Free versus total fumonsins in corn. Mark Busman USDA- ARS Peoria, Illinois

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1 Free versus total fumonsins in corn Mark Busman USDA- ARS Peoria, Illinois

2 Fungi in corn Aspergillus Fusarium

3 Mycotoxins in corn Aflatoxins H H CH 3 Tricothecenes Fumonisins H H H H H H H H H H H H CH 3 H 3 C CH3 CH 3 H NH 2 H H

4 Fumonisin effects Carcinogenic Pulmonary edema (swine) Leukoencephalomalacia (horse) Neural tube defects (human) Disrupts sphingolipid pathways

5 DeterminaMon of fumonisins Biological assays Chemical analysis

6 Problem Why is there open a discrepancy between biological and chemical toxin analysis? Answer Toxins that are masked or hidden from convenmonal chemical analysis.

7 Example (Extruded corn grits): Samples (measured by HPLC) Contaminated corn grits: 31 ppm FB1 Extruded: 28 ppm FB1 Extruded w/ 10% glucose: 8 ppm FB1 Where did the FB1 go? N- deoxyfructosyl FB1: 17 ppm Bullerman, JAFC, 56, 2008

8 What is a masked fumonisin molecule? Contains the fumonisin base molecule Chemically masked by a covalent chemical bond to a masking agent Masking open can be readily cleaved to produce the base mycotoxin

9 Masked mycotoxin

10 Masked mycotoxin H H H H CH 3 H 3 C CH3 CH 3 H NH 2 H H H H H H CH 3 H 3 C CH3 CH 3 H NH 2 H H

11 What does masking do? DetoxificaMon? Interferes with detecmon of fumonisin in food / feed materials. Achieves a change in chemical idenmty Can result in a change in toxic behavior

12 What is the nature of the chemical masking in fumonisins? GlucosylaMon (Poling, Plaener, NCAUR) ther modificamons Hydrolysis Starch Proteins, pepmdes, amino acids Lipids

13 Fumonisin FB1 H H H H CH 3 H 3 C CH3 CH 3 H NH 2 H H

14 N- (1- Deoxy- D- fructos- 1- yl) Fumonisin FB1 H H H H CH 3 H 3 C CH3 CH 3 H NH H H H H H H

15 Fumonisin analysis Liquid chromatography Mass spectrometry

16 Fumonisin glucoside product Glucoside producmon N- deoxyfructosyl- fumonisin Glucoside chromatographic behavior Glucoside mass spectrometric behavior Molecular weight Collision induced dissociamon: fragmentamon paeern Produced by Fusarium ver+cillioides in presence of glucose Can be chemically synthesized

17 Fructosyl FB1 mass spectrometry FB1 Fruct- FB1 Poling, JAFC, 2002, 50, 1318

18 Fructosyl FB1 fragmentamon Fruct- FB1 Seefelder, JAFC, 2003, 51, 5567

19 LC- MS results for FB1, fructosyl FB1 mixture Fruct- FB1 FB1 Seefelder, JAFC, 2003, 51, 5567

20 What causes the masking of fumonisins? Produced by fungi? Plants Free sugars, lipids Plant enzymes Food processing

21 Should we care about masked toxins? - aper all they are masked Recent research shows that fumonisin glucosylamon can readily be reversed by mammalian digesmve systems. Recent studies provide toxicology informamon for the fructosyl FB1 and the co- occurring hydrolyzed FB1 However, much of the current informamon is conflicmng

22 Can we see masked fumonisins? - aper all they are masked Will convenmonal toxin analysis see the masked toxins? Maybe. Biological analysis: Probably yes. Chemical analysis: Probably no. SAMPLE PREPARATIN!!

23 Analysis of masked fumonisins? Biological assays have not been developed or characterized for fructosyl fumonisins Standard materials have not been developed for the fructosyl fumonisins SAMPLE PREPARATIN!! Methods will need to be developed to effec=vely extract both free and masked forms

24 Masked fumonisins and plant breeding Will masked toxins increase in plants bred for fungal / toxin resistance? Masking is thought to be a defense mechanism of resistant plants Will plants bred for higher linoleic and oleic acid content have higher levels of hidden toxins? Some evidence indicates yes

25 Fumonisin levels vs. linoleic acid Dall Asta, Food Addit Contam A, 2013, 30, 1606

26 Fumonisin levels vs. linoleic acid leic acid ester FB1 linoleic acid ester FB1 Dall Asta, Food Addit Contam A, 2013, 30, 1606

27 Should we look to modify toxin analymcal methodology to make sure we include the masked toxins? Do we need to talk in terms of total toxin levels versus free toxin levels How do we resolve the disagreements between first Mer (biological) tests and second Mer (chemical) tests

28 Masked fumonisin vs. hidden fumonisin Masked fumonisins are covalently modified with a masking agent Hidden fumonisins are non- covalently bound to make them difficult to extract Bound to: Proteins Starches Subject to how the corn kernel is treated

29 Fumonisin binding to proteins and starches Seefelder, JAFC, 2003, 51, 5567

30 How do you look for total fumonisin Look for more effecmve ways to extract fumonisins Use strong detergents to help with the extracmon EnzymaMcally break down the corn kernel to free the fumonisin Modify the fumonisin chemically to help free it from the Mght associamon with the kernel components EnzymaMc hydrolysis Alkaline hydrolysis

31 MS approach to total fumonisin AlternaMve way to chemically analyze fumonisins Perform a hydrolysis of fumonisins to drive fumonisins to hydrolyzed fumonisins Assume that all chemical modificamons will be uniformly and completely hydrolyzed to hydrolysis products (HFB)

32 MS approach to total fumonisin Chemically analyze for HFB s Level of HFB s should indicate total fumonisin Free FB + hidden FB = Total FB

33 Fumonisin hydrolysis H H H H CH 3 H 3 C CH3 CH 3 H NH 2 FB1 H H H H H CH 3 H 3 C CH3 H CH 3 H NH 2 HFB1

34 Free fumonisin mass spectrometry FB1 FB3 FB2

35 Hydrolyzed fumonisin MS: total FB HFB1 HFB3 HFB2

36 Total fumonisin analysis Assumes complete hydrolysis of fumonisins Can be compared to free fumonisin levels Requires access to HFB standards

37 Masked fumonisins and plant breeding For six hybrids: Toxin level changes vs. corn drying temperatures: 20% to 12% moisture Dall Asta, J.Cereal Sci., 2015, 61, 78

38 Fumonisin levels vs. linoleic acid Dall Asta, JAFC, 2012, 60, 3800

39 Where are we now with fumonisin? Fumonin- glucoside and hydrolyzed fumonisin standards are not commonly available Numerous, but conflicmng studies regarding metabolic fate of toxin glucosides in animals have been published Cross reacmvity of fumonisin glucosides in anmbody- based tests is not well characterized

40 Future research Characterize the cross- reacmvity of exismng biological based screening methods to masked fumonisins. Develop anmbodies leading to direct biological based screening methods for masked fumonisins. Characterize the acmvity of masked fumonisins in toxicity assays. Provide standards for the masked fumonisins. Characterize binding processes responsible for hiding of fumonisins in corn kernel.

41 Acknowledgements NCAUR Peoria: Chris Maragos Susan McCormick ISPA Bari, Italy Veronica Laeanzio Thank you for your aeenmon

42 Various forms of fumonisins and their transformamon paths during technological processes.(8, 10, 16, 19, 20) Published in: Marcin Bryła; Marek Roszko; Krystyna Szymczyk; Renata Jędrzejczak; Elżbieta Słowik; Mieczysław W. biedziński; J. Agric. Food Chem. 2014, 62, DI: /jf504077m Copyright 2014 American Chemical Society