Review pubs.acs.org/JAFC
Reduction of Fumonisin Toxicity by Extrusion and Nixtamalization (Alkaline Cooking) Kenneth Voss,*,† Dojin Ryu,§ Lauren Jackson,# Ronald Riley,† and Janee Gelineau-van WaesΔ †
Toxicology and Mycotoxin Research Unit, U.S. National Poultry Research Center, Agricultural Research Service, U.S. Department of Agriculture, 950 College Station Road, Athens, Georgia 30605, United States § School of Food Science, University of Idaho, 875 Perimeter Drive, MS 2312, Moscow, Idaho 83844, United States # Division of Food Processing Science and Technology, Center for Food Safety and Nutrition, U.S. Food and Drug Administration, 6502 South Archer Road, Bedford Park, Illinois 60501, United States Δ Department of Pharmacology, Creighton University School of Medicine, 2500 California Plaza, Omaha, Nebraska 68178, United States ABSTRACT: Fumonisins are mycotoxins found in corn. They are toxic to animals and cause cancer in rodents and neural tube defects in LM/Bc mice. Reducing their concentrations in corn-based foods is therefore desirable. Chemical analysis or in vitro bioassays of food extracts might not detect toxic fumonisin reaction products that are unknown or unextractable from food matrices, thus potentially underestimating in vivo toxicity. The effectiveness of two common cooking methods, extrusion and nixtamalization (alkaline cooking), to reduce the toxicity of fumonisin-contaminated corn grits (extrusion) and whole kernel corn (nixtamalization) was shown by means of rat feeding bioassays using fumonisin-specific kidney effects as indicators of potential toxicity. A third bioassay showed that in contrast to fumonisin B1 (FB1), hydrolyzed fumonisin B1 (HFB1; formed from FB1 during nixtamalization) did not cause neural tube defects in LM/Bc mice. The findings indicate that extrusion and nixtamalization reduce the potential toxicity of FB1-contaminated corn. KEYWORDS: fumonisins, toxicity, nixtamalization, extrusion cooking, corn (maize)
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INTRODUCTION Fumonisin mycotoxins are produced by Fusarium verticillioides (formerly F. moniliforme Sheldon), a common fungal contaminant of corn (maize) worldwide.1 These mycotoxins are relatively heat stable under many cooking and processing conditions and are therefore found in corn-based food products.2 Fumonisin B1 (FB1), 1 (Figure 1), is the most common and most intensely studied congener. In addition to its “free” form, FB1 and other fumonisins exist in “matrixassociated forms”. The latter are bound to, physically trapped within, or otherwise interact with matrix constituents so that they are not extractable until the matrix has been subjected to alkaline hydrolysis, hydrolysis following sodium dodecyl sulfate extraction, or simulated digestion.3−6 Fumonisin reaction products such as hydrolyzed FB1 (HFB1), 2 (Figure 1), or N(1-deoxy-D-glucos-1-yl) FB1 (NDFB1),7 3 (Figure 1), and a series of N-acylated FB1 metabolites 4 (Figure 2) have been characterized in rat liver and kidney.8 Fumonisins are toxic to animals, causing hemorrhagicliquifactive brain lesions in Equidae (equine leukoencephalomalacia), pulmonary edema in swine, and kidney and/or liver toxicity in most species.9 FB1 is a liver and kidney carcinogen in rodents.1,10,11 Fumonisins such as FB1 that have a primary amino function inhibit the enzyme ceramide synthase, increasing tissue concentrations of the upstream metabolite sphinganine (Sa) and its metabolite, Sa 1-phosphate (Sa1P). Concentrations of sphingosine (So; which is salvaged from ceramide) and its 1-phosphate metabolite (So1P) also increase, but less so than Sa and Sa1P. Downstream complex © 2017 American Chemical Society
sphingolipid concentrations decrease. Consequently, overall sphingolipid metabolism and sphingolipid-dependent functions are disrupted, which ultimately leads to cytotoxicity and tissue damage.12−14 A correlation between elevated tissue Sa, Sa1P, and Sa/So ratios in tissues and the ultimate toxicological and tissue pathological effects of fumonisins in animals is well established.1,15 No human health effects attributable to fumonisin exposure have been shown with certainty; however, there is evidence suggesting that they are risk factors for cancer, neural tube birth defects, and stunted growth in children, particularly among persons residing in “high-exposure” areas. High-exposure areas (or communities) are those in which the population is dependent on corn contaminated with relatively high fumonisin concentrations as a diet staple.1,14,16−18 In this regard, urinary FB1 (a biomarker of exposure) has been positively correlated with Sa1P concentrations and Sa1P/So1P ratios in whole blood spots collected from volunteers living in Guatemala:18 the basis of the latter is that erythrocytes take up Sa and So from serum and kinases therein convert them to Sa1P and So1P. Sa1P concentrations and Sa1P/So1P in blood spots of persons living in “high-exposure” communities were higher than in persons from “low-exposure” communities, a pattern that is consistent Special Issue: Public Health Perspectives of Mycotoxins in Food Received: Revised: Accepted: Published: 7088
December 24, 2016 January 25, 2017 January 27, 2017 February 7, 2017 DOI: 10.1021/acs.jafc.6b05761 J. Agric. Food Chem. 2017, 65, 7088−7096
Review
Journal of Agricultural and Food Chemistry
reduced bioavailability through sequestering of fumonisins to food matrix components; conversion to less toxic forms) or to maintain or enhance toxicity (formation of modified fumonisins or breakdown products that retain or augment biological activity) that occur. Interactions between the intestinal microbiome and fumonisins, especially those that might affect bioavailability and toxicity, are not understood, although transient changes in the intestinal microbiota of pigs fed fumonisins have been reported.19 Thus, microbiome−food matrix interactions might have an effect on fumonisin bioavailability. In vivo bioassays using fumonisin-specific biochemical and pathological outcomes as experimental end points offer an advantage over analytical chemistry-based procedures or in vitro bioassays for evaluating the effectiveness of cooking and processing methods for reducing fumonisin toxicity in food products. First, the in vivo approach accounts for unknown and potentially toxic forms for which standards are not available and therefore are not detected by chemical analysis. The in vivo approach provides an intact physiological model that is based on fumonisin-specific effects of relevance for risk assessment. In this presentation, three examples of the use of in vivo bioassay are considered. First, extrusion cooking with and without the addition of glucose reduced fumonisin concentrations and toxicity of contaminated corn grits. Second, nixtamalization (alkaline cooking) was shown to detoxify fumonisin-contaminated whole kernel corn. The efficacy of nixtamalization to detoxify fumonisins was further shown. Specifically, the bioassay demonstrated that, in contrast to FB1, the alkaline hydrolysis product HFB1 does not cause neural tube defects (NTD) or significantly disrupt sphingolipid metabolism in the LM/Bc mouse model.
Figure 1. Structures of fumonisin B1, 1; hydrolyzed fumonisin B1, 2; and N-(1-deoxy-D-fructos-1-yl) fumonisin B1, 3.
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EXTRUSION REDUCES FUMONISIN CONCENTRATIONS AND TOXICITY OF CONTAMINATED CORN GRITS Background. Extrusion is a commonly used cooking method used to produce a variety of foods such as ready-toeat cereals and snacks. It utilizes high heat, pressure, and torque, which are applied to an uncooked preparation, such as cereal-based dough, by passing it over a single rotating or two counter-rotating screws housed in a heated nozzle.20,21 Extrusion has been shown to reduce fumonisin concentrations in corn-based foods, although reductions vary depending on the matrix (whole corn, grits, flour, etc.), recipe, and specific cooking conditions: in the absence of additives such as sugar or salt, reported reductions have ranged from as low as 2% to as high as 99%.2,22 Reduction of FB1 in corn grits by extrusion is enhanced when glucose is added to the recipe before cooking. Bullerman et al.20 reported that extrusion reduced FB1 in three grits preparations by 21−37%, whereas reductions of 77−83% were found with the addition of glucose. Negligible amounts of N-(carboxymethyl) FB1 (CMFB1), 5 (Figure 2), were produced, whereas NDFB1 was the major form of fumonisin recovered after extrusion with glucose, accounting up for up to 66% of the total FB1 species on a mass balance basis. In another experiment, addition of glucose before extrusion led to an additional 20−25% reduction of FB1 compared to extrusion alone.21 Enhancement is achieved partly by FB1 reacting with glucose through its amino group to form NDFB1 by means of the Maillard reaction with subsequent Amadori rearrangement.7,23 NDFB1 can undergo further transformation to CMFB1, N-methyl FB1, 6 (Figure 3), or
Figure 2. Structures of N-acylated fumonisins B1, 4; N-(carboxymethyl) fumonisin B1, 5; and N-methyl fumonisin B1, 6.
with the concept that fumonisins inhibit ceramide synthase in more highly exposed individuals in the same manner as in animals. Because of their possible implications for human health, it is desirable to reduce fumonisins in foods to their lowest levels possible. The effectiveness of a cooking or processing method to reduce fumonisins in foods, and thus exposure in consumers, is determined by the balance of events expected to detoxify (e.g., 7089
DOI: 10.1021/acs.jafc.6b05761 J. Agric. Food Chem. 2017, 65, 7088−7096
Review
Journal of Agricultural and Food Chemistry
Figure 3. Nominal concentrations of free fumonisin B1 (FB1-Free), matrix-associated FB1 (FB1-Matrix), and free fumonisins B2 (FB2) and B3 (FB3) in diets prepared with Fusarium verticillioides fermented corn grits: (A) batch 1 = uncooked batch 1 grits, 1-E = extruded batch 1 grits, 1-EG = glucose-supplemented extruded batch 1 grits; (B) batch 2 = uncooked batch 2 grits, 2-E = extruded batch 2 grits, 2-EG = glucose-supplemented extruded batch 2 grits; ∗ = not detected. Adapted from Jackson et al.21 and Voss et al.27
Figure 4. Pie chart showing mass balance estimates of fumonisin B1 (FB1) species in diets prepared with Fusarium verticillioides fermented corn grits: (A) mass balance of free FB1 (black), matrix-associated FB1 (dark gray), hydrolyzed FB1 (medium gray), and N-(1-deoxy-D-fructos-1-yl) FB1 (light gray with asterisk) in diets prepared from batch 1 and batch 2 fermented grits (see legend to Figure 3); (B) pie chart showing recovery of FB1 species (free FB1 plus matrix-associated FB1 plus HFB1 plus NDFB1, indicated in black) in diets prepared from extruded (1-E; 2-E) or glucosesupplemented, extruded (1-EG; 2-EG) F. verticillioides fermented corn grits as a fraction of the total FB1 species found in the diets prepared from uncooked batch 1 or batch 2 grits. Adapted from Jackson et al.21 and Voss et al.27
matrix-associated FB1 concentrations, the solids remaining after extraction of free fumonisins were hydrolyzed under alkaline conditions (KOH) to convert matrix-associated FB1 to HFB1 for quantitation by HPLC as reported by Jackson et al.21 The uncooked and cooked grits products were fed (50% w/ w in a standard rodent chow) to male Sprague−Dawley rats for 3 or 8 weeks. Kidney Sa, Sa1P, So, and So1P were quantified by liquid chromatography−mass spectrometry (LC-MS). Histopathological evaluations of kidney were done, and severity of lesions consistent with fumonisin exposure was subjectively scored on a four-point scale of 0 (no evidence of toxicity) to 3 (moderate apoptotic and regenerative lesions). Results. The nominal (calculated on the basis of the analysis of the grits) concentrations of FB1 species in the diets prepared from the uncooked and cooked batch 1 and batch 2 grits are shown in Figures 3 and 4. Free FB1 in the diets was reduced from 4.9 (batch 1) to 1.4 mg/kg (batch 1E) and from 25 (batch 2) to 9.0 mg/kg (batch 2E) by extrusion. Further reductions were found in extruded grits supplemented with glucose: respective free FB1 concentrations in diets made from batches 1EG and 2EG were 0.3 and 2.9 mg/kg. A similar pattern of
other compounds, depending on conditions. NDFB1 was less toxic than FB1 to rats24 and swine,25 and CMFB1 was less toxic than FB1 when fed to mice26 due to the absence of the primary amine group of the molecule. However, other fumonisin-matrix or degradation products, some possibly toxic, were formed during extrusion as mass balance recoveries of FB1 species after extrusion were incomplete.21 Bioassay. Materials, grits preparation, analytical procedures, and bioassay protocols have been described.21,27 Briefly, two batches of degermed corn grits were fermented with F. verticillioides. Each batch (20% moisture content) was divided into three portions: the first portion of each remained unprocessed (designated batches 1 and 2); the second portions of each, designated batches 1E and 2E, were extruded (160 °C, 40 rpm at 100 g/min feed rate); and the third portions, designated batches 1EG and 2EG, were supplemented with 10% w/w glucose and then extruded (same conditions). Unfermented grits and unfermented extruded grits served as controls. HPLC and LC-MS was used to quantitate free FB1, FB2, FB3, HFB1, and NDFB1 in the unprocessed grits and the grits extruded with and without glucose.21 To determine 7090
DOI: 10.1021/acs.jafc.6b05761 J. Agric. Food Chem. 2017, 65, 7088−7096
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Journal of Agricultural and Food Chemistry reduction was found for matrix-associated FB1 as well as free FB2 and FB3 (Figure 3). Mass balance calculations (μmol/kg diet) of individual FB1 species within the diets showed that considerable amounts of the FB1 species in the unprocessed grits could not be accounted for after extrusion (Figure 4). Matrix-associated FB1 concentrations in the diets ranged from 40 to 55% that of the total FB1 (free plus matrix-associated), thereby accounting for 23−38% of the total FB1 species (total FB1 plus HFB1 plus NDFB1). The FB1−matrix interaction products were not characterized. In the batch 1EG and batch 2EG diets, NDFB1 made up 35− 40% of the total FB1 species. In contrast, NDFB1 amounts were batch 1E > batch 1EG and batch 2 ≥ batch 2E > batch 2EG. When all groups and both of the 3 and 8 week times were considered, lesion severity scores decreased with decreasing FB1 intake (point 1 above) in the order batches 2 > 2E > 1 ≥ 2E > 1E > 1EG, with no histopathological evidence of exposure being found in the group fed batch 1EG grits. 4. Kidney Sa, So, and Sa(So)1P concentrations were elevated (Figure 5) in a dose-related manner (based on FB1 intake), and the increases were correlated with histopathology severity scores. Kidney sphingoid base concentrations of rats fed batch 1E did not differ from those of rats in the control groups.
Table 1. Kidney Histopathology in Rats Fed Uncooked Fumonisin-Contaminated Grits (Batches 1 and 2), Extruded Contaminated Grits (Batches 1E and 2E), or GlucoseSupplemented Contaminated Grits (Batches 1E and 2E)a diet uncooked controlc extruded control batch 1 (4.9)d batch 1E (1.4) batch 1EG (0.3) batch 2 (25) batch 2E (9.0) batch 2EG (2.9) controls uncooked control extruded control batch 1 (4.9) batch 1E (1.4) batch 1EG (0.3) batch 2 (25) batch 2E (9.0) batch 2EG (2.9)
individual scoresb (n = 5)
mean (±SD)
Week 00 00 32 01 00 33 33 20 Week
3 0 0 3 2 1 3 3 1 8
0 0 2 2 0 3 3 1
1 0 2 1 0 3 3 1
0.2 0 2.4 1.2 0.2 3.0 3.0 1.0
0 0 2 2 0 3 0 0
0 0 2 1 0 3 3 3
0 0 0 0 0 3 3 0
0 0 2 0 0 3 0 3
0 0 1.6 0.6 0 3.0 1.8 1.2
0 0 2 0 0 3 3 0
(±0.4) (±0.5) (±0.8) (±0.04)
(±0.7)
(±0.9) (±0.9)
(±1.6) (±1.6)
a From Voss et al.27 bLesion severity score definition: 0 = no evidence of fumonisin exposure; 1 = minimal; 2 = mild; 3 = moderate. c Uncooked and extruded control diets were prepared using corn grits not fermented with Fusarium verticillioides. dConcentration of free fumonisin B1 (mg/kg) in the test diets based on the results of analysis of the F. verticillioides fermented corn grits used to make test diets.
The bioassay showed that extrusion and, more effectively, extrusion with glucose supplementation reduced fumonisin intakes of the animals and in vivo toxicity; extrusion, especially with the addition of glucose, is a potentially useful cooking method for reducing fumonisin concentrations in foods.
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NIXTAMALIZATION DETOXIFIES FUMONISIN-CONTAMINATED WHOLE KERNEL CORN Background. Nixtamalization is the traditional alkaline cooking method for making masa and masa-based foods such as tortillas. It involves cooking and steeping corn kernels in alkaline water, rinsing the cooked kernels (known as nixtamal), and further processing them to masa flour, tortillas, or other food products.28 Fumonisins are removed from corn during nixtamalization by a combination of extraction and conversion to their hydrolyzed forms or perhaps other degradation or matrix interaction products.29−34 The alkaline cooking process also liberates matrix-associated fumonisins as shown by the studies of De Girolamo et al.34 According to their mass balance calculations, recovery of FB1 and FB2 species from the masa, steeping liquid, and wash water combined was increased 53− 83% compared to the raw corn depending on cooking conditions. Nonetheless, the overall effect of alkaline cooking was beneficial as fumonisins (FB1 plus FB2) in the masa were reduced by 32−52%. Lesser reductions of 14−36% were found in masa based on mass balance for total fumonsins, defined as FB1 plus FB2, plus their partially and fully hydrolyzed forms. In vitro bioassays of extracts of nixtamalized corn showed that both fumonisin concentrations and the ceramide synthase inhibition activity critical for fumonisin toxicity are reduced by 7091
DOI: 10.1021/acs.jafc.6b05761 J. Agric. Food Chem. 2017, 65, 7088−7096
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Journal of Agricultural and Food Chemistry
Figure 5. Box plots indicating Sa, Sa1P, So, and So1P concentrations in kidney of rats fed diets prepared with Fusarium verticillioides contaminated grits (batches 1 and 2), the grits after extrusion (batches 1E and 2E), or the grits after extrusion with glucose supplementation (batches 1EG and 2EG) for 3 weeks. Results for the combined control groups fed uncooked or cooked uncontaminated grits are also shown. (a−c) Control and batch 1 groups not sharing letters are significantly different; (a, d, e) control and batch 2 groups not sharing letters are significantly different; significance was judged at p < 0.05. Adapted from Voss et al.27
alkaline cooking.31,35 However, these in vitro assays did not account for the potential biological activity of residual free and matrix-associated fumonisins or their reaction products in the cooked corn. Experimental. Experimental procedures have been reported in detail elsewhere.36 In summary, F. verticillioidesinfected whole kernel corn was mixed with different amounts of sound corn to produce three batches of contaminated corn designated low (L), medium (M), or high (H) level corn on the basis of their free fumonisin content. Half of each batch was cooked by nixtamalization using a published recipe.37 After freeze-drying and grinding, the nixtamalized and uncooked corn was mixed (50% equivalent weights) with a standard rodent ration to make the six test diets, which were designated LU, LC, MU, MC, HU, and HC, where L, M, and H are defined as above and U and C indicate whether the corn was uncooked (U) or nixtamalized (C). A control diet was prepared by blending uncooked sound corn and rodent chow in the same manner. FB1 concentrations, determined by LC-MS/MS in the formulated diets, were LU = 1.8 mg/kg, LC = 0.08 mg/kg, MU = 3.6 mg/kg, MC = 0.13 mg/kg, HU = 4.2 mg/kg, and HC = 0.37 mg/kg. FB1 (0.22 mg/kg) was found in the control diet. The diets were fed to male Sprague−Dawley rats for 3 weeks, and renal toxicity was assessed by microscopic examination of the kidney and LC-MS/MS quantitation of renal Sa and Sa1P.
Results. Nixtamalization reduced free FB1 concentrations by ≥90%. That the amount of free FB1 in the control diet prepared with uncooked sound corn was slightly higher than in the LC and MC diets is a further indication that nixtamalization effectively reduces free fumonisin concentrations. The bioassay revealed no evidence of toxicity in the LC and MC groups as no differences among the LC, MC and control groups were found by microscopic tissue examinations or sphingoid base analysis of the kidney36 (Figure 6; Table 2). Fumonisin-attributable effects were also significantly reduced, although not totally eliminated, in the HC group. Taken together, the findings showed that nixtamalization of contaminated corn significantly reduces its free FB1 concentrations and in vivo toxicity.
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THE HYDROLYSIS PRODUCT OF FB1 (HFB1) FOUND IN ALKALINE-COOKED FOODS DOES NOT CAUSE NEURAL TUBE DEFECTS IN MICE Background. The reproductive effects of fumonisins have been reviewed in detail elsewhere.38,39 Results of the initial studies in rodents,40−42 rabbits,43 and hamsters44 indicated FB1 was fetotoxic but not teratogenic. The few malformations, variations, or developmental retardations found were considered secondary effects of maternal toxicity. FB1 was, however, shown to cause neural tube defects (NTDs), exhibiting as 7092
DOI: 10.1021/acs.jafc.6b05761 J. Agric. Food Chem. 2017, 65, 7088−7096
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Journal of Agricultural and Food Chemistry
fumonisins are hydrolytically removed from the molecule’s hydrocarbon “backbone”. The hydrolyzed reaction product, HFB1, and other hydrolyzed fumonisins are found in tortillas. The amounts depend upon the amount of fumonisins in the raw corn and the exact cooking conditions. As a result, pregnant women consuming tortillas as a diet staple are undoubtedly exposed to HFB1 in addition to residual FB1 remaining in the tortillas after nixtamalization of the corn.28−31 A study was therefore done to test the relative potencies of HFB1 and FB1 using NTDs in the LM/Bc mouse model as the experimental end point. Experimental Section. Experimental details are found in Voss et al.48 Pregnant LM/Bc mice were given intraperitoneal injections of 2.5, 5, 10, or 20 mg/kg maternal body weight (corresponding to 6, 12, 25, or 49 μmol/kg body weight) HFB1 on embryonic day (E) days E7 and E8. A negative control group was treated with the vehicle only, whereas a positive control group was treated with 10 mg/kg (= 14 μmol/kg) maternal body weight FB1. The fetuses were weighed and examined for NTD on E16 and maternal liver (the main target organ in mice), placenta, and fetal liver samples collected for sphingolipid analyses. To evaluate the effects of HFB1 and FB1 on maternal toxicity at the critical time for neural tube closure, additional dams from the negative control, high-dose HFB1, and positive controls groups were euthanized on E9 for evaluation of liver sphingolipids and histopathology. Results. HFB1 did not cause NTDs (n = 8−9 litters/group), and none were found in the vehicle-treated group (n = 8 litters).48 In contrast, all litters (n = 10) of FB1-treated females examined at E16 had at least one fetus exhibiting NTD, and 67% of the fetuses in this group had an NTD. A significant increase in the number of in utero deaths and decreased weight of viable fetuses compared to all other groups was also found in the FB1-exposed litters at E16. In contrast, the number of embryo resorptions and late deaths were not increased and fetal weight was not decreased at E16 in any group treated with HFB1. The livers of FB1-treated dams examined at E9 showed moderately severe liver lesions typical of those caused by FB1 in mice. This maternal toxic effect was reversible as no significant liver histopathology was found in the FB1-exposed dams 8 days following the final dose. Females treated with HFB1 did not exhibit microscopic evidence of fumonisin-induced liver pathology. Sphingolipid analysis results were consistent with the microscopic liver findings. Relative to the vehicle-treated group, a slight (
