Food and Chemical Toxicology 49 (2011) 711–721
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Food and Chemical Toxicology
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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 during processing of corn, which can also involve the use of harsh acids. Generally, this leads to orientation of hydrophilic amino conditions, such as the extreme alkali conditions encountered dur- acids on the exterior of a protein and hydrophobic amino acids ing nixtamalization, an ancient process first developed in Meso- packed into the interior to exclude water from the protein core. america to improve the nutritional quality of maize [discussed This hydrophobic effect, or the exclusion of water from the protein further in Appendix A, Supplementary data. interior, causes a large increase in entropy with the close packing Depending on the desired outcome of a process, changes in con- of hydrophobic residues reducing enthalpy; these thermodynamic ditions can be used to manipulate the physical properties of a pro- changes yield a stable system with low free energy (Kilara and tein. Precipitation is often used to remove proteins from other food Sharkasi, 1986). The combination of various forces maintains a fractions like lipids, as changes in the physical properties of a pro- protein’s three-dimensional structure and allows it to perform a tein can reduce its solubility leading to aggregation or precipitation biological function. or cause multimeric proteins to dissociate into monomers also resulting in loss of function (Schultz and Liebman, 2002; Meade 2.1. Changes in the environment of proteins can alter their structures et al., 2005). Cooking proteins aids their digestion in the gastroin- testinal tract as proteases (e.g. pepsin and trypsin), are able to The balance between large and opposing entropic and enthalpic cleave the random coil of a denatured protein more quickly and effects, both of which are highly temperature dependent, main- efficiently compared with the same protein in its native conforma- tains protein structures (Kristjansson and Kinsella, 1991). In gen- tion (Herman et al., 2006). eral, protein structures are only marginally stable under a limited range of physiological conditions and are easily disrupted by any 3. Processing of corn and soy into human foods environmental changes, such as shifts in temperature, variation of pH, or physical disruption, that overcome the forces keeping Food and food ingredients derived from corn and soy are them folded (Creighton, 1993). Denaturation of proteins occurs subjected to a variety of processing conditions to improve their as a drastic change in structure that invariably results in a com- sensory and functional qualities, e.g. flavor, texture, taste, and plete loss of biological function, as the denatured polypeptide is appearance (Rooney and Serna-Saldivar, 2003). Digestibility of more like a random coil than a folded protein. As the environment soy is improved by cooking soybean meal to inactivate natural of a protein is gradually altered toward conditions that favor antinutrients such as trypsin inhibitors and lectins that interfere unfolding, the folded structure initially changes very little, if at with digestion of dietary proteins (Rackis, 1974). Cooking (pasteur- all, but suddenly, the protein will unfold. The abruptness of the ization) also destroys microbes that reduce storage stability and protein denaturation transition results because the process is can impart desired properties to processed fractions formulated highly cooperative. For example, as pH varies, multiple charged into various foods (Thomas et al., 2007). Both ground corn grain amino acids change ionization state simultaneously. Similarly, in- and soy beans can also be extracted with solvents (e.g. hexane) creased temperature affects all the forces that maintain a stable to remove oils that are used in food applications; the defatted meal protein structure at the same time. Denaturation can also result is heated (toasted) to remove the solvents (Berk, 1992). from other changes to a protein, such as altered oxidation state or the removal of cofactors and prosthetic groups. Typically, dena- turation does not involve changes in the primary structure of a pro- 3.1. Processing of corn grain into human food tein (i.e., degradation of the polypeptide chain). The environmental conditions that cause denaturation may differ for each protein According to a 2009 survey (NCGA, 2010), there were 13,070 (Pearce, 1989), but proteins that function under normal physiolog- million bushels of corn produced in the United States in 2009. Of ical conditions tend to have similar stabilities to unfolding, even this number, approximately 42.5% was used for feed and residual though they have different amino acid sequences and uses, 31.2% for fuel ethanol, 15.7% for export (gluten feed and meal three-dimensional structures (Creighton, 1993). For purposes of etc.), 3.5% high-fructose corn syrup, and 6.2% other uses (starch, this review, all of the proteins involved in food processing are from sweeteners, cereal/other, beverage alcohol and seed). Cereals used mesophiles (organisms that grow at moderate (15–40 °C) temper- for human food represent approximately 193 million bushels or atures). It is recognized that proteins from organisms adapted to 1.5% of total corn production. Since biotechnology-derived corn extreme environments (high or low temperature – psychrophiles varieties were planted on approximately 85% of the land planted and thermophiles; high salt – halophiles; low pH – acidophiles) in corn in the US in 2009 (NCGA, 2009), then approximately 193 are intrinsically resistant to harsh environmental conditions be- million  85% or approximately 164 million bushels of grain from cause of thermodynamic interactions unique to those proteins that biotech corn were used for cereal and related human food uses. provide additional structural stability. But the types of proteins Industrial processing of corn grain in North America is carried intended for use in agricultural biotechnology crops would not typ- out primarily by dry milling, dry-grind ethanol and wet milling. ically be derived from extremophile organisms. Dry milling is used primarily to generate products used for human Changes in the environment of proteins from food sources rou- food applications as well as fuel ethanol production (dry-grind tinely occur. For example, during the processing of corn and soy- milling) and the remaining by-products used for animal feeds beans into food fractions, heating, extrusion under high pressure, (Khullar et al., 2009). Prior to milling, corn grain is dried and sub- mechanical shearing, changes in pH, and the use of reducing agents jected to extensive cleaning to remove any extraneous materials. 714 B.G. Hammond, J.M. Jez / Food and Chemical Toxicology 49 (2011) 711–721
Table 1 Traditional foods made with corn grain in various countries (from Rooney and Serna-Saldivar, 2003).
Food Country Process Whole grain Hominy US Kernels cooked in lye (alkaline pH) at 100 °C for 25–40 min Pozole, posole Mexico, Central Kernels cooked in lime or lye (alkaline pH) to make hominy, add to soup, bring to a boil and simmer for America 45 min Thin porridges, unfermented Atole Mexico, Central Kernels ground or cooked in lime or water to produce masa. This is boiled for 15–20 min America Pinole Mexico, Central Same as atole except kernels are first roasted for 3–15 min on griddle America Mingau Brazil Corn grits or immature kernels are cooked in water to produce porridge like atole Canjica munguca Brazil Degermed corn kernels cooked with sugar and milk Pamonha Brazil Immature corn that is steam cooked in a sac of corn husks Fermented thin porridge Ogi Nigeria Kernels are steeped and fermented for several days. Fermented sediment is boiled in water to produce porridge Uji, ugali East Africa Corn slurry is fermented for several days until it becomes acidic. Slurry boiled and flavored with sugar Mahewu South Africa Corn meal mixed with water and boiled for 1.5 h Thick porridges Hanchi South America Corn grits cooked in water with sugar, fruit for 1 h. Lemon juice added while cooling Mazamorra South America Soaked corn in water overnight, cook in water (medium heat) or milk for 15 min to 2 h Humita South America Green corn is mixed into dough, mixed with meats or cheese and spread on corn husks, cooked steamer for 20–40 min Tô, tuwo, asida, ugali, sadza, pap, corn Africa Corn meal is cooked in water until it is completely gelatinized. Acid (lemon) or alkali (ashes) is added to the meal mush porridge. Or bring to a boil, and then reduce heat and let sit for 10–15 min, then let cool Polenta South America, Degerminated corn grits cooked in water (bring to boil, let simmer for 30–40 min) until gelatinized, mixed Italy with sauces, cheeses and meats and baked Snack foods Popcorn Worldwide Special flint corns with vitreous endosperm are popped in hot oil, hot air or hot surface, microwave Corn on the cob Worldwide Corn is boiled, broiled or barbecued Tostadas, totopos Central and Tortillas are fried in hot oil or tostada shells are baked at 176 °C for 5–7 min North America Steamed foods Couscous, cuzcuz Africa, Brazil Corn flour is mixed with boiling water or steam cooked for 5–10 min. Maybe further cooked when added to other foods Tamales Latin America Lime cooked corn is ground into dough, mixed with other foods and wrapped in leaves and steamed for 60– 90 min Unfermented breads Quesadillas, Rosquillos, rosquetes Central and Baked at 66 F for 60 min North America Tortillas, tacos, enchiladas, sopes, Central and Corn is cooked in lime for 15–60 min at near boiling temperature, steeped for 12–16 h, baked on a hot joroch, gorditas, papusas etc. North America griddle Arepas, hallaquitas, hallacas, Venezuela, Meal from partially degerminated corn is cooked in boiling water. Masa is formed into discs that are baked empanadas etc. Colombia 2 min on each side Roti, chapati India Corn flour is mixed with hot water into dough, hand formed into a thin disk and baked on a griddle at 210 °C for 25 s or longer until it puffs Corn bread Worldwide Corn flour is mixed with water and/or milk into a dough and baked at 218 °C for 22–25 min Fermented bread Ethiopia Corn flour/water is fermented for 17–72 h. A portion of the batter is mixed with water and boiled, returned to the main batter and fermented for another 0.5–2 h. The leavened acidic batter is baked as a thin layer for 2–3 min on a very hot fire Fermented dough Kenkey Ghana, West Corn is soaked in water for 12–48 h, ground into dough and fermented for 2–3 days. A portion of the Africa fermented corn is half-cooked, blended with the remaining uncooked portion which is molded into balls and boiled until done Pozol, akasa, koko, banku, akple, abcle, S. Mexico, Lime-cooked masa is shaped into balls, wrapped in banana leaves and fermented for 1–14 days and kpekle Central America Alcoholic beverages Urwaga, mwenge Kenya, Uganda Corn flour roasted over fire, fermented for 12–24 h Chicha, chichi morada South and Salivated or germinated corn flour is heated in water to 75 °C for 60 min. Further processed Central America Tesguino Mexico Corn soaked in water for several days, germinated, ground, and steeped in water and fermented to bring alcohol content to 3–4% Pito, burokuto Nigeria Corn soaked in water two days, germinated, mashed and fermented Talla Ethiopia Slurry of toasted, ground and cooked corn is mixed with other breads/malt, fermented for several days Busas Kenya Corn flour mixed with water to form dough, held for 4 days, toasted, and fermented for 3–4 days Opaque beer Zambia Corn is germinated for 3 days, sprouts dried and mashed. Corn meal cooked into porridge, mixed with malt, fermented Munkoyo Zambia Corn cooked into porridge, soaked root plant added, may or may not be further fermented B.G. Hammond, J.M. Jez / Food and Chemical Toxicology 49 (2011) 711–721 715
In Asia, Latin America, Africa and the Balkans, white maize is While most of the defatted soy flakes are further processed into generally preferred for foods, but yellow maize is preferred in Bra- soybean meal for animal feeding, the flakes can also be ground to zil, China, Argentina, and some other areas. In Latin America, corn produce soy flour, sized to produce soy grits or texturized to pro- is processed into tortillas, arepas, couscous, polenta, porridges, and duce textured vegetable protein (TVP) for human food uses. Fur- various meals and gruels. These are the basis for many traditional ther processing can produce high protein food ingredients such foods. In Africa and Asia, corn is generally milled into grits, meals as soy protein concentrates and isolated soy protein. These ingre- and flours for production of flat breads, i.e., roti, corn bread, unfer- dients have functional and nutritional applications in various types mented and fermented porridges, steamed foods (couscous, rice- of bakery, dairy and meat products, infant formulas and other soy like corn grits), snacks, popcorn, and alcoholic and nonalcoholic foods (NSRL, 2010). beverages (Rooney and Serna-Saldivar, 2003). In Asian countries, soybeans are processed into fermented (soy The use of fresh or immature (green) corn on the cob is prac- sauce, miso, natto, yogurts and sufu) and non-fermented products ticed worldwide. Corn cobs with or without husks are boiled in (kinako, protein crisp, desserts, baby food, and soy milk which is water, steamed or cooked over a fire, flavored with salt, cream, but- further processed into tofu, aburage, yuba etc.). Roasted whole soy- ter, margarine, and sauces. In most areas, field corn is used for beans and their flour are used as ingredients of traditional confec- green corn. Sweet corn is widely used only in the US (Rooney tionery products and snacks in China, Japan, Korea and Indonesia. and Serna-Saldivar, 2003). These ‘‘vegetable’’ soybeans are used for special food purposes in As shown in Table 1, which was largely derived from Rooney Asia such as immature whole green soybeans that are consumed and Serna-Saldivar (2003). Corn grain is processed in many differ- as a vegetable after boiling in salt water (edamame). The vegetable ent ways around the world into various food fractions. A common soybeans are produced on a much smaller scale than commercial feature to all of the foods derived from corn grain is cooking the varieties and are different from varieties used for large scale pro- grain by boiling, baking, or frying. The temperatures and duration duction of soybean meal which are often biotechnology-derived. of cooking will vary considerably. Corn grain is also steeped in Mature dry vegetable soybeans are seldom used as cooked legumes alkaline or other aqueous environments and can be fermented (e.g., navy beans, black beans, chick peas or lentils) even in the tra- for production of other foods (Rooney and Serna-Saldivar, 2003). ditional areas of soybean consumption. The reason for this may be More details regarding the processing conditions used to convert attributable to the persistent bitterness and ‘‘green beany taste’’ of corn grain into various foods can be found in Appendix A, Supple- soybeans, the low starch content, the relatively low water adsorp- mentary data. tion (swelling) capacity, long cooking time and poor digestibility (Berk, 1992). In Western countries, the use of soy-based foods 3.2. Processing of soybeans into human foods has only recently become popular due in part to published reports of the health benefits of soy. A variety of soy based food products Most of the commercial varieties of soybeans are processed into are now consumed in Western countries such as soy milk, tofu, meal to feed farm animals, and soybean oil is often recovered from soy sprouts, edamame, and soy protein products that are textured processed meal for human food uses. During processing, the soy- and used in soy and tofu burgers, soy sausages and chicken nug- beans are cracked to remove the hull and then rolled into full-fat gets, soy ice cream, yogurt and numerous other products (Liu, flakes. The rolling process disrupts the oil cells, facilitating solvent 2004a,b; Golbitz, 1995). extraction of the oil. After the oil has been extracted, the solvent is Industrial production of processed meal from commercial soy- removed, and the flakes are dried, creating defatted soy flakes. beans involves cleaning and drying of soybeans that are subjected
• • •
Fig. 2. Industrial scale soybean processing. http://www.foodtechinfo.com/FoodPro/FacilityTypes/311222_Soybean_Processing.htm. 716 B.G. Hammond, J.M. Jez / Food and Chemical Toxicology 49 (2011) 711–721 to cracking and aspiration to remove hulls from the seed meat ous solution at different temperatures ranging from 25 to 100 °C for (cotyledons) which are subsequently broken into pieces. The seed a duration of 15–30 min. A protocol for measuring the heat stability meat is conditioned to 10–11% moisture at temperatures ranging of proteins has been proposed by the Indian Department of Biotech- from 71 to 79 °C and flaked using smooth rolls to increase surface nology (DBT, 2010). Once the protein solution has returned to room area for hexane extraction of soybean oil. The hexane is removed temperature, the functional activity of the protein can be assessed by steaming the soybean flakes which toasts the defatted meal to through measurement of catalytic activity (enzyme) or biological inactivate trypsin inhibitors and lectin antinutrients and also dena- activity, such as an insect bioassay that measures insect survival tures other soy proteins (Hettiarachchy and Kalapathy, 1999). The and growth after exposure to an insect control protein. It is impor- seed meats can also be run through an expander/extruder to make tant to assess functional activity at room temperature to rule out collets (95–120 °C) that increases surface area to improve the the possibly that the protein might re-nature when cooled, which extraction efficiency for removing soybean oil. Extraction of soy- has been reported for certain proteins (Albillos et al., 2009). SDS– bean oil from the collets is accomplished with hexane (49–60 °C PAGE can also be used to assess degradation and/or post-heating for 30 min), repeated two more times for durations of 15–30 min solubility. Enzymologists traditionally use similar in vitro methods each cycle. The oil is further processed and contains very little soy- to determine the optimum temperature for enzyme function as bean protein. Oil spent collets are placed in mixer, steam injected well as the melting temperature when an enzyme denatures and to reach 105–114 °C for 20–30 min to remove hexane (Berk, loses functional activity (Kristjansson and Kinsella, 1991). For pro- 1992). An example flow diagram for industrial scale soybean pro- teins where there is no bioassay to measure biological function, cessing is presented in Fig. 2. such as for structural proteins, it may not be possible to ascertain Commercial soy flours and grits are classified according to their whether heat treatment causes a loss of function, although the bio- Nitrogen Solubility Index (NSI), or their Nitrogen Dispersibility In- physical properties may be indicative of denaturation (e.g., shift in dex (NDI). These parameters indicate the extent of protein solubil- protein solubility, migration on chromatography). ity which is significantly affected by treatments used for its Several introduced proteins have been subjected to in vitro tests production. Moist heat treatments used to inactivate (denature) to measure heat stability; a few processing studies have also been trypsin inhibitors and lipoxygenase will also denature and precip- carried out to assess the impact on the functional activity of intro- itate other soy proteins leading to lower protein solubility (Hetti- duced proteins (Table 2). Results from these studies indicate that arachchy and Kalapathy, 1999). Flours made from ‘‘white flakes’’ the introduced proteins tested are effectively denatured when ex- have NSI values of about 80%, while those made from toasted flakes posed to temperatures/processes similar to those used to prepare show NSI levels of 10–20%. Other grades are available over the en- food fractions from corn and soybeans. The impact of food process- tire range of intermediate NSI values. The specification of a precise ing on the functional activity of introduced proteins may be rele- value of NSI reflects a compromise between the need to maintain vant for other processed crops such as wheat and rice that are the functional properties of the soy proteins or some enzyme activ- also typically cooked before consumption. It would not apply to ity, and the desire to inactivate anti-nutritional factors and elimi- food crops that are not processed and/or are consumed raw, such nate the beany taste (Berk, 1992). Additional details on soybean as certain fruits and vegetables. processing conditions used to make human food fractions can be As one might expect, the denaturation of introduced proteins found in Appendix A, Supplementary data. during processing of corn and soybeans into food fractions has made it difficult to detect these proteins in a functionally active 4. Testing introduced proteins for potential denaturation by form in final food products. Detection methods identifying foods heat treatment that occurs during food processing derived from biotech-derived crops is mandated in some countries for purposes of labeling. Protein detection methods are immuno- The potential for a protein to be denatured by heat treatment logically-based that use antibodies that bind to epitopes on the can be readily assessed in vitro where the protein is heated in aque- introduced protein. Processing can denature the introduced
Table 2 Impact of heating and food processing on functional activity of introduced proteins.
Introduced protein In vitro heat Functional activity References CP4 EPSPS enzyme 65–75 °C 30 min Enzymatica ND EFSA, 2009b 2 m EPSPS enzyme 65 °C 30 min Enzymatica ND EFSA, 2007a PAT enzyme 55 °C 10 min Enzymaticb ND Hérouet et al., 2005 GAT enzyme 56 °C 15 min Enzymaticc ND Delaney et al., 2008b Cry1Ab insect control 80 °C 10 min Insecticidald ND De Luis et al. 2009 Cry1F insect control 75–90 °C 30 min Insecticidal ND EFSA, 2005 Cry9C insect control 90 °C 10 min No loss activity De Luis et al. 2009 Cry34Ab1, Cry35Ab1 insect control 60–90 °C 30 min Insecticidal ND EFSA, 2007b Acetolactate synthase enzyme 50 °C for 15 min Enzymatice ND Mathesius et al., 2009 b-Glucuronidase enzyme 60 °C 15 min Enzymaticf 50% loss Gilissen et al., 1998 Proteins Food processing conditions Functional activity References Malt a-amylase enzyme Bake from 68 to 83 °C 4 min Enzymatic activity ND Pyler and Gorton, 2009 b-Amylase enzyme Bake from 57 to 72 °C 2 min Enzymatic activity ND Pyler and Gorton, 2009 CP4 EPSPS enzyme Toasted soy meal Enzymatic activitya ND Padgette et al., 1993 CP4 EPSPS enzyme Soy protein isolate Enzymatic activitya ND Padgette et al., 1993 CP4 EPSPS enzyme Soy protein concentrate Enzymatic activitya ND Padgette et al., 1993
ND: not detected. a Conversion of phosphoenolpyruvate to 5-enolpyruvylshikimate-3-phosphate. b Conversion of L-phosphinothricin, to N-acetyl L-phosphinothricin. c Cleave the thioester bond of acetyl-CoA. d Measured against target lepidopteran pests in an insect bioassay. e Conversion of pyruvate to acetolactate. f Catalyzes the hydrolysis of b-D glucuronides to D-glucuronic acid and the aglycone. B.G. Hammond, J.M. Jez / Food and Chemical Toxicology 49 (2011) 711–721 717 protein resulting in the loss of antibody binding epitopes; aggrega- of introduced proteins have been estimated making highly conser- tion of proteins during food processing can also reduce the ability vative assumptions about intake (e.g., that all grain consumed by to extract the introduced protein from the food matrix (Terry et al., humans is derived only from the biotech variety and there is no 2002; Grothaus et al., 2006; Margarit et al., 2006; Thomas et al., loss of the functionally active introduced protein during processing 2007; de Luis et al., 2009; Codex, 2010). It is possible to develop of the grain into human foods). antibodies to epitopes on a denatured introduced protein as it ex- Using these highly conservative assumptions, the chronic intake ists in the processed food if the test is ‘‘validated and fit for the pur- of corn grain containing Cry1Ab protein and/or CP4 EPSPS protein pose’’ (Grothaus et al., 2006). This had been applied to detect in the United States was estimated to be 0.005 mg/person/day and certain denatured introduced proteins in processed soybean meal 0.27 mg/person/day respectively for adults (Hammond and and for food and feed ingredients derived from corn grain. How- Cockburn, 2008). However, these introduced proteins are effec- ever, in consideration of the technical challenges in detecting tively denatured when corn grain is processed into human foods, denatured introduced proteins in processed food, protein-based as no functional activity of Cry1Ab and CP4 EPSPS proteins was de- immunologic detection methods are mostly reserved for testing tected in processed foods (Table 2). Therefore, the level of function- raw agricultural commodities or crops that are minimally pro- ally active proteins in these foods was assumed to have been cessed to make food or feed (Bogani et al., 2008; de Luis et al., reduced by at least two orders of magnitude to levels below detect- 2009). An example of this is the Cry1Ab protein that has been ability. Thus the aforementioned dietary intakes could be reduced introduced into corn to protect against corn borer pests. While this proportionally to 0.0027 mg/person/day for CP4 EPSPS and protein can be readily detected in grain and plant tissues (raw agri- 0.0005 mg/person/day for Cry1Ab protein. When converted to cultural commodities) from Bt corn, it has not been detected in mg/kg body weight exposures (assuming average adult body processed food products (Margarit et al., 2006; de Luis et al., weight of 60 kg), the dietary exposure rates were 0.000045 and 2009) nor in wet milled fractions and in corn mash from the dry- 0.000008 mg/kg body weight respectively. Corn grain intakes in grind ethanol process (Dien et al., 2002). The monitoring for bio- Central and Latin America, and in Africa may be 20 times higher tech-derived crops in processed food and feed rely more routinely than that in North America and Europe, but 20 times a very small on DNA based detection methods (Alderborn et al., 2010). number is still a very low intake. For soybeans, the least processed food fraction is full fat flour that is added to wheat flour at levels ranging from 1% to 5% and 5. Implications for dietary risk assessments is subsequently cooked to make breads and cakes etc. (Liu and Limpert, 2004). If CP4 EPSPS protein were present at the highest re- The levels of introduced proteins in raw agricultural commodi- ported level in soybeans (400 ppm or 0.4 mg/g), and assuming that ties such as corn grain and soybeans can range from as low as there was negligible loss in full fat soybean meal, the meal would 0.04 ppm (CSPB protein) in drought tolerant corn grain (FSANZ, be subsequently cooked at high temperatures conditions that 2010) to 14 ppm for CP4 EPSPS protein in herbicide tolerant corn would effectively denature CP4 EPSPS. If the levels of functionally grain and up to 400 ppm in herbicide tolerant soybeans active CP4 EPSPS were reduced by two orders of magnitude by bak- (Hammond and Cockburn, 2008). Potential human dietary intake ing, the estimated human exposure would be 0.004 mg/gram full
Table 3 Summary of NOAELs in acute high dose studies with different proteins.
Protein Function NOAEL⁄ (mg/kg) References Cry1Ab Insect control 4000 Betz et al., 2000 Cry1Ab/Cry1Ac fusion protein Insect control 5000 Xu et al., 2010 Cry1A.105 Insect control 2072 EPA, 2008a Cry 1Ac Insect control 4200 Betz et al., 2000 Cry1C Insect control 5000 Cao et al., 2010 Cry2Aa Insect control 4011 Betz et al., 2000 Cry2Ab Insect control 1450 Betz et al., 2000 Cry2Ab2 Insect control 2198 EPA, 2008b Cry3A Insect control 5220 Betz et al., 2000 Cry3Bb Insect control 3780 Betz et al., 2000 Cry1F Insect control 576 EPA, 2001 Cry34Ab1 Insect control 2700 Juberg et al., 2009 Cry35Ab1 Insect control 1850 Juberg et al., 2009 VIP3A Insect control 3675 EPA, 2004 ACC deaminase Enzyme 602 Reed et al., 1996 Alkaline cellulase Enzyme 10,000 Greenough et al., 1991 Dihydrodipicolinate-synthase (cDHDPS) Enzyme 800 FSANZ, 2006 b-Galactosidase Enzyme 20,000 Flood and Kondo, 2004 CP4 EPSPS) Enzyme 572 Harrison et al., 1996 b-Glucanase Enzyme 2000 Coenen et al., 1995 Glutaminase Enzyme 7500 Ohshita et al., 2000 Glyphosate acetyltransferase Enzyme 2000 Delaney et al., 2008b Hexose oxidase Enzyme 2000 Cook and Thygesen, 2003 Laccase Enzyme 2700 Brinch and Pedersen, 2002 Lactase Enzyme 10,000 Coenen et al., 2000 Lactose oxidase Enzyme 900 Ahmad et al., 2004 Lipase Enzyme 2000 Ciofalo et al., 2006 Lipase Enzyme 5000 Coenen et al., 1997 Neomycin phosphotransferase Enzyme 5000 Fuchs et al., 1993 Phosphinothricin acetyl transferase Enzyme 2500 Hérouet et al., 2005 Phosphomannose isomerase Enzyme 3030 Reed et al., 2001 Xylanase Enzyme 2000 Harbak and Thygesen, 2002 718 B.G. Hammond, J.M. Jez / Food and Chemical Toxicology 49 (2011) 711–721
flat flour  5.0% w/w (highest full fat soybean flour incorporation change the potential allergenicity of known protein allergens rate)  3.25 g/kg/day (chronic consumption of flour from baking (Thomas et al., 2007). However, the impact of heat treatment on products – Bui et al., 2005) = 0.0007 mg/kg body weight. This is protein allergens is not addressed further in this review since the also a very low dietary intake. proteins introduced into biotech-derived crops to date do not fit These dietary intakes are many orders of magnitude below the the profile of known protein allergens. Through the use of bioinfor- doses of various proteins administered to rodents in numerous matics comparisons, only proteins that show no meaningful struc- acute and subchronic toxicity studies which produced no treat- tural or functional similarity to known toxins or allergens have ment-related adverse effects as summarized in Tables 3 and 4. been introduced into food crops. In regards to allergens, population Many of the proteins tested were functionally active enzymes pro- threshold doses for elicitation of allergic reactions to peanut aller- duced by fermentation. These studies with fermentation-derived gens were recently proposed based on the review of clinical re- enzymes were not conducted to address the safety of the enzyme cords for 286 peanut allergic patients in France (Taylor et al., but rather to rule out the theoretical presence of adventitious tox- 2010). The threshold dose was in the low mg protein/person for ins from the microorganisms used to produce the enzyme (Pariza the most sensitive population and considerably higher doses for and Johnson, 2001). The actual doses of proteins administered var- those whose allergic reactions were less severe. The potential die- ied as the purity of the enzyme in fermentation batches varied tary exposures to the Cry1Ab and CP4 EPSPS proteins in processed from 2% to 70% (Spok, 2006), whereas other proteins introduced food crops derived from corn are far below these observed thresh- into biotech-derived food crops and prepared heterologously in old levels. These levels of exposure are also considerably below the E. coli for confirmatory safety testing were generally of higher pur- proposed threshold of toxicological concern (TTC) level (1.8 mg/ ity and were also functionally active. It has been reported that as of person/day) for chemicals considered to have a low potential for 2001, over 800 toxicity tests have been carried out on 180 different toxicity based on metabolism and mechanistic data (Vermeire enzymes made by fermentation and the studies found no evidence et al., 2010). Proteins are not known to be capable of genotoxic of safety concerns (Spok, 2006). interactions (Pariza and Johnson, 2001), nor are they known to It is recognized that certain proteins are toxic or allergenic if be carcinogenic or teratogenic when consumed in the diet consumed (Delaney et al., 2008b). Moreover, heat treatment dur- (Delaney et al., 2008b). Proteins are structurally quite different ing normal food processing can either reduce, increase, or not from chemicals since they are large macromolecules and their size
Table 4 Summary of NOAELs in subchronic feeding studies with different proteins.
Protein Function Study NOAEL⁄ References Bovine somatotropin Hormone 13 week 50 mg/kg Hammond et al., 1990 Dipel Bt microbial spray Insect control 13 week 8400 mg/kg Betz et al., 2000 Dipel Bt microbial spray Insect control 2 years 8400 mg/kg Betz et al., 2000 Teknar Bt microbial spray Insect control 13 week 4000 mg/kg Betz et al., 2000 Bt Berliner microbial spray Insect control 5 day (human) 1000 mg/adult Betz et al., 2000 Cry1Ab Insect control 28 day 0.45 mg/kg/day Betz et al., 2000 Cry34Ab1 and Cry35Ab1 Insect control 28 day 195 and 8 mg/kg Juberg et al., 2009 Amylase Enzyme 90-days 17.5 mg/kg/day Bui et al., 2005 Amylase Enzyme 90-days 890 mg/kg Landry et al., 2003 Amyloglucosidase Enzyme 14-days 1640 mg/kg Van Dijck et al., 2003 Amylomaltase Enzyme 90-days 1230 mg/kg Tafazoli et al., 2010 Amino peptidase Enzyme 90 days 2000 mg/kg Coenen and Aughton, 1998 Arabinofuranosidase Enzyme 14-days 103 mg/kg Van Dijck et al., 2003 Chymosin Enzyme 90 days 1000 mg/kg Lin, 2006 Chymosin Enzyme 90 days 11.9 mg/kg Lin 2006 b-Galactosidase Enzyme 6 months (rat) 4000 mg/kg Flood and Kondo, 2004 Glucanase Enzyme 90 days 1258 mg/kg Elvig and Pedersen, 2003 Glutaminase Enzyme 90-days 9000 mg/kg/day (yeast CK) Ohshita et al., 2000 1200 mg/kg/day (yeast CKD10) Glyphosate acetyltransferase Enzyme 28 days 1000 mg/kg Delaney et al., 2008b Hexose oxidase Enzyme 90-days 5000 HOX units/kg Cook and Thygesen, 2003 Laccase Enzyme 90-days 1720 mg/kg Brinch and Pedersen, 2002 Lactase Enzyme 28-days 1540 mg/kg Coenen et al., 2000 Lacterimin (whey growth factor extract) Enzymes, growth factors etc. 90-days 3000 mg/kg Dyer et al., 2008 Lactose oxidase Enzyme 90-days 900 mg/kg Ahmad et al., 2004 Lipase Enzyme 90-days 658 mg/kg Coenen et al., 1997 Lipase Enzyme 90-days 1680 mg/kg Ciofalo et al., 2006 Lipase G Enzyme 90-days 1516 mg/kg Kondo et al., 1994 Lipase AY Enzyme 90-days 2500 mg/kg Flood and Kondo, 2001 Pectin methylesterase Enzyme 14-days 133 mg/kg Van Dijck et al., 2003 Phosphodiesterase Enzyme 28-days 165 mg/kg Steensma et al., 2004 Phospholipase-A Enzyme 90-days 1350 mg/kg Van Dijck et al., 2003 Phytase Enzyme 90-days 1260 mg/kg Van Dijck et al., 2003 Pullulanase Enzyme 28-days 5000 mg/kg Moddeerman and Foley, 1995 Tannase Enzyme 91-days 660 mg/kg Lane et al., 1997 Xylanase Enzyme 90-days 1850 mg/kg Van Dijck et al., 2003 Xylanase Enzyme 90-days 4095 mg/kg Van Dijck et al., 2003 Lactoferrin (human) Iron transport 90 days 2000 mg/kg/d Appel et al., 2006 Lactoferrin (bovine) Iron transport 90 days 2000 mg/kg/d Yamauchi et al., 2000 Silkworm pupae protein Not defined 30 days 1500 mg/kg/d Zhou and Han, 2006 Thaumatins Sweetner 90 days 2696 mg/kg/d Hagiwara et al., 2005 Ice-structuring protein Cryo-preservation 90 day 580 mg/kg/d Hall-Manning et al., 2004
* In all cases, the NOAELs were the highest dose tested. B.G. Hammond, J.M. Jez / Food and Chemical Toxicology 49 (2011) 711–721 719 limits systemic absorption from the GI tract. Unlike most chemi- tions. Characterization of the potential denaturation of introduced cals, proteins are also degraded by proteases which cleave peptide proteins during normal food preparation processes should be con- bonds that hold the protein together. Independent of the differ- sidered as part of the ‘‘reliable information’’ to assess their safety. ences between proteins and chemicals, the estimated exposure levels for CP4 EPSPS and Cry1Ab proteins after food processing Conflict of Interest (0.0027 and 0.0005 mg/person/day respectively) are comparable to, or below the level of no toxicological concern for most The authors declare that there is no conflicts of interest. chemicals. The impact of processing on the loss of functional activity of Acknowledgement introduced proteins has particular relevance to recent recommen- dations from the European Food Safety Authority (EFSA). ‘‘Unless Appreciation is expressed to Kevin C. Glenn for his helpful re- reliable information is provided which demonstrates the safety of view and comments on this manuscript. the newly expressed protein, the safety assessment of proteins with no history of safe use (for consumption as food) should nor- mally include a repeated-dose toxicity test (normally 28 days) Appendix A. Supplementary data and not rely on acute toxicity testing’’ (EFSA, 2009a). If there is evi- dence that the introduced protein is heat labile when tested in vitro Supplementary data associated with this article can be found, in and is, therefore, unlikely to survive functionally intact during the online version, at doi:10.1016/j.fct.2010.12.009. normal food processing, there would be negligible, if any, human dietary exposure to the functionally active protein in typically References consumed foods. This could obviate the need to carry out a 28 day toxicity study on the functionally active protein if human Ahmad, S.K., Brinch, D.S., Friis, E.P., Pedersen, P.B., 2004. Toxicological studies on lactose oxidase from Microdochium nivale expressed in Fusarium venenatum. dietary intake is negligible and it is not structurally or functionally Regul. Toxicol. Pharmacol. 39, 256–270. related to proteins known to be toxic to mammals. Furthermore, Albillos, S.M., Menhart, N., Fu, T.J., 2009. 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