Food and Chemical Toxicology 49 (2011) 711–721 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox Review Impact of food processing on the safety assessment for proteins introduced into biotechnology-derived soybean and corn crops ⇑ B.G. Hammond a, , J.M. Jez b a Monsanto Company, Bldg C1N, 800 N. Lindbergh Blvd., St. Louis, Missouri 63167, USA b Washington University, Department of Biology, One Brookings Drive, Campus Box 1137, St. Louis, Missouri 63130, USA article info abstract Article history: The food safety assessment of new agricultural crop varieties developed through biotechnology includes Received 1 October 2010 evaluation of the proteins introduced to impart desired traits. Safety assessments can include dietary risk Accepted 10 December 2010 assessments similar to those performed for chemicals intentionally, or inadvertently added to foods. For Available online 16 December 2010 chemicals, it is assumed they are not degraded during processing of the crop into food fractions. For intro- duced proteins, the situation can be different. Proteins are highly dependent on physical forces in their Keywords: environment to maintain appropriate three-dimensional structure that supports functional activity. Food Biotech crops crops such as corn and soy are not consumed raw but are extensively processed into various food frac- Introduced proteins tions. During processing, proteins in corn and soy are subjected to harsh environmental conditions that Processing soy and corn Denaturation proteins drastically change the physical forces leading to denaturation and loss of protein function. These condi- Dietary exposure tions include thermal processing, changes in pH, reducing agents, mechanical shearing etc. Studies have shown that processing of introduced proteins such as enzymes that impart herbicide tolerance or pro- teins that control insect pests leads to a complete loss of functional activity. Thus, dietary exposure to functionally active proteins in processed food products can be negligible and below levels of any safety concerns. Ó 2010 Elsevier Ltd. All rights reserved. Contents 1. Introduction . ...................................................................................................... 712 2. Relationship of structure to protein function . ................................................................ 712 2.1. Changes in the environment of proteins can alter their structures . ...................................................... 713 3. Processing of corn and soy into human foods . ................................................................ 713 3.1. Processing of corn grain into human food . ......................................................................... 713 3.2. Processing of soybeans into human foods . ......................................................................... 715 4. Testing introduced proteins for potential denaturation by heat treatment that occurs during food processing . .......... 716 5. Implications for dietary risk assessments. ................................................................ 717 6. Conclusions. ...................................................................................................... 719 Conflict of Interest . ................................................................................... 719 Acknowledgement . ................................................................................... 719 Appendix A. Supplementary data . ................................................................................... 719 References. ...................................................................................................... 719 Abbreviations: Codex, Codex Alimentarius Commission; EFSA, European Food Safety Authority; FAO, Food and Agricultural Organization; OECD, Organization for Economic Cooperation and Development; ELISA, Enzyme-Linked Immunosorbent Assay; WHO, World Health Organization; RTE, ready to eat; NDI, nitrogen dispersibility index; NSI, nitrogen solubility index; SO2, sulfur dioxide; NaOH, sodium hydroxide; HCl, hydrochloric acid; TVP, textured vegetable protein; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Bt, Bacillus thuringiensis; DNA, deoxyribonucleic acid; FSANZ, Food Safety Agency Australia New Zealand; CP4 EPSPS, CP4 5- enolpyruvylshikimate-3-phosphate synthase. ⇑ Corresponding author. Tel.: +1 314 694 8482; fax: +1 314 694 5071. E-mail addresses: [email protected] (B.G. Hammond), [email protected] (J.M. Jez). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.12.009 712 B.G. Hammond, J.M. Jez / Food and Chemical Toxicology 49 (2011) 711–721 1. Introduction proteins introduced into biotechnology-derived crops and the implications for dietary risk assessment. All foods derived through biotechnology must undergo a com- prehensive safety evaluation as part of the regulatory approval 2. Relationship of structure to protein function process before entering the market and becoming part of the food supply (Codex, 2003; EFSA, 2004; FAO, 1996; FAO/WHO, 2000; Proteins are large macromolecules composed of combinations OECD, 1997; WHO, 1995). As a part of this assessment, the safety of the 20 amino acids commonly found in nature. These amino of the proteins encoded by the introduced genes are evaluated. acids are linked by covalent peptide bonds into polypeptide chains This includes a bioinformatic analysis of the amino acid sequence consisting of tens to thousands of amino acids (Branden and Tooze, to confirm that the protein is not related to known mammalian 1991). The specific sequence of amino acids in a protein dictates toxins and allergens, an assessment of the protein’s potential for the formation of secondary structure (i.e., a-helices, b-strands, ran- digestion when incubated in vitro with proteases, and an evalua- dom coil) and how those are arranged into the stable tertiary or tion of the protein’s history of safe use in food (Delaney et al., three-dimensional structure of a protein. For certain proteins, the 2008a; Rice et al., 2008). Where appropriate, a dietary risk assess- sequence contributes to the quaternary structure of multi-subunit ment may also be carried out with the introduced protein to esti- proteins. These higher order structures are essential for protein mate potential human dietary intake (Hammond and Cockburn, function, whether it is a structural, enzymatic, immunologic, neu- 2008). In the past, dietary risk assessments for biotechnology- ronal, or hormonal (Fig. 1)(Branden and Tooze, 1991). Proteins derived corn and soybeans have made the highly conservative that perform either similar or identical biological functions in dif- assumption that introduced protein(s) do not lose functional ferent organisms typically share related amino acid sequences and activity during processing of corn grain or soybeans into food. Corn tertiary structures and can be grouped into the same protein fam- grain and soybeans are not consumed raw by humans, but are ilies. Thus, conserved sequence motifs and/or structural features processed into various food fractions using conditions that often offer important information about the possible biochemical role denature and degrade proteins. It has been recommended that of a given protein. Many proteins also contain multiple functional the stability of introduced proteins to food processing conditions regions, also known as domains. Different combinations of do- be explored since the default use of the aforementioned highly mains can give rise to a diverse range of proteins. The identification conservative assumptions may significantly overestimate potential of domains that occur within proteins can also yield insights into human dietary exposures (EFSA, 2008). Therefore, this paper their physiological function (Buljan and Bateman, 2009; Moore reviews the impact of corn and soy food processing activities on et al., 2008; Thornton et al., 1999; Ganfornina and Sánchez, 1999). Fig. 1. Variety in protein structure. B.G. Hammond, J.M. Jez / Food and Chemical Toxicology 49 (2011) 711–721 713 The proper folding of any amino acid sequence into a functional are all employed and will unfold a native protein structure and/or protein involves a combination of physical forces; short-range alter the primary structure of a protein by hydrolysis of peptide repulsions, electrostatic forces (i.e., charge–charge interactions bonds (Kilara and Sharkasi, 1986; Meade et al., 2005). In typical and dipole moments), van der Waals interactions, hydrogen bonds, processing of soybeans into food fractions, temperatures of 95– and hydrophobic interactions (Branden and Tooze, 1991; 100 °C for several minutes are commonly encountered (Kilara Creighton, 1993). The laws of thermodynamics require a protein and Sharkasi, 1986). These elevated temperatures can lead to irre- to assume a configuration that expends the least amount of free versible denaturation and loss of protein function (de Luis et al., energy to maintain it. Electrostatic, hydrogen bond, and van der 2009; Thomas et al., 2007), although the nutritional value of the Waals interactions in aqueous environments, such as the cell, are denatured protein as a source of dietary amino acids is not lost, weak compared to interactions with the water surrounding a pro- and may be enhanced. Similar denaturation of proteins can occur tein; however, proteins also contain regions of hydrophobic amino