2 to 90.5% ± 1.1, and 66.1% ± 4.3 to 92.7% ± 1.7, respectively (Figure 3). Thus, DON levels increased in the hull-enriched fractions and decreased in the endosperm-enriched fractions as precision milling time increased. For example, the endosperm-enriched fraction of Nomini, Atlantic, and VA96-44-304 had DON concentrations that decreased from 12.2 ± 2.9 to 8.1 ± 3.8, from 9.9 ± 4.0 to 3.1 ± 0.1, and from 19.8 μg g−1 ± 1.9 (2 min) 4208



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Figure 3. Percentage of DON removed by dehulling barley genotypes Nomini (triangle, y = 29.73 ln(x) + 21.82, R2 = 0.84), Atlantic (circle, y = 24.82 ln(x) + 50.79, R2 = 0.97), and VA96-44-304 (square, y = 30.13 ln(x) + 47.22, R2 = 0.94) in the precision mill from 2 to 5 min. Each time point for a given genotype was run in duplicate, and values given are mean ± standard deviation. The percentage of DON removed by milling an individual kernel was determined by multiplying the DON values (μg g−1) of each fraction by the appropriate mass fraction value (Figure 2A, B) and then calculating the proportion of DON in the hull-enriched tissue that was removed relative to the total DON.

Figure 4. Percent starch lost (% d.w.b.) from dehulling kernels of barley genotypes Nomini (triangle, y = 2.08 ln(x) + 0.83, R2 = 0.97), Atlantic (circle, y = 3.10 ln (x) + 4.44, R2 = 0.81), and VA96-44-304 (square, y = 7.82 ln(x) + 3.36, R2 = 0.98) in the time course precision milling experiment. Each time point for a given genotype was run in duplicate, and values given are mean ± standard deviation. The percentage of starch lost by milling an individual kernel was determined by multiplying the starch compositional values (%) of each fraction by the appropriate mass fraction (Figure 2A, B) and then calculating the proportion of starch in the hull-enriched tissue that was removed relative to the total starch percentage.

to 5.1 μg g−1 ± 0.4 (5 min), respectively (data not shown). Correspondingly, the hull-enriched fraction had DON concentrations that went from 110.6 ± 6.8 to 88.9 ± 2.1, from 150.8 ± 10.5 to 108.2 ± 10.5, and from 178.7 μg g−1 ± 6.4 (2 min) to 152.0 μg g−1 ± 10.2 (5 min), respectively (data not shown). There were significant differences (P ≤ 0.05) in DON concentration between time points in the endosperm-enriched fractions and significant differences (P ≤ 0.05) between genotypes and between time points in the hull-enriched fractions (Table S2). When precision milling time increased (2 to 5 min), so did the proportion of starch lost when the hull-enriched tissue was removed likely due to simultaneous removal of small amounts of endosperm (Figure 4). As more and more hull was removed, eventually the precision mill blades struck the dehulled kernel and removed more endosperm.28 Significant differences (P ≤ 0.05) were observed for % starch loss between time points and genotypes (Table S2 in the SI). Starch loss ranged from 2.3% ± 0.3 to 4.1% ± 0.1 for Nomini, 6.2% ± 0.9 to 8.8% ± 0.1 for Atlantic, and 9.0% ± 0.6 to 15.9% ± 2.3 for VA96-44-304 (Figure 4). Barley genotype VA96-44-304 had the largest loss of starch during dehulling in the precision mill. Although starch loss increased from 2 to 5 min, precision milling led to increases in the starch concentration for the endospermenriched fractions. The starch composition (increasing in milling time) for the endosperm-enriched fractions of Nomini, Atlantic, and VA96-44-304 increased from 55.6% ± 0.3 to 60.0% ± 1.5, 57.6% ± 3.1 to 61.5% ± 1.0, and 55.9% ± 0.5 (2 min) to 61.2% ± 0.0 (5 min), respectively (data not shown). The starch composition (increasing in milling time) for the hulled-enriched fractions ranged from 14.8% ± 0.1 to 13.4% ± 0.3, 26.9% ± 2.4 to 21.4% ± 0.4, and 25.6% ± 0.1 (2 min) to 26.7% ± 2.2 (5 min), respectively (data not shown). There were significant differences (P ≤ 0.05) in % starch between time points in the endosperm-enriched fractions and significant

differences between genotypes and genotype ∗ time interactions in the hull-enriched fractions (Table S2). The percent starch lost by dehulling and the percent DON removed in the precision mill was significantly correlated with the % hull mass fraction (r = 0.95, P < 0.0001 and r = 0.87, P < 0.0001, respectively) (Figure 5). When a linear model was fit to the trend lines, the slope (rate) of starch loss and DON removal were 0.63 and 2.26, respectively (Figure 5). However, the logarithmic model was a better fit for the DON removal (R2 of 0.81, Figure 5).

Figure 5. Percent DON removed (y = 40.84 ln(x) − 43.05, R2 = 0.81) and % starch loss (y = 0.63x − 3.59, R2 = 0.91) as a function of the % hull mass fraction during time course precision milling. The amount of DON removed and the amount of starch lost increased as the mass of the hull fraction increased. 4209



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Time Course Precision Milling-Aspirated Samples. The aspirated hull-enriched fractions possessed components enriched in endosperm that contained DON levels ranging from 98.6 μg g−1 (Nomini, 5 min) to 131.4 μg g−1 (VA96-44304, 4 min) and components enriched in hulls that contained DON ranging from 4.8 μg g−1 (Nomini, 5 min) to 9.2 μg g−1 (VA96-44-304, 5 min) (Table 3). The aspirated endospermenriched fractions possessed components enriched in hulls that contained DON levels that ranged from 2.6 μg g−1 (Atlantic, 3 min) to 7.7 μg g−1 (VA96-44-304, 2 min) and components enriched in endosperm that contained DON ranging from 1.8 μg g−1 (Atlantic, 3 min) to 6.5 μg g−1 (VA96-44-304, 2 min) (Table 3). The hull-enriched fractions contained endosperm-enriched components that could be removed by aspiration, and these isolated subfractions had starch levels that ranged from 52.0% (Nomini, 5 min) to 65.6% (Atlantic, 5 min) and the hullenriched components remaining after aspiration had starch composition that ranged from 4.7% (Nomini, 5 min) to 11.2% (Atlantic, 5 min) (Table 3). The endosperm-enriched fractions contained hull-enriched components that were isolated by aspiration and these isolated subfractions had starch levels that ranged from 0.8% (Nomini, 3 min) to 3.2% (Atlantic, 2 min). The endosperm-enriched components remaining after aspiration had starch levels that ranged from 55.6% (VA96-44-304, 2 min) to 66.1% (Atlantic, 3 min) (Table 3).

roller mill (Table 2), which is important for fuel ethanol production. Hicks et al.28 reported that Thoroughbred, a hulled barley cultivar, processed in the precision mill resulted in hull mass fractions of ∼14% that were composed of less than 6% starch, which corresponded to less than 1.5% starch loss from the original kernel. However, in our study with five other barley genotypes, the precision mill removed much more mass than seen with Thoroughbred. On average, the precision mill removed a mass of 23.1% (hull-enriched fraction) while the roller mill resulted in only a 12.2% mass removal (Figure 1). Therefore, while the precision mill resulted in much lower DON levels in the endosperm-enriched fractions by removing more hull-enriched tissue (Table 1), the precision mill resulted in higher starch losses (Table 2). Hull-enriched fractions collected from the roller mill and precision mill, contained starch levels that ranged from 7.2% to 11.1% and 13.5% to 30.0% (Table 2), respectively. To calculate the amount of starch lost when the hull is removed, the starch contents in the hull-enriched fraction and endosperm-enriched fraction were adjusted on the basis of their respective percent mass fraction, and then the proportion of starch in the hull (i.e., the adjusted starch value in the hull-enriched fraction) relative to total starch was calculated. An evaluation of starch levels adjusted on the basis of the mass fraction showed that grain processed in the roller mill yielded an average of 49.5 g of starch per 100 g of kernel versus 47.1 g of starch per 100 g of kernel for grain processed in the precision mill. The average starch lost to the hull was 2.1% from grain processed in the roller mill and an average of 9.6% starch loss from grain processed in the precision mill (Table 2). This level of starch loss was larger than the previously reported starch loss of 1.5%.28 Thus, comparison of the two milling strategies certainly demonstrated that the precision mill system is able to remove more DON (Table 1) than the roller mill (Table 1), but at the expense of starch loss (Table 2) for these genotypes. Consequently, to make up for starch loss, additional grain may have to be processed; however, using grain containing little DON would improve the quality of the DDGS and obviously be favored over starch losses observed when using the precision mill system. Time Course Precision Milling. Since more endospermenriched tissue and starch were removed from the kernel during precision milling (Figure 1 and Table 2) than expected, a time course experiment was conducted by running the precision mill at increments of one min (2, 3, 4, or 5 min) using whole kernels from three of the five barley genotypes tested in the first experiment (Nomini, Atlantic, and VA96-44-304). Time course precision milling conducted at shorter run times provided fractions with improved percent mass values for which mass loss was reduced from the endosperm-enriched fraction into the hull-enriched fraction (Figure 2). A 2 min run time produced hull-enriched mass fractions that were closest to those reported in Hicks et al.28 In the first precision mill experiment (Figure 1), the hull-enriched percent mass fraction for Nomini (16.0%), Atlantic (29.2%), and VA96-44-304 (29.2%) were reduced to 8.2%, 12.3%, and 17.8% at 2 min, respectively (Figure 2). Other examples of using milling to reduce DON include work using a semi-industrial semolina mill, which demonstrated a 45% loss of DON when 10% of the grain tissue was removed22 and 66% DON reduction after 15 s of pearling using an abrasive type dehulling procedure when 15% of the grain mass was removed.25 In our study, hullenriched mass fractions of 10% and 15% corresponded to a

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DISCUSSION Previous interest in using barley as a potential feedstock for fuel ethanol production6 led to the development of methods to create more efficient, high-starch ethanol feedstocks from hulled barley using a precision dehulling process.28 Occasionally, barley kernels may contain high levels of the mycotoxin DON, and while such kernels can be used to make fuel ethanol, the DON concentrates in the ethanol coproduct DDGS, making it unmarketable. DDGS sales are critical for an ethanol plant’s economic viability. In this report, we compared the abilities of two milling strategies (roller mill and FitzMill precision mill) to remove DON-contaminated hull-enriched tissue from winter barley, while preserving starch levels in the kernel. This work was based on the report that the FitzMill offers a simple process for dehulling hulled barley to produce a dehulled kernel with higher starch content28 and other reports that have shown DON accumulating in the hull and peripheral regions of the kernel.17−20 Roller Mill vs Precision Mill. In our study, the roller mill removed a smaller mass of hull-enriched tissue from the kernel (Figure 1) and yielded endosperm-enriched fractions with larger concentrations of DON than the precision mill (Table 2). This result can be explained by the mechanics of the roller mill and precision mill systems. The roller mill is a common milling technique27,34 that involves running kernels between multiple corrugated rolls and does not result in localized hull tissue removal, resulting in ground grain (Figure S1a, in the SI) that requires multiple flows and/or sieving steps27 to separate hull from endosperm. In contrast, the precision mill system uses sharp blades that shear the barley, swiping the kernels parallel to the central axis and lifting the hull from the kernel resulting in an intact, dehulled kernel (Figure S1d and e [SI], kernel and hull processed in the precision mill, respectively).28 The precision mill had a 2-fold effect of reducing DON (Table 1) and increasing the starch concentration in the endosperm-enriched fractions relative to the results with the 4210



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enriched fraction are highly contaminated with DON, while the purified hulls contained DON at relatively low levels (Table 3). This was in contrast to our initial understanding that DON accumulates in the barley hull, which was based on our first set of dehulling experiments and previous work17,18 but is consistent with the results from Rios et al.35 This group reported that the finest particles of semolina from durum wheat have a 3−5-fold increase in DON concentration and may indicate the need to control the distribution of these fine particles during milling.35 Thus, while aspiration of endospermenriched tissue yielded a more pure endosperm-enriched fraction with reduced DON, it is unlikely that the aspiration process will be used in a dry grind ethanol plant to salvage starch lost to the hull-enriched fractions, which we found to be highly contaminated with DON. A more likely scenario is dehulling for roughly 6 min in the precision mill, using the more than 75% mass of endosperm for ethanol production (Figure 1), and burning the remaining DON contaminated hull-enriched fraction to generate energy for the plant. This process would improve the resultant DDGS by generating a coproduct with little DON and would improve the economics of fuel ethanol production. Also relevant to DDGS production is protein content in the milled grain. Prior work, reported that after 10 min using the precision mill system, the amount of protein in the processed kernel stayed relatively constant at about 8%.28 Similarly, the protein content in our time course experiment stayed relatively even from two to 5 min with endosperm-enriched fractions containing protein on average of about 9.5% (data not shown). Future research efforts should focus on elucidating the distribution of DON in barley and methods to isolate and remove the highly contaminated endosperm-enriched tissue, while maximizing starch retention in barley and other cereal grains. Commercial mills may be able to leverage the milling strategies described here for mitigating DON. When DON levels are of concern or exceed acceptable limits, millers could follow the milling guidelines presented in this study and modify their mill settings or separate products from different mill streams to minimize DON content in their finished products. Such strategies, however, may be linked in part to knowledge of the genotype/variety of barley that will be processed. If mills are primarily interested in using barley as a feedstock that is high in starch (e.g., for fuel ethanol production), then the comparative information presented here should be of value for maximizing the amount of desired product (flour or dehulled grain) versus byproducts (hulls), regardless of DON.

51% and 68% reduction of DON, respectively (Figure 5). Although we were able to reduce the mass lost during milling by reducing the run time, DON levels correspondingly increased, and the starch concentration decreased. DON levels in the endosperm-enriched fractions were as much as 4-fold higher (5.1 μg g−1 (5 min) vs 19.8 μg g−1 (2 min) for VA96-44304) (data not shown) and starch concentrations were as much as 8.7% lower at 2 min (55.9%) versus 5 min (61.2% for VA9644-304) (data not shown). Starch lost during milling was reduced with lower run times; however, even at 2 min the percent starch lost was still higher than 1.5%, with values of 2.3%, 6.2%, and 9.0% for barley genotypes Nomini, Atlantic, and VA96-44-304, respectively (Figure 4). The results of this work were unexpected as we were not able to produce endosperm-enriched fractions from the precision mill with reduced losses in grain mass and reduced starch loss as those reported previously.28 Hicks’ group conducted their analysis using the Virginia winter barley genotype Thoroughbred, which was harvested in 2005, and may explain some of the differences between studies. Phenotypic differences between genotypes,14,15 seasonal differences in FHB disease levels,15 and grain quality and composition6 may also impact kernel characteristics such as starch content and hull removal potential. Years with low FHB may yield unadulterated kernels with unaltered starch concentrations. In contrast, years with high FHB may yield kernels with reduced starch and brittle hulls that may fall off easily in the mill, leading to unintended removal of endosperm tissue.3 These reasons, as well as differences in disease levels that may occur between plots of the same genotype in a given year, may help explain any high standard deviations seen with some of the data, such as the observations for the Atlantic genotype in the first milling experiment. A correlation analysis of data in this study revealed that concentrations of DON in the whole kernels were significantly correlated with the hull percent mass fractions (roller mill: r = 0.81, P < 0.0001; precision mill: r = 0.86, P < 0.0001). We also wanted to test the hypothesis that during precision milling some components of the endosperm-enriched tissue were included in the hull-enriched fraction and that some hull-enriched components were included in the endospermenriched fraction. Therefore, we aspirated these samples in an attempt to improve fraction purity. Following aspiration of time course, precision-milled samples, hull-enriched fractions were found to contain endosperm-enriched components at percent mass fractions that ranged from 18.5% (Nomini, 5 min) to 30.1% (VA96-44-304, 5 min) and endosperm-enriched fractions were found to contain hull-enriched components at percent mass fractions that ranged from 3.3% (VA96-44-304, 3 min) to 10.0% (Nomini, 3 min) (Table 3). This confirmed that cross contamination of milling fractions occurs when the precision mill is abrading the surface of the kernel to remove the hull, it is also removing some endosperm. The hypothesis that milling will reduce DON levels was demonstrated with the removal of the hull-enriched tissue in the first experiment (Table 1, Figure 3), and we were able to further reduce the concentration of DON by aspirating the endosperm-enriched fractions to remove contaminating hullenriched tissue. At the 2 min mark, DON was reduced in aspirated samples from 12.2 μg g−1 to 2.8 μg g−1 (Nomini), 9.9 μg g−1 to 3.5 μg g−1 (Atlantic), and 19.8 μg g−1 to 6.5 μg g−1 (VA96-44-304) (Table 3). Aspiration of the hull-enriched fractions from the time course, precision mill experiment revealed that the endosperm-enriched components in the hull-

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ASSOCIATED CONTENT

* Supporting Information S

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Email: [email protected]. Telephone: (540) 231-6943. Fax: (540) 231-7477. Author Contributions

For correspondence about DON analyses conducted in this work, please contact D.G.S. For correspondence about milling methods used in this work, please contact K.B.H. For correspondence about specific barley genotypes used in this work, please contact C.A.G. 4211



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Funding

(11) Diaz-Llano, G.; Smith, T. K. Effects of feeding grains naturally contaminated with Fusarium mycotoxins with and without a polymeric glucomannan mycotoxin adsorbent on reproductive performance and serum chemistry of pregnant gilts. J. Anim Sci. 2006, 84, 2361−6. (12) Stepanik, T.; Kost, D.; Nowicki, T.; Gaba, D. Effects of electron beam irradiation on deoxynivalenol levels in distillers dried grain and solubles and in production intermediates. Food Addit. Contam. 2007, 24, 1001−6. (13) Magan, N.; Aldred, D. Post-harvest control strategies: minimizing mycotoxins in the food chain. Int. J. Food Microbiol. 2007, 119, 131−9. (14) Khatibi, P. A.; Berger, G.; Liu, S.; Brooks, W. S.; Griffey, C. A.; Schmale, D. G. Resistance to Fusarium head blight and deoxynivalenol accumulation in Virginia barley. Plant Dis. 2011, 96, 279−284. (15) Berger, G.; Green, A.; Khatibi, P.; Brooks, W.; Rosso, L.; Liu, S.; Griffey, C.; Schmale, D. Characterization of Fusarium head blight (FHB) resistance and deoxynivalenol accumulation in hulled and hulless winter barley. Plant Dis. 2014, 98, 599−606. (16) Taketa, S.; Amano, S.; Tsujino, Y.; Sato, T.; Saisho, D.; Kakeda, K.; Nomura, M.; Suzuki, T.; Matsumoto, T.; Sato, K.; Kanamori, H.; Kawasaki, S.; Takeda, K. Barley grain with adhering hulls is controlled by an ERF family transcription factor gene regulating a lipid biosynthesis pathway. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 4062−7. (17) Clear, R. M.; Patrick, S. K.; Nowicki, T.; Gaba, D.; Edney, M.; Babb, J. C. The effect of hull removal and pearling on Fusarium species and trichothecenes in hulless barley. Can. J. Plant Sci. 1997, 77, 161− 166. (18) Legzdina, L.; Buerstmayr, H. Comparison of infection with Fusarium head blight and accumulation of mycotoxins in grain of hulless and covered barley. Cereal Sci. 2004, 40, 61−67. (19) Lee, U.-S.; Jang, H.-S.; Tanaka, T.; Oh, Y.-J.; Cho, C.-M.; Ueno, Y. Effect of milling on decontamination of Fusarium mycotoxins nivalenol, deoxynivalenol, and zearalenone in Korean wheat. J. Agric. Food Chem. 1987, 35, 126−129. (20) Trigo-Stockli, D. M.; Deyoe, C. W.; Satumbaga, R. F.; Pedersen, J. R. Distribution of deoxynivalenol and zearalenone in milled fractions of wheat. Cereal Chem. 1996, 73, 388−391. (21) Fandohan, P.; Ahouansou, R.; Houssou, P.; Hell, K.; Marasas, W. F.; Wingfield, M. J. Impact of mechanical shelling and dehulling on Fusarium infection and fumonisin contamination in maize. Food addit. contam. 2006, 23, 415−21. (22) Rios, G.; Pinson-Gadais, L.; Abecassis, J.; Zakhia-Rozis, N.; Lullien-Pellerin, V. Assessment of dehulling efficiency to reduce deoxynivalenol and Fusarium level in durum wheat grains. J. Cereal Sci. 2009, 49, 387−392. (23) Dexter, J. E.; Marchylo, B. A.; Clear, R. M.; Clarke, J. M. Effect of Fusarium head blight on semolina milling and pasta-making quality of durum wheat. Cereal Chem. 1997, 74, 519−525. (24) Dexter, J. E.; Clear, R. M.; Preston, K. R. Fusarium head blight: effect on the milling and baking of some Canadian wheats. Cereal Chem. 1996, 73, 695−701. (25) House, J. D.; Nyachoti, C. M.; Abramson, D. Deoxynivalenol removal from barley intended as swine feed through the use of an abrasive pearling procedure. J. Agric. Food Chem. 2003, 51, 5172− 5175. (26) Trenholm, H. L.; Charmley, L. L.; Prelusky, D. B.; Warner, R. M. Two physical methods for the decontamination of four cereals contaminated with deoxynivalenol and zearalenone. J. Agric. Food Chem. 1991, 39, 356−360. (27) Flores, R. A.; Hicks, K. B.; Eustace, D. W.; Phillips, J. Highstarch and high-β-glucan barley fractions milled with experimental mills. Cereal Chem. 2005, 82, 727−733. (28) Hicks, K. B.; Wilson, J.; Flores, R. A., Progressive hull removal from barley using the Fitzpatrick comminuting mill. Appl. Eng. Agric. 2011, 27. (29) Mirocha, C. J.; Kolaczkowski, E.; Xie, W.; Yu, H.; Jelen, H. Analysis of deoxynivalenol and its derivatives (batch and single kernel) using gas chromatography/mass spectrometry. J. Agric. Food Chem. 1998, 46, 1414−1418.

This work was supported primarily by a grant to David G. Schmale (D.G.S.), Carl A. Griffey (C.A.G.), and Kevin B. Hicks (K.B.H.) from the Biodesign and Bioprocessing Research Center at Virginia Tech (Project #208-11-110A-012-331-1). Grants to D.G.S. by the Maryland Grains Producers Utilization Board (Proposal #10121612), the Virginia Agricultural Council (Proposal #10183402), the Virginia Small Grains Board (Proposal #10278306) and the United States Wheat and Barley Scab Initiative (Proposal #07185403) also provided support for the work. The conclusions presented here are those of the authors and do not necessarily reflect the views of the United States Department of Agriculture. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

The authors thank Michael Kurantz and Robyn Moten for conducting the compositional analysis of the barley fractions.

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ABBREVIATIONS ANOVA: analysis of variance; DDGS: distillers dried grains with solubles; DON: deoxynivalenol; DWB: dried weight basis; FHB: fusarium head blight; GC-MS: gas chromatography− mass spectrometry; LOQ: limit of quantitation; SIM: selected ion monitoring; TMCS: trimethylchlorosilane; TMSI: Ntrimethylsilylimidazole

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REFERENCES

(1) Goswami, R. S.; Kistler, H. C. Heading for disaster: Fusarium graminearum on cereal crops. Molecular Plant Pathology 2004, 5, 515− 525. (2) Jansen, C.; von Wettstein, D.; Schafer, W.; Kogel, K. H.; Felk, A.; Maier, F. J. Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16892−7. (3) McMullen, M.; Jones, R.; Gallenberg, D., Scab of Wheat and Barley: A Re-emerging Disease of Devastating Impact. Plant Disease 1997, 81. (4) McLaughlin, C. S.; Vaughn, M. H.; Campbell, J. M.; Wei, C. M.; Stafford, M. E.; Hansin, B. S. Inhibition of protein synthesis by trichothecenes. Mycotoxins Hum. Anim. Health, Proc. Conf. 1977, 19, 263−273. (5) Peska, J. J. Deoxynivalenol: toxicity, mechanisms and animal health risks. Animal Feed Sci. Technol. 2007, 137, 283−298. (6) Griffey, C.; Brooks, W.; Kurantz, M.; Thomason, W.; Taylor, F.; Obert, D.; Moreau, R.; Flores, R.; Sohn, M.; Hicks, K. Grain Composition of Virginia winter barley and implications for use in feed, food, and biofuels production. J. Cereal Sci. 2010, 51, 41−49. (7) Khatibi, P. A.; Montanti, J.; Nghiem, N. P.; Hicks, K. B.; Berger, G.; Brooks, W. S.; Griffey, C. A.; Schmale, D. G., III. Conversion of deoxynivalenol to 3-acetyldeoxynivalenol in barley-derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases. Biotechnol. Biofuels 2011, 4, 26. (8) Schaafsma, A. W.; Limay-Rios, V.; Paul, D. E.; Miller, J. D. Mycotoxins in fuel ethanol co-products derived from maize: a mass balance for deoxynivalenol. J. Sci Food Agric. 2009, 89, 1574−80. (9) Madson, P. W.; Monceaux, D. A. Fuel Ethanol Production; KATZEN International Inc: Cincinnati, OH, 1995. (10) Binder, E. M. Managing the risk of mycotoxins inmodern feed production. Anim. Feed Sci. Technol. 2007, 133, 149−166. 4212



Article

(30) Tacke, B. K.; Casper, H. H. Determination of deoxynivalenol in wheat, barley, and malt by column cleanup and gas chromatography with electron capture detection. J. AOAC Int. 1996, 79, 472−5. (31) Lagana, A.; Curini, R.; D’Ascenzo, G.; De Leva, I.; Faberi, A.; Pastorini, E. Liquid chromatography/tandem mass spectrometry for the identification and determination of trichothecenes in maize. Rapid Commun. Mass Spectrom. 2003, 17, 1037−43. (32) Rodrigues-Fo, E.; Mirocha, C. J.; Xie, W.; Krick, T. P.; Martinelli, J. A. Electron ionization mass spectral fragmentation of deoxynivalenol and related tricothecenes. Rapid Commun. Mass Spectrom. 2002, 16, 1827−35. (33) AACC, Approved method 76-13. In Total Starch Assay Procedure (Megazyme Amyloglucosidase/alpha/Amylase Method), AACC International: 10th ed. St. Paul, MN, 2000. (34) Izydorczyk, M. S.; Dexter, J. E.; Desjardins, R. G.; Rossnagel, B. G.; Lagasse, S. L.; Hatcher, D. W. Roller milling of Canadian hull-less barley: Optimization of roller milling conditions and composition of mill streams. Cereal Chem. 2003, 80, 637−644. (35) Rios, G.; Zakhia-Rozis, N.; Chaurand, M.; Richard-Forget, F.; Samson, M. F.; Abecassis, J.; Lullien-Pellerin, V. Impact of durum wheat milling on deoxynivalenol distribution in the outcoming fractions. Food Addit. Contam. 2008, 26, 487−496.

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A Comparison of Two Milling Strategies To Reduce the Mycotoxin

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