Protein interference on aflatoxin B1 adsorption by smectites in corn fermentation solution

Applied Clay Science 144 (2017) 36–44

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Research paper

Protein interference on aflatoxin B1 adsorption by smectites in corn fermentation solution

MARK

Sabrina Sharmeen Alam⁎, Youjun Deng Department of Soil and Crop Sciences, Texas A & M University, College Station, TX 77843-2474, USA

A R T I C L E I N F O

A B S T R A C T

Keywords: Aflatoxin B1 Smectite Fermentation solution Zein Protein

Corn is the main feedstock used for ethanol production in the United States. To reduce wastage and toxicity to human and animal, using aflatoxin contaminated corn in biofuel industry is thought to be rational. Yet up to three-fold of increment of the mycotoxins in the co-product have detrimental impact on animal health. It would be desirable to inactivate or to remove aflatoxins during fermentation of corn. Smectites were previously found to be highly efficient for aflatoxin B1 adsorption in ethanol and glucose solution, two major compounds in corn fermentation solution. The primary objective of the present study was to evaluate the aflatoxin B1 adsorption efficiency by smectites in real corn fermentation solution. The secondary objective was to identify any interfering compound that might hinder aflatoxin B1 adsorption. Aflatoxin B1 adsorption by smectites in fermentation solution was found to be low. A calcium smectite (3MS) had aflatoxin B1 adsorption capacity (Qmax) of 0.22 mol kg− 1in the fermentation solution but 0.54 mol kg− 1 in the aqueous solution. The Fourier transform infrared (FTIR) analyses indicated that some compounds from fermentation solution were adsorbed on the smectites and had irreversible bonding with the clay minerals. Those compounds competed with aflatoxin B1 for the adsorbing sites of smectites. The major infrared bands due to interfering compounds were at ~1653, 1532, 1451, and 1235 cm− 1. These bands appeared when smectites were added to either clean or aflatoxin B1 spiked fermentation solutions. Similar spectral bands were obtained after treating the smectites with zein, a major protein in corn. Thus, the major interfering compounds in fermentation solution were believed to be proteins. The XRD results proved the adsorption of the proteins in the interlayer of smectites. After heating at 300 °C, smectites reacted with fermentation solution had d-spacing of at least 15 Å, whereas the pure smectites collapsed to ~10 Å. This reflected great interferences of the compounds, most possibly proteins on aflatoxin B1 adsorption by the smectites. However, despite of strong interferences, adsorption experiments suggested that smectites were still able to adsorb aflatoxin B1 to some extent. Presence of characteristic aflatoxin B1 bands at ~1595, 1383, 1362, 1304, 1272, and 1205 cm− 1 on smectite complexes treated in fermentation solution revealed the existence of the mycotoxins on the clay minerals. Strategies should be taken to enhance the selectivity of smectites for the aflatoxins in corn fermentation solution.

1. Introduction Aflatoxins are carcinogenic metabolites of fungi, principally produced by the species of Aspergillus flavus. Aflatoxin B1, B2, G1, and G2 are the most common aflatoxins, among which aflatoxin B1 is the most toxic form. Many researchers have demonstrated the toxicity of aflatoxins that caused various diseases and disorders both in animals and humans (Coppock et al., 1989; Richard et al., 1978; Wogan, 1973). For example, the aflatoxin contaminated staple diets of people altered the normal nutritional pattern, suppressed growth, weakened the immunity, and eventually caused death, especially to children (Denning et al., 1995; Gong et al., 2003; Oyelami et al., 1997). The

⁎

Food and Agriculture Organization (FAO) estimated that 25% of the world's food crops are affected by mycotoxins (Richard et al., 1989). Though aflatoxin toxicity is a global problem, researches have investigated that population living between 40°N and 40°S of the equator in developing countries are extremely susceptible to the risk of chronic and largely uncontrolled aflatoxin exposure (Williams et al., 2004). The outbreaks of aflatoxicosis in Kenya in 2004 drew more attention of the mycotoxin researchers. Studies on animal experiments proved that animal health and reproductive systems could be adversely affected by the ingestion of mycotoxin contaminated feeds, where severe doses caused aflatoxicosis. The young poultry were extremely susceptible to the toxicity effects (Diaz et al., 2008; Doerr et al., 1976; Doerr and

Corresponding author. E-mail addresses: [email protected] (S.S. Alam), [email protected] (Y. Deng).

http://dx.doi.org/10.1016/j.clay.2017.04.024 Received 30 August 2016; Received in revised form 8 February 2017; Accepted 27 April 2017 0169-1317/ © 2017 Elsevier B.V. All rights reserved.

Applied Clay Science 144 (2017) 36–44

S.S. Alam, Y. Deng

because 1) DDG is a dry material, there would be less chance to have interaction between the aflatoxins and smectite, and 2) on the other hand, aflatoxin in ethanol solution would be more soluble and hence, there would be greater chance of reaction between the mycotoxin and adsorption domains of smectites. A study showed that the addition of sorbents such as brewers dried yeast anti-caking binder containing glucomannan to DDG was found to reduce aflatoxin toxicity significantly compared to positive control (Johnston et al., 2012). The detoxification of aflatoxin during fermentation process is thought to be a more effective strategy to reduce toxicity effects on animals because the toxins are removed or at least reduced to much lower concentration before reaching to DDG. Moreover, the consumers would be more interested in clean feed rather than mycotoxin contaminated feed though treated by clays for detoxification purpose. The ultimate practical goal of our study was to apply the smectites during biofuel production to reduce the aflatoxin hazards in animal feed from which both the biofuel and animal industry can be benefited. Some questions need to be addressed: a) what are the major interfering compounds in fermentation solution that might compete with aflatoxin B1 for clay's adsorption sites? b) how to minimize their interferences and optimize the clay's selectivity for aflatoxin adsorption? c) how to introduce the clays in fermentation system?, and d) finally, how to separate and remove the clay from the system already loaded with mycotoxins? The specific objectives of the current research were: 1) to evaluate aflatoxin B1 adsorption capacity of the smectites in corn fermentation solution, 2) to investigate the bonding pattern between aflatoxin B1 and the smectites in corn fermentation solution, 3) to evaluate whether interlayer aflatoxin B1 adsorption occurred in this solution, and 4) to identify any interfering compound that blocked aflatoxin B1 adsorption by the smectites.

Hamilton, 1981; Giambrone et al., 1985). Bentonites, in which smectites are the major minerals, have long been used as clay additives in animal feed and proved to improve the overall production of the poultry (Phillips, 1999; Quisenberry, 1968). In addition to some beneficial uses of bentonite for human and animal, they have showed their high efficiency to bind aflatoxin in many studies (Desheng et al., 2005; Dixon et al., 2008; Kannewischer et al., 2006). Our research on aflatoxin-smectite interaction proved that some calcium smectites had up to 0.6 mol kg− 1 of aflatoxin B1 adsorption capacity in water, which was equivalent to ~20% of the clay on dry mass basis (Deng et al., 2012a, 2012b). A study confirmed most of the adsorptions occurred in the interlayer, though some adsorptions were also found at basal surfaces and edges (Grant and Phillips, 1998). In the drought prone regions of the states, aflatoxin contamination of corn is unavoidable despite of taking many preventive strategies. In addition to climatic influences, other biotic and abiotic factors also accelerate the mold growth during crop production, harvest, and storage. The U.S. Food and Drug Administration (FDA) regulated the acceptable limit for aflatoxin to 20 ppb in common foods and feeds. Corn (Zea mays) is the main feedstock used for ethanol production in the rapid rising biofuel industry of the states. According to independent statistics and analysis of U.S. Energy Information Administration (EIA), the country produced 14.8 billion gal of ethanol in 2015. To meet such a high demand on sustainable energy, more than one third of the corn produced in the USA is utilized in biofuel industry for ethanol production. Due to high market price of corn, frequent mycotoxin contamination, and rapid growing of biofuel industry, it seemed to be rational to use aflatoxin contaminated corn for ethanol production by which wastage of corn can be reduced. Three fold enrichment of the mycotoxin occurs in the co-products of ethanol production after the fermentation process. The co-products are known as dried distiller's grains (DDG), which are extensively used as animal feeds worldwide due to their large availability, low cost, and high nutritive value. Mycotoxin contamination often threatens their suitability of using as animal feeds. The aflatoxins were found to be degraded during fermentation process in two studies (Dam et al., 1977; Okoye, 1986) while other researchers concluded that they were not degraded (Lillehoj et al., 1979; Schaafsma et al., 2009). The economical sustainability of biofuel industries at least partially depends on the marketability of their DDG (Wu and Munkvold, 2008). The ethanol industry as well as the animal industry in close proximity to aflatoxin contamination susceptible regions might have greater concern. Not all the DDG in the United States were detected to have mycotoxins level beyond the FDA regulatory limit (Zhang et al., 2009). Due to three times increment of aflatoxin in DDG, it is desirable to have the mycotoxins removed, decomposed, or inactivated during the fermentation system. The alternative use of aflatoxin contaminated DDG were suggested to be crop fertilizer in a study (Kelly et al., 2009). However, it's not surprising that the Aspergillus spores in the DDG would be further risk for soil and crop contamination with mycotoxins. Ethanol is produced from the corn biomass through the fermentation process with the addition of yeast. Corn fermentation solution is a complex organic solution which typically contains various organic compounds such as ethanol, sugar, oil, lipid, lactic acid, acetic acid, glycerol, proteins, and amino acids. In our previous study, we discovered that the two major soluble compounds e.g., ethanol and glucose had minimal interference on aflatoxin B1 adsorption by the smectites (Alam et al., 2015). This discovery led us to expect that smectites would also have high aflatoxin adsorption capacity in real corn fermentation solution. However, the compounds in fermentation solution other than ethanol and glucose, might compete with aflatoxin B1 for the clay's adsorption sites. Maintaining the clay's ability to detoxify aflatoxins from such a complex solution could be a challenge. A question may arise why using clays in corn fermentation solution but not in DDG directly? This is

2. Materials and methods 2.1. Extraction of < 2 μm calcium clays from bentonite Five calcium bentonites 3MS, 1MS (Mississippi), MBBO1 (Alabama), 37GR (Greece), and 8TX (Texas) were used for the present study. These bentonites were previously reported as highly efficient aflatoxin B1 binders in aqueous solution (Alam et al., 2015; Deng et al., 2012a, 2012b). Bentonites were size fractionated and the clays (< 2 μm) were extracted (Deng et al., 2012b). The Na ions of the fractionated clays were exchanged with Ca ions before the aflatoxin adsorption experiment. 2.2. Collection of corn fermentation solution (FS) About 6 L of aflatoxin-free corn fermentation solutions containing corn mash were supplied by a local ethanol plant White Energy Inc. in Plainview of Texas. The fermentation solutions with corn mash contained ethanol as they were collected before the distillation process. The fermentation process was still going on even after the solutions were stored at ~4 °C in a refrigerator, and CO2 gas bubbling was observed. For the aflatoxin adsorption experiment, the fermentation solution was filtered by the slow flow rate filter paper with fine porosity (diameter is 15.0 cm) of Fisher brand®, and then centrifuged to discard any solid materials. Thus, the clean solution was obtained. The pH of fermentation solution was recorded as 4.78. The solution was preserved in the refrigerator for long term use. 2.3. Smectite preparation for FTIR and XRD analyses To understand if any compound from fermentation solution would adsorbed into the interlayer of smectites, and thus if they impacted the bonding pattern between smectite and aflatoxin B1, smectite complexes for each of 3MS, 8TX, 37GR, MBBO1, and 1MS were prepared in the filtered fermentation solutions without and with spiked aflatoxin B1. To 37

Applied Clay Science 144 (2017) 36–44

8TX-FS

2959

AlMgOH

Al2-OH

~0% humidity

MBBO1-FS

3MS-FS

2926 2850

3623 3290

790

1451 1233 1538 1652

1743

837

3294

1451 1235 1532 1653

1743

2925 2854

1544

3%

37GR-FS

1237

2959 2872

~1

00 ~0 % %

1MS-FS

1629

~2

T r a n s m i t t a n c e (%)

MBBO1

amorphous SiO2

S.S. Alam, Y. Deng

915 // // 1800 1600 1400 4000 3600 3200 2800 1800 1600 1400 1200 1000 750 3600 3200 -1 -1 W a v e n u m b e r (cm ) W a v e n u m b e r (cm-1) W a v e n u m b e r (cm )

1200

Fig. 1. FTIR spectra of one smectite and five smectite complexes formed in fermentation solution.

34% from the corn fermentation co-product such as DDG (Paraman and Lamsal, 2011). Suspecting that the interfering compound in fermentation solution could be protein, smectite-zein and smectite-AfB1-zein complexes were prepared following the procedure described in Section 2.3 for XRD and FTIR. For this experiment, the zein was purchased from Sigma Aldrich. Zein was investigated in its powder form by the ATR accessory. To saturate smectites with zein, zein was dissolved in 90% ethanol (v/v), and in water at pH 12 by adding 2 M NaOH solution until the zein was completely dissolved. The solution was then filtered by the Fisher brand® filter paper with fine porosity (diameter is 15.0 cm) and slow flow rate to remove any particles not dissolved.

compare the adsorption of aflatoxin B1 in fermentation solution with the adsorption in aqueous solution, smectite-AfB1 complexes were also prepared in aqueous solution. Therefore, the four samples for each of smectites were (a) smectite, (b) smectite-FS, (c) smectite-AfB1 (AfB1 in water), and (d) smectite-AfB1-FS. Here, AfB1 indicated aflatoxin B1, and FS indicated fermentation solution. The last three complexes were prepared as below: One milligram of Ca-smectite was added to each 50 mL Falcon® tube containing 35 mL of fermentation or aqueous solution artificially contaminated with 8.0 ppm aflatoxin B1. The clay suspensions were shaken for 24 h at 200 rpm, and centrifuged at 4000 rpm for 57 min. The treatment was repeated one more time by replacing the supernatant with additional 30 mL of respective solution. The complexes were washed with de-ionized water twice to remove excess aflatoxins and any materials that were not adsorbed by the clays. The clay residues were dispersed in ~0.5 mL water to avoid drying and kept in refrigerator at 4 °C. Same procedure was followed to saturate the clays in fermentation solution where no aflatoxin was added. The FTIR spectra of the clay or complex films on ZnS discs (ClearTran, International Crystal Labs, Garfield, New Jersey, USA) were recorded in the transmission mode on a Spectrum 100 Fourier transform infrared spectrometer (Perkin-Elmer) at ~0% humidity, room humidity (~ 23%), and ~100% humidity. A Dewar accessory was used for FTIR analyses. For the XRD analyses, samples were analyzed at room temperature (~ 25 °C) and after heated at 300 °C by XRK900 (Anton Paar). The methodology required for FTIR and XRD analyses were described in detail by Alam et al. (2015).

3. Results 3.1. FTIR investigations of smectite complexes in aqueous and corn fermentation solution The FTIR spectra of the smectite-FS complexes had similar responses to humidity. The spectra under three moisture conditions were shown only for the MBBO1-FS as an example (Fig. 1). Many IR bands generated due to the interaction between smectite and the compounds in fermentation solution. These bands were recorded at ~3290, 2926, 2850, 1743, 1652, 1538, 1451, and 1233 cm− 1 in the range from 4000 to 1200 cm− 1. They were absent in the spectrum of pure smectite MBBO1. The IR band intensities or positions of MBBO1-FS complex did not change remarkably upon humidity variations. Only the bands 1538 cm− 1 and 1233 cm− 1 at 0% moisture condition shifted slightly to 1544 and 1237 cm− 1, respectively upon 100% moisture treatment. As no remarkable variation was observed due to moisture treatment, only the IR spectra recorded at 0% humidity condition for 8TX, 1MS, 37GR, and 3MS treated with fermentation solution were presented (Fig. 1). All the smectite-FS complexes showed similar spectra. The similar IR bands that were present in MBBO1-FS complex were also appeared in the four spectra of other smectite-FS complexes. Again, the bands were recorded at ~ 3294, 2925, 2854, 1743, 1653, 1532, 1451, and 1235 cm− 1. These bands were completely absent in the respective pure smectites (spectra were not shown). The IR bands at the region between 1800 and 1200 cm− 1 were known to be the most common bands to indicate the bonding between aflatoxin B1 and smectite. Within this range, bands at ~1653 and 1532 cm− 1 were the most intensive and broad bands due to the adsorption of the interfering unknown compounds from fermentation solution in the interlayer of smectites. FTIR analyses for five smectite complexes treated in aflatoxin B1 spiked fermentation solution showed that IR bands found due to interaction between smectite and fermentation solution (presented in the Fig. 1), were also present in each of the five smectite-AfB1-FS complexes (Fig. 2). The bands for compounds adsorption from fermen-

2.4. Procedures of aflatoxin B1 adsorption isotherm in fermentation solution Aflatoxin B1 from A. flavus was purchased from Sigma Chemical Co. (St. Louis, MO 63118, USA), CAS No. 1162-65-8. The procedure for isothermal adsorption of aflatoxin B1 by smectites was described thoroughly by Kannewischer et al. (2006). The exception was that in the present study the 8 ppm aflatoxin B1 working solution was prepared in diluted fermentation solution rather than in simplified 100% aqueous solution. Batch solution containing 0.0, 0.4, 1.6, 3.2, 4.8, 6.4 and 8.0 ppm aflatoxin B1 was prepared in the corresponding solutions. For single point aflatoxin adsorption experiment, only 8.0 ppm aflatoxin B1 solution was used. The supernatant after adsorption of the toxin by the clay was analyzed by the Beckman Coulter DU800 UV/visiblespectrophotometer at 365 nm wavelength. 2.5. Preparation of smectite-zein complexes Zein is the major storage protein in corn, in fact it is the mixture of different peptides, which comprises about half of the protein in corn (Shukla and Cheryan, 2001). Zein was also isolated and recovered up to 38

Applied Clay Science 144 (2017) 36–44

a

Sm-AfB1(AfB1 in water)

1659

a b

1239

1303

1532

1532

1659

MBBO1-AfB1-FS a b

1304 1272 1236 1205

1383 1362

1595 1545

1743

1443

c

1304 1272 1236 1205

1383 1362

1443

1595

1545

c

1743

T r a n s m i t t a n c e (%)

1800 1700 1600 1500 1400 1300 1200 37GR-AfB1-FS

~0% humidity

1205

1592

1727

1304 1272 1236 1205

1383 1362

1443

1595

1545

c

1271

b

1546 1506 1483 1462 1443 1417 1384 1361

1659

1532

3MS-AfB1-FS

1743

T r a n s m i t t a n c e (%)

S.S. Alam, Y. Deng

1659

a b c

1304

1383

1443

1545

1743

1304 1272 1236 1205

1383 1362

1443

1236 1205

1532

b

1532

8TX-AfB1-FS a

c

1595 1545

1659

1MS-AfB1-FS

1743

T r a n s m i t t a n c e (%)

1800 1700 1600 1500 1400 1300 1200 1800 1700 1600 1500 1400 1300 1200

1800 1700 1600 1500 1400 1300 1200 1800 1700 1600 1500 1400 1300 1200

W a v e n u m b e r (cm-1)

W a v e n u m b e r (cm-1)

a = ~0% humidity, b = ~23% humidity, c = ~100% humidity Fig. 2. FTIR spectra of five smectite complexes under three moisture conditions. Solid lines are the AfB1 bands.

tation solution were recorded at 1743, ~1659, 1532/1545, 1443, and 1236 cm− 1 in the all spectra. No remarkable differences were noticed in the spectral position or intensity upon moisture variations, except a minor shifting of the band from 1532 to 1545 cm− 1 when moisture level converted from ~0% to room or ~100% humidity. Comparison of the spectra of smectite-AfB1-FS complexes with the spectra of smectiteAfB1 (AfB1 in water) revealed that the other minor bands 1595, 1383, 1362, 1304, 1272, and 1205 cm− 1 in almost all of the smectites appeared because of the adsorption of aflatoxin B1 molecules onto the smectites. It was obvious that the number and intensity of aflatoxin B1 bands decreased in the smectites-AfB1-FS complexes compared to the smectites-AfB1 (AfB1 in water) complex. More than fifty percent of the aflatoxin B1 characteristic bands were absent in each of three spectra of the five smectite-AfB1-FS complexes, which suggested lower amounts of aflatoxin B1 intercalation in those complexes prepared in the fermentation solutions.

example, the d-spacing of 3MS expanded from 15.2 to 18.2 Å, of 37GR from 14.2 to 17.8 Å, of 8TX from 13.7 to 17.7 Å, of MBBO1 from 14.2 to 18.1 Å, and of 1MS from 14.7 to 18 Å at room temperature after exposed to fermentation solutions. After heating at 300 °C, d-spacing of the smectites after treatment with fermentation solutions remained up to 14.8, 15.2, 15.0, 14.8, and 15.9 Å for 3MS, 37GR, 8TX, MBBO1, and 1MS, respectively. These d values were even higher than the d values of all smectite-AfB1 (AfB1 in water) samples. Though at room temperature, the d-spacing were very close or similar between the smectites and the respective smectite-AfB1 (AfB1 in water) complexes, remarkably higher d-spacing between 12.1 and 13.3 Å were recorded after heating treatment for each smectite-AfB1 complexes. Negligible differences in the d-spacing, for example, from 14.8 to 15.1 Å, 15.2 to 16.2 Å, 15.0 to 15.4 Å, and 14.8 to 15.5 Å for 3MS, 37GR, 8TX, and MBBO1, respectively were observed between the smectite-FS and smectiteAfB1-FS complexes after the 300 °C heating treatment. No difference e.g., 15.9 to 16 Å was found in case of 1MS.

3.2. XRD analyses revealed smectite's interlayer adsorption of compounds from fermentation solution

3.3. Aflatoxin B1 adsorption of smectites in aqueous and fermentation solution

The basal d-spacing for the five pure smectites at room temperature varied from 13.7 Å (8TX) to 15.2 Å (3MS), however, all of them collapsed to ~ 10 Å after heating at 300 °C (Fig. 3). On the other hand, the d-spacing of the smectite-FS or smectite-AfB1-FS complexes increased largely when they were treated in fermentation solutions. For

As XRD and FTIR analyses indicated strong interlayer adsorption of one or more organic compounds from fermentation solution onto the smectites, the aflatoxin B1 adsorption capacity of the smectites in the presence of such complex solutions would possibly be low. Pilot tests were conducted to investigate aflatoxin B1 adsorption 39

Applied Clay Science 144 (2017) 36–44

S.S. Alam, Y. Deng

20

20 18.5

18.2 18

18.2 16

15.8 15.2

Basal d-spacing (Å)

Basal d- spacing (Å)

18

15.1 14.8

14 13.2 12

3MS 3MS -FS

10

3MS -AfB1

17.8 16.2

16 14

14.2 12.4

12 37GR 37GR-FS 37GR-AfB1 37GR-AfB1-FS

10

9.8

15.2

14.4

3MS -AfB1-FS

10

8

8 0

100

200 Temperature (°C)

0

300

100

200

300

Temperature (°C) 20

20

18.5 18.3

18

18.1 16

Basal d-spacing (Å)

Basal d-spacing (Å)

18

15.5 14.8

14.4 14 14.2

12.1

12

MBBO1 MBBO1-FS MBBO1-AfB1 MBBO1-AfB1-FS

10

18 14.5 14

15.9

14.7 12.9

12 1MS 1MS-FS 1MS-AfB1 1MS-AfB1-FS

10

10

16

16

10

8

8 0

100

200

0

300

100

200

300

Temperature (°C)

Temperature (°C) 20 17.9

Basal d-spacing (Å)

18

17.7 16

15.4 15

15.6 14

13.3

13.7 12 8TX 8TX-FS 8TX-AfB1 8TX-AfB1-FS

10

10

8 0

100

200

300

Temperature (°C) Fig. 3. The d-spacing of smectites and their complexes in different solutions analyzed by heating XRD.

smectite in fermentation solution were much lower compared to the adsorption of the smectite in aqueous solution (Fig. 5). The adsorption of aflatoxin B1 in fermentation solution was even remarkably lower than the adsorption in 10% glucose (w/v) and 20% ethanol (v/v) as described in our previous study (Alam et al., 2015). The isotherm adsorption in water and fermentation solution was fitted using the modified Langmuir equation (known as QKLM) with q-dependent affinity as shown below:

onto the smectites in aqueous and fermentation solution. As the UV absorbance of undiluted fermentation solution was too high, the solution was diluted to 1:3 (fermentation solution:water) ratio. The diluted fermentation solution yielded a manageable spectrum of aflatoxin B1 that could be used to quantify aflatoxin B1 with the UV method (Fig. 4). The absorbance of AfB1 standard solutions at wavelength 365 nm showed a linear relationship with concentrations (R2 of 0.999). The linearity of dilutions was done only once. Due to the similar responses of the smectites to the fermentation solutions, only 3MS was used for the full batch isothermal aflatoxin adsorption experiment. This smectite previously showed higher aflatoxin B1 adsorption in aqueous, ethanol, and glucose solutions (Alam et al., 2015). Both aflatoxin B1 adsorption capacity and affinity of

⎡ Ke(−2bq) C ⎤ q = Q max ⎢ ⎥ ⎣ 1 + Ke(−2bq) C ⎦ The parameters of the above equation were described in a study (Deng et al., 2012a). The fitting curve in fermentation solution was 40

Applied Clay Science 144 (2017) 36–44

S.S. Alam, Y. Deng

0.8

b c

1.5

a = AfB1 in water b = AfB1 in undiluted FS c = AfB1 in diluted FS

1

AfB1 peak

0.5 a

0 200

255

310

365

420

475

Absorbance @ 365 nm

Absorbance

2

y = 0.0675x + 0.2856 R² = 0.9999

0.7 y = 0.0675x - 0.002 R² = 0.9999

0.6 0.5 0.4 0.3 0.2

Uncorrected absorbance

0.1

Corrected absorbance

Wavelength (nm)

0 0.0

2.0

4.0

6.0

8.0

AfB1 concentration (ppm) Fig. 4. Left: (a) UV spectra of AfB1 in aqueous solution, (b) AfB1 prepared in the undiluted, and (c) diluted fermentation solutions (FS). Right: Standard curves of AfB1 in diluted FS.

0.6

in aqueous solution

3MS AfB1 adsorption capacity Qmax (mol kg-1)

0.5 Media

AfB1 adsorption parameters Modified Langmuir, QKLM 2 K Qmax b (mol kg-1) (µM-1) Aqueous solution 0.54 0.408 -1.05 0.98 Fermentation 0.22 0.031 -3.89 0.92 solution

0.4 0.3 0.2 in fermentation solution

0.1 0 0

5

10

15

20

25 -1

AfB1 equilibrium concentration (µmol L ) Fig. 5. AfB1 adsorption isotherms of smectite in aqueous and fermentation solution.

better (linear R2 = 0.93) when the QKLM was used rather than using original Langmuir model (linear R2 = 0.89). The data derived from adsorption in aqueous solution was also fitted by using QKLM model for ideal comparison to the adsorption values in fermentation solution. The aflatoxin B1 adsorption capacity of 3MS in aqueous solution was 0.54 mol kg− 1, whereas the adsorption capacity was reduced to 0.22 mol kg− 1 in fermentation solution. Smectites 8TX, 37GR, MBBO1, and 1MS were tested for their adsorption efficiencies using only single point concentration of aflatoxin B1 solution (Fig. 6). Results showed that for each of the smectites, aflatoxin B1 adsorption in fermentation solution was much lower than the adsorption in aqueous solution.

AfB 1 adsorbed (mol kg-1)

0.6 In aqueous solution

0.5

In fermentation solution

0.4 0.3 0.2 0.1 0

3.4. Predicting the interfering compounds in fermentation solution by FTIR and XRD analyses of smectite-zein complexes

8TX

37GR

MBBO1

1MS

Fig. 6. AfB1 adsorbed by four smectites in aqueous and fermentation solution.

Zein is the major protein in corn and soluble in ethanol. As the fermentation solution contains an appreciable quantity of ethanol, some zein proteins could be expected in the fermentation solution. The presence of the bands 1744, 1646, 1521, 1456, and 1235 cm− 1 in the ATR spectra of zein were very similar to the bands for interfering compounds appeared in the smectite-FS complexes (Fig. 7). Again, very similar position and intensity of the interfering bands such as 1744/ 1739, 1656/1622/1652, 1530/1538, 1443/1447, and 1235 cm− 1 were obtained from 3MS-AfB1-FS and 3MS-AfB1-zein complexes, irrespective of the nature of the solution where zein was dissolved (in ethanol or

pH 12 water solution). A study showed that alcohol solubilization of α zeins did not affect its conformation (Forato et al., 2003). The smectiteAfB1-zein complexes were also intercalated by the aflatoxin B1 as was evident by its characteristic bands, however, the bands were weaker in those complexes (Fig. 7). High temperature XRD analyses of the 3MS-zein complex (zein dissolved in ethanol) revealed that the zein protein went into the interlayer of that smectite (Fig. 8). Smectite treated in very high 41

Applied Clay Science 144 (2017) 36–44

S.S. Alam, Y. Deng

1

-Z ei n 1235

1447 1538 1622

-Z ei n

1739

(z ei n

1 et ha no l)

3M SA in fB 90 %

1530 1656

3M S

1739

1447 16521538 1800 1600 1400 1200 1000

1600

1400

1303

1521 1800

1235

(z ei n

1361

in

1456

1235

-A wa te fB ra tp 1 H 12 )

1238

T r a n s m i t t a n c e (%)

1744

1646

T r a n s m i t t a n c e (%)

1443

3M SA fB

1744

1271 1206

1389

1595

solid zein

-F S

Dewar-IR ATR

1200

W a v e n u m b e r (cm-1)

W a v e n u m b e r (cm-1)

Fig. 7. ATR spectra of zein (left) and Dewar-IR spectra of smectite complexes (right). The dotted lines are AfB1 bands.

18 17 16 15 14 13 12 11 10 9

The overall FTIR patterns of the five smectite-FS complexes suggested that one or more compounds from fermentation solution entered into the interlayer of smectites. The adsorption of those interfering compounds by the smectites reduced the chance of bonding between aflatoxin B1 and smectites as indicated by the reduced number of characteristic aflatoxin B1 bands as well as their decreased intensities. Early research showed that the band at 1742 cm− 1 was a characteristic band of aflatoxin B1 in Ba saturated smectite-AfB1 complexes when treated under 100% humidity but later shifted to 1725 cm− 1 when the samples were analyzed under ~0% moisture (Deng et al., 2012a). Again, in another study it was observed that band 1743 cm− 1 shifted much to lower bands when the moisture changed from ~100% to 0% humidity irrespective of the exchange cations (Deng et al., 2010). On the contrary, in the present study this band did not change or disappeared upon humidity variation. However, the presence of the minor band 1743 cm− 1 in all smectite-FS complexes revealed that it appeared due to adsorption of compound, most likely lipids from fermentation solution. The specific IR bands such as ~ 1744, ~1653, ~1532, ~1451, and ~1235 cm− 1 that were generated due to reacting the smectites with fermentation solution, were not found in our previous studies where smectites-AfB1 complexes were treated in high concentrations of glucose or ethanol solutions (Alam et al., 2015). The current investigations confirmed that those particular five bands were not due to the adsorption of ethanol or glucose from the fermentation solution but were due to the adsorption of other organic compounds by the smectites. The presence of the bands due to the adsorption of interfering compounds on the smectite-FS or smectiteAfB1-FS spectra even after repeated washing of the clay residues with distilled water suggested that the compounds from fermentation solution had strong attraction to the smectites. The persistence of the compounds on smectite's interlayer suggested they were either positively charged, had great molecular weight, or both. It seemed to be difficult to dissociate those compounds from the clay. Any strategy that block the adsorption of the interfering compounds by the smectites might indirectly reduce their interferences on aflatoxin B1 adsorption. In the current study, the infrared investigations of the zein protein suggested that the new bands appeared on the clay spectra due to reaction of fermentation solution with smectites were most probably

Basal d-spacing (Å)

16.89 15.2

3MS-zein 13.07 3MS 9.74

0

100 200 Temperature (°C)

300

Fig. 8. The d-spacing of smectite after treated in pure ethanol (lower line) and in ethanol containing zein (upper line).

concentration of ethanol (90%, v/v) collapsed to 9.74 Å after heating at 300 °C but the smectite-zein complex did not collapsed, rather had higher d-spacing of 13.07 Å. 4. Discussion The adsorption data of the present study demonstrated that aflatoxin B1 adsorption capacity of smectites from corn fermentation solution was reduced at least 50% compared to the adsorption in aqueous solution. This suggested strong interferences of some compounds from fermentation solution that competed greatly with aflatoxin B1 molecules to occupy the adsorbing sites of the clays, and thus drastically impeded interlayer access of aflatoxin B1. A study showed that aflatoxin B1 adsorption capacity of smectite in corn meal extracted by 60% methanol was also found to be significantly lower than the adsorption in water (Jaynes et al., 2007). Another study showed that aflatoxin B1 adsorption by a Ca-smectite in simulated gastric fluid was very low due to the intercalation of pepsin on smectite (BarrientosVelázquez et al., 2016). The same study demonstrated that the Nasmectite had greater pepsin adsorption than the Ca-smectite. 42

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The overall FTIR and XRD data confirmed that the interfering compounds were more potential than the aflatoxin molecules to occupy the interlayer exchange sites of the clays, which subsequently reduced aflatoxin B1 adsorption by the smectites. Very similar bands position and intensities between the spectra of smectite-AfB1-zein and smectiteAfB1-FS indicated that the interfering compounds in fermentation solution were most likely the dissolved proteins. The XRD result of Fig. 8 revealed that the zein proteins could be adsorbed onto the smectite. The greater d-spacing of the 3MS-FS (14.8 Å) than the 3MSZein (13.07 Å) indicated a greater adsorption of the compounds from fermentation solution. There might be some compounds other than zein in such solution, for example, corn proteins namely albumin, globulin, or glutelin as well as the yeast enzyme (zymase) that catalyze corn during fermentation could also be a barrier for optimal adsorption. Future investigations are suggested regarding the effects of these compounds on aflatoxin B1 adsorption by smectites. The major constituents of corn include starch, protein, oil, fiber, where protein comprises about 9–12% (w/w) of corn kernel (Earle, 1977). A study showed that protein content of ground corn (~ 7.5%) increased more than three times (~ 28%) in the fermented mass produced in three different dry-grind ethanol plants, and remained almost unchanged in dried distiller's grain with soluble (Han and Liu, 2010). The presence and persistence of the proteins in the smectites revealed that they were nonionic or cationic in charge properties, and possibly had irreversible bonding with smectites. A study suggested that the adsorption of cationic and nonionic polymers on smectites was irreversible (Deng et al., 2006). Proteins are mainly adsorbed by the clays through electrostatic interactions and entropy gain. Adsorption of proteins and immobilization of enzymes by clays from various sources were found in many studies (Bajpai and Sachdeva, 2002; Larsson and Siffert, 1983; Ralla et al., 2010; Sinegani et al., 2005). Therefore, the proteins were believed to be the major interfering compounds for reduced aflatoxin B1 adsorption by smectites in corn fermentation solution, though some other organic compounds might have minor interferences.

due to the intercalation of the proteins by the smectites. Similar IR bands were recognized as bands of protein substances in many other studies. For example, a medium intensity band in the 3300 to 3000 cm− 1 region was reported due to intermolecular hydrogen bonding of primary amines (both aliphatic and aromatic) and secondary amines exhibited a single band in the 3450 to 3310 cm− 1 region (Conley, 1966). In our study, band at ~3290 cm− 1 could be such band from fermentation solution that had medium intensity and possibly originated as a result of nitrogen-hydrogen (NeH) stretching vibration. The CeH stretching vibrations showed a relatively large contribution of CH3 groups (2959 and 2872 cm− 1) as typical for proteins. Again, the imino group was generally confirmed by the weakly absorbing C]N stretching vibration from 1690 to 1640 cm− 1. Interestingly, the most intensive and broad band ~1656 cm− 1 from fermentation solution appeared within this region. It should be mentioned that this region was also recognized as carbonyl (C]O) stretching bands. Differentiation could be made as the C]O stretching bands were more intense then the C]N stretching bands. Furthermore, the bands in the region from 1580 to 1490 cm− 1 were considered as the bending vibrations of single NeH group of secondary amines. The second broad band ~1532 cm− 1 in the smectite-FS complexes appeared within this wave number. A review article mentioned that the NeH stretching vibration gave rise to the amide A band from 3310 to 3270 cm− 1, and a weak amide B band from 3100 to 3030 cm− 1 (Barth, 2007). In the present study, band ~ 3294 cm− 1 most probably indicated the presence of amino groups in proteins from fermentation solution. Again, the infrared bands at ~ 1650, ~1550, and from ~1400 to 1200 cm− 1 were suggested as the amide I, amide II, and amide III bands of proteins, respectively. The bands for water absorption in the mid-infrared spectral region such as 1629 cm− 1 overlapped with the broad and intensive amide I band of proteins recorded at ~1652 cm− 1. In another study, bands at 1653 and 1530 cm− 1 were suggested for amide I and amide II, respectively (Sepelyak et al., 1984). In this study, the band at 1232 cm− 1 might be due to DNA and RNA coming from the residue of fermentation solution that was mixed with clays. A study showed that the higher intensity backbone PO2 asymmetric stretching band at 1225 cm− 1 of free DNA was shifted towards a lower frequency at 1210 cm− 1 when interacted with human serum albumin, whereas band 1240 cm− 1 for free RNA didn't shifted remarkably (Tajmir-Riahi et al., 2009). The XRD analyses of the smectite-FS complexes revealed that compounds from fermentation solution went into the interlayer of smectites, and were adsorbed in large amounts. This was proved by the remarkably greater d-spacing of the five smectite-FS complexes than the d-spacing of smectites both at low and high temperatures. Even after heating at 300 °C, the d− spacing of ~15 to 16 Å of the smectite-FS complexes suggested that the interfering compounds were not decomposed fully by heating. The noticeably high d-spacing of the clay complexes after repeated washing again reflected the strong interlayer adsorption of the compounds from fermentation solution. The relatively higher d-spacing of smectite-FS complexes than that of smectite-AfB1 (AfB1 in water) complexes demonstrated that the biological molecules such as proteins from fermentation solution were much more potential than the aflatoxin molecules to occupy the interlayer spaces of smectites, and thus they did interfere with aflatoxin B1 adsorption. A negligible increase in basal spacing of smectite-AfB1-FS complexes relative to smectite-FS complexes indicated a very low interlayer adsorption of aflatoxin B1 onto the smectites in fermentation solution. The XRD result was in accordance with the FTIR investigations, where very few aflatoxin B1 bands were present in the smectite-AfB1-FS complexes. The remarkably higher basal spacing of the smectite-AfB1FS than the smectite-AfB1 (AfB1 in water) complexes was again attributed to the intensive interlayer access of compounds from fermentation solution but surely along with some aflatoxin B1 adsorption. It could be possible that zein proteins in fermentation solution can aggregate the clay particles and hence, reduced the surface of the clays for aflatoxin B1 binding.

5. Conclusions Compared to the adsorption in simplified aqueous solutions, smectites had much lower aflatoxin B1 adsorption capacities in corn fermentation solution. The presence of FTIR bands at ~1635, 1532, 1451, and 1235 cm− 1; and higher d-spacing of 13.07 Å of the zeinsmectite complex compared to smectite alone at elevated temperature suggested the intercalation of proteins from corn fermentation solution. Thus, the proteins were believed to be the major interfering compounds in corn fermentation solution. They occupied the interlayer space of smectites, and consequently, blocked the accessibility of the interlayer for aflatoxin B1. The affinity of aflatoxin B1 for the smectite's adsorption sites became weaker in the presence of such complex organic solution, as indicated by the reduced number of particular IR bands for aflatoxin B1 adsorption. A considerably higher basal d-spacing of the smectite complexes in fermentation solution than pure smectites suggested that the interfering compounds had very strong affinity for the smectites. Despite of strong interferences of the proteins, the smectite's efficiency in adsorbing aflatoxin B1 in fermentation solution could still be adequate as smectites was evidenced to reduce aflatoxin toxicity in human and animal body in the presence of various biological components. Strategies should be developed to minimize the interferences of the proteins or other compounds in fermentation solution for optimal aflatoxin B1 adsorption. Acknowledgements This research was financially supported by Aflatoxin Mitigation Center of Excellence (AMCOE) (Grant 406893) and Texas A & M AgriLife Bioenergy Initiative Program (Grant 11473, 12473). 43

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