Source: TEXAS WOMAN'S UNIVERSITY submitted to NRP
CHEMICAL AND TOXICOLOGICAL EVALUATION OF FUMONISIN B1 IN EXTRUDED CORN GRITS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0204751
Grant No.
2005-35201-16329
Cumulative Award Amt.
(N/A)
Proposal No.
2005-02053
Multistate No.
(N/A)
Project Start Date
Sep 1, 2005
Project End Date
Aug 31, 2009
Grant Year
2005
Program Code
[32.0]- Food Safety
Recipient Organization
TEXAS WOMAN'S UNIVERSITY
304 ADMINISTRATION DRIVE, P.O. BOX 425589
DENTON,TX 76204
Performing Department
(N/A)
Non Technical Summary
Toxigenic Fusarium species pose major agricultural and food safety problems by invading cereal grains in the field. Among a large number of toxic compounds by the fungi, fumonisin B1 (FB1) has been found to be the most commonly occurring toxin in corn or maize. Surveys have shown that FB1 occurs not only in crops but also in finished corn-based processed food products due to its marked heat stability. Previous studies in our laboratory have also shown that extrusion processing, especially in the presence of glucose, may significantly (over 90%) reduce concentrations of this mycotoxin. These previous studies have shown that extrusion processing resulted in the greatest reduction of FB1 in corn-based foods compare to other thermal processes such as baking and canning. However, these results are based on analyses showing reduction of FB1 by extrusion processing, that were done by chemical and immunological assays that are based on the chemical structure of the toxin. Therefore it has not yet been proven conclusively that the toxicity or biological action of FB1 is likewise destroyed and not converted to another form that may remain toxic that may not be detected by above methods. Consequently, it is important to determine if residual toxicity of FB1 remains after extrusion processing using suitable bioassay techniques, particularly animal feeding trials, to assess the true efficacy of extrusion processing. The results will provide useful information to help assess health risks of FB1 in corn and thermally processed corn based foods.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121510100050%
2121510110225%
2121510115025%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1510 - Corn;

Field Of Science
1102 - Mycology; 1000 - Biochemistry and biophysics; 1150 - Toxicology;
Goals / Objectives
The goal of this study is to assess chemical and toxicological changes of FB1 during extrusion cooking of contaminated corn grits using high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), liquid chromatography-mass spectroscopy (LC-MS), and in vivo rat feeding trials. The LC-MS analyses will help to identify degradation products. Studies indicate that FB1 is rather heat stable and can be found with its derivatives in thermally processed corn products. Our central hypothesis is that the toxicity of FB1 can be reduced significantly by extrusion processing, which utilizes higher temperatures, high pressures and shear forces. If our hypothesis is correct, the toxicity or biological activity of FB1 in corn products can be significantly reduced by extrusion processing under previously determined optimal conditions for maximum reduction. We expect to test our central hypothesis and to achieve the goal of this study by pursuing three objectives. We will 1. Determine reduction of FB1 and presence of hidden FB1 using HPLC and ELISA methods. 2. Determine the identity of any degradation products by LC-MS. 3. Determine residual toxicity of FB1 in the extruded corn grits by rat feeding tria
Project Methods
The grits contaminated with FB1 will be produced by growing a toxigenic strain of mold on the corn grits and extruded prior to carrying out objective 1. The grits will then be extruded under conditions that maximum destruction of FB1, as determined by routine HPLC methods. These extruded products will be further analyzed to determine the concentration of residual FB1 and its degradation products by HPLC, ELISA, LC-MS and rat feeding trials to accomplish above objectives. Improved method of detection will be used to detect protein bound or hidden FB1 for a mass balance approach.

Progress 09/01/05 to 08/31/09

Outputs
OUTPUTS: Two batches of flaking corn grits were prepared by growing Fusarium verticillioides to contain low and high levels of fumonisin B1 (FB1), Batch-1 at 9.7 ppm and Batch-2 at 50 ppm FB1 as determined by HPLC. These two batches were extruded (Batch-1E; Batch-2E) or extruded with 10% w/w glucose supplementation (Batch-1EG; Batch-2EG) using a conical twin-screw extruder with 3 mm nozzle. Uncontaminated corn grits (C) served as a control in addition to extruded uncontaminated grits (CE). Applied extrusion parameters include moisture content (20% dry weight basis), screw speed (40 rpm), and temperature (160 degree C). FB1 concentrations after extrusion were: Batch-1E = 2.7 ppm; Batch-1EG = 0.6 ppm; Batch-2E = 18 ppm; and Batch-2EG = 5.7 ppm. These values corresponded to FB1 reductions of 72% (Batch-1E), 94% (Batch-1EG), 64% (Batch-2E) and 89% (Batch-2EG). Similarly, extrusion alone resulted in 26-73% reduction in fumonisin B2 (FB2) and fumonisin B3 (FB3) levels, while levels of both toxins were reduced by >89% in extruded corn containing 10% glucose. The mass balance indicates that hydrolyzed fumonisin B1 (HFB1) was a minor species in extruded corn with or without added glucose, representing <15% of the total FB1 species present. The analysis also found different levels of N-(1-Deoxy-D-fructos-1-yl) FB1 (NDF-FB1) in extruded corn without glucose (1.9-2.4 % of total FB1 species) and with added glucose (38-46% of total FB1 species). These results corroborate our previous study that found 57-66% of FB1 species detected in fermented, extruded corn with glucose was NDF-FB1. For toxicological evaluation, Batch-1, Batch-1E, Batch-1EG, Batch-2, Batch-2E, Batch-2EG as well as the control group (C or CE) were mixed (1:1) with basal rodent chow and fed to young male rats for 3 (n=5/group) or 8 (n=5/group) weeks. No significant differences in general appearance, body weights or detailed hematology and serum chemistry profiles were found. The kidney is the most sensitive target organ of fumonisins in male rats and evidence of renal toxicity was found in five of the six test groups. Specifically, relative kidney weights were decreased in groups fed the Batch-1 or Batch-2 diets while apoptotic tubule lesions consistent with fumonisin-induced nephropathy were found upon microscopic exanimation of the kidneys from animals fed the Batch-1, Batch-1E, Batch-2, Batch-2E, or Batch-2EG diets. Lesions were most severe in the groups fed diets having the highest FB1 concentrations: that is, Batch-1 (diet FB1 = 4.9 ppm), Batch-2 (25 ppm) and Batch-2E (9.0 ppm). Less severe, minimal to mild changes were evident in the Batch-1E (1.4 ppm) or Batch-2EG (2.9 ppm) groups whereas no lesions were found in the rats fed the Batch-1EG (0.3 ppm) diet, which contained the lowest levels of residual FB1. Overall, the findings indicate that extrusion with glucose using a twin-screw configuration reduces FB1 concentrations of contaminated corn grits and confirm that glucose supplementation enhances FB1 reductions as well as the in vivo toxic potential of the extrusion cooked grits PARTICIPANTS: Individuals: Dojin Ryu (PD), Lloyd B. Bullerman, Milford A. Hanna, Kenneth A. Voss, Lauren S. Jackson. Partner Organizations: USDA/ARS (Kenneth A. Voss), FDA/CFSAN (Lauren S. Jackson) Training: Andreia Bianchini, Aplil Hollub, Lauren Lopez TARGET AUDIENCES: Target audiences - academia, industry, regulatory agency PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This conclusive study showed that extrusion processing reduced detectable level of fumonisin and its toxicity with HPLC, LC-MS, and rat bioassay. Mass balance approach indicated that fumonisin B1 was reduced significantly during extrusion with single or twin-screw configuration and identified N-(1-Deoxy-D-fructos-1-yl) FB1 as a major reaction product. Rat bioassay confirmed that the reduction of FB1 during extrusion cooking can result in reduced toxicity particularly when glucose was supplemented in extrusion. Therefore, extrusion processing can reduce both detectable level and toxicity of FB1 in corn grits. These findings are applicable in food industries and regulatory agencies.

Publications

  • Voss, K.A., L.B. Bullerman, A. Bianchini, M.A. Hanna, L.S. Jackson, and D Ryu. 2009. Toxicological evaluation of fumonisin B1 in corn grits extruded with added glucose. In Preparation.
  • Jackson, L.S., J. Jablonski, Bullerman, L.B., A. Bianchini, M.A. Hanna, and D Ryu. 2009. Fate of fumonisin B1 in corn grits during twin-screw extrusion with added glucose. In Preparation.
  • Voss, K.A., L.B. Bullerman, A. Bianchini, M.A. Hanna, and D Ryu. 2008. Reduced toxicity of fumonisin B1 in corn grits by single-screw extrusion with added glucose. J. Food Prot. 71(10):2036-2041.
  • Bullerman, L.B., A. Bianchini, M.A. Hanna, L.S. Jackson, J. Jablonski, and D Ryu. 2008. Reduction of fumonisin B1 in corn grits by single-screw extrusion. J. Agric. Food Chem. 56(7):2400-2405.
  • Voss, K.A., Jackson, L.S., Jablonski, J., Bianchini, A., Hanna, M.A., Bullerman, L.B., and Ryu, D. 2009. Concentrations and toxicity of fumonisin B1 in fermented corn grits are reduced by extrusion cooking with glucose supplementation. Joint Panel on Toxic Microorganisms, United States-Japan Cooperative Program on Development and Utilization of Natural Resources, Tokyo, Japan.
  • Voss, K.A., Jackson, L.S., Jablonski, J., Bianchini, A., Bullerman, L.B., Hanna, M.A., and Ryu, D. 2007. The effect of extrusion on the fumonisin content and toxicity of corn grits. Gordon Research Conference, Waterville, ME.
  • Voss, K.A., Jackson, L.S., Bianchini, A., Bullerman, L.B., Hanna, M.A., and Ryu, D. 2007. Chemical and toxicological evaluation of fumonisin B1 in extruded corn grits. Society of Toxicology Meeting, Charlotte, NC.
  • Bianchini, A., Ryu, D., Jackson, L.S., Voss, K.A., Hanna, M.A., and Bullerman, L.B. 2007. Toxicity of fumonisin degradation products produced by extrusion. Fourth Annual Nebraska Food Research Symposium, Lincoln, NE.
  • Voss, K.A., .Jackson, L.S., Jablonski, J., Bianchini, A., Bullerman, L.B., Hanna, M.A., and Ryu, D. 2006. The effect of extrusion on the fumonisin content and toxicity of corn grits. The 10th International Symposium on Toxic Microorganisms, College Park, MD.
  • Bianchini, A., Ryu, D., Jackson L.S., and Bullerman, L.B.. 2006. Fate of fumonisin B1 during extrusion cooking of corn grits with and without glucose. Third Annual Nebraska Food Research Symposium, Lincoln, NE.


Progress 09/01/07 to 08/31/08

Outputs
OUTPUTS: The second extrusion study using twin-screw extruder has been completed. The reduction of fumonisin B1 (FB1) during extrusion of contaminated corn grits averaged to 73 and 60% for low (11 ppm) and high (70 ppm) levels of FB1, respectively. Addition of glucose at 10% (w/w) increased the reduction of FB1 to 95 and 90 % for low and high levels of FB1, respectively. Based on the results of our first trial, the protocol for the bioassay (rat feeding trial, Objective 3) has been modified from 4 weeks to 8 weeks. This bioassay and analyses of unknown degradation products by LC-MS-MS are in progress. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our data showed that the efficacy of extrusion processing in reducing FB1 may be different depending on the level of contamination in corn grits. The reduction of FB1 increased significantly by addition of glucose to corn grits regardless of contamination level. Conclusive studies on the toxicity and degradation products formed in grits with different contamination levels are in progress.

Publications

  • Voss, K.A., L.B. Bullerman, A. Bianchini, M.A. Hanna, and D Ryu. 2008. Reduced toxicity of fumonisin fumonisin B1 in corn grits by single-screw extrusion with added glucose. J. Food Prot. In Press.
  • Bullerman, L.B., A. Bianchini, M.A. Hanna, L.S. Jackson, J. Jablonski, and D Ryu. 2008. Reduction of fumonisin fumonisin B1 in corn grits by single-screw extrusion. J. Agric. Food Chem. 56(7):2400-2405.


Progress 09/01/06 to 08/31/07

Outputs
Since the last progress report, additional flaking corn grits were artificially contaminated with fumonisin B1 (FB1) by growing a toxigenic strain of Fusarium verticillioides. Two batches of contaminated grits with high (60 ppm) and low (10 ppm) levels of FB1. The concentration of each batch was adjusted by mixing contaminated grits with clean grits. Prepared batches were extruded using a twin screw extruder at 160 degree C and screw speed of 40 rpm with or without addition of glucose at 10% (dry weight basis, db). Clean corn grits were also processed under same extrusion conditions. Moisture content of all samples was adjusted to 20% (db) prior to extrusion cooking. Analysis of FB1 by HPLC and ELISA is in progress. Analyses of unknown degradation products by LC-MS-MS and rat feeding trial are under way.

Impacts
We expect to generate data on the efficacy of extrusion processing in reduction of FB1 at different levels of contamination. The results will be used to better estimate actual exposure and human health risks from fumonisins in corn and corn based foods at varying levels of contamination.

Publications

  • Voss, K.A., Jackson, L.S., Bianchini, A., Bullerman, L.B., Hanna, M.A., Ryu, D. 2007. Chemical and toxicological evaluation of fumonisin B1 in extruded corn grits. Society of Toxicology Meeting, Charlotte, NC, March 25-29.
  • Voss, K.A., Jackson, L.S., Jablonski, J., Bianchini, A., Bullerman, L.B., Hanna, M.A., and Ryu. D. 2006. The effect of extrusion on the fumonisin content and toxicity of corn grits. The 10th International Symposium on Toxic Microorganisms. College Park, MD, November 7-9.


Progress 09/01/05 to 09/01/06

Outputs
Corn grits were artificially contaminated with FB1 by (i) adding purified FB1 in an aqueous solution, and (ii) by growing a toxigenic strain of Fusarium verticillioides. Contaminated grits were extruded using a laboratory scale twin screw extruder at 160 C and screw speed of 40 rpm with or without addition of glucose at 10% (dry weight basis, db). Clean corn grits were also processed under same extrusion conditions. Moisture content of all samples was adjusted to 20% (db) prior to extrusion cooking. Concentrations of FB1 ranged 25-39 ppm (µg/g) in artificially contaminated corn grits prior to extrusion and reduced by up to 86% when extruded with 10% glucose. In rat feeding trial, food consumption of the FC2 (132+5.9 (SD) g) and FC2-E group (129+13.7 g) was decreased 12-14% compared to the negative control (149+5.5 g) during week 2. However, food consumption values for the FC2-E and FC2-EG groups remained 5-10 % less than those of the negative and extrusion control groups during week 3. No differences in absolute (g) or relative (% Final Body Wt) liver weights were found. In contrast, a number of significant differences were found for the kidney. Relative kidney weights of the SC group were less than those of the negative control while relative kidney weights of the SC-E and SC-EG groups were less than those of both the negative and extrusion control groups. Relative kidney weights of the FC1 and FC1-E groups were significantly decreased compared to the negative controls and the FC1-EG groups, suggesting that the addition of glucose to the extrusion mixture was in this case beneficial. This was not the case for Batch 2 (FC2) of fermented corn. Relative kidney weights of the FC2, FC2-E and FC2-EG groups were similar and significantly less than that of the negative control group. Microscopic examinations revealed no evidence of a test-related effect on liver. No noteworthy microscopic lesions were found in the kidneys of the negative or extrusion control groups. However, kidney lesions typically found in Sprague-Dawley rats fed diets containing fumonisins were found. Among the affected groups, lesions in the FC1-EG group were least severe, suggesting that extrusion plus glucose reduced the toxicity of FC1. One FC1-EG kidney was judged "normal" and two were scored as having minimal ("1") lesions. The remaining kidneys from this group were considered mildly affected ("2"). Thirty-six of the remaining 40 animals, 3-5 per group, fed diets prepared from FB1-spiked corn or the two fermented corn preparations had more extensive, moderate ("3") kidney lesions. Thus, the histopathology findings, along with the relative kidney weight results, indicate that the combination of extrusion and glucose reduced the toxicity of the first batch of Fusarium-fermented corn (FC1). Additional experiments incorporating dose and time-course parameters are needed to better understand the feasibility of using extrusion and glucose supplementation for reducing fumonisins and fumonisin-toxicity of extruded products.

Impacts
We expect to generate conclusive evidence of whether or not reduction in toxicity of FB1 in corn after extrusion processing occurs by using a rat feeding trial. We will determine if the reduction in toxicity of FB1 as measured by the rat feeding trial compares to that determined by chemical and immunochemical analyses. This information will help industry to establish processing conditions to assure safer food products. This information is needed for both regulatory agencies and the food industry, and will allow a better estimation of actual exposure and human health risks from fumonisins in corn and corn based foods.

Publications

  • No publications reported this period