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Antioxidants: Science, Technology, and Applications

  1. P. K. J. P. D. Wanasundara1,
  2. F. Shahidi2

Published Online: 15 JUL 2005

DOI: 10.1002/047167849X.bio002

Bailey's Industrial Oil and Fat Products

Bailey's Industrial Oil and Fat Products

How to Cite

Wanasundara, P. K. J. P. D. and Shahidi, F. 2005. Antioxidants: Science, Technology, and Applications. Bailey's Industrial Oil and Fat Products. 1:11.

Author Information

  1. 1

    Agriculture and Agri-Food Canada Saskatoon Research Center, Saskatoon, Saskatchewan, Canada

  2. 2

    Memorial University of Newfoundland, St. John’s, Newfoundland, Canada

Publication History

  1. Published Online: 15 JUL 2005

1 An Antioxidant—Definition

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References

In a biological system, an antioxidant can be defined as “any substance that when present at low concentrations compared to that of an oxidizable substrate would significantly delay or prevent oxidation of that substrate” (1). The oxidizable substrate may be any molecule that is found in foods or biological materials, including carbohydrates, DNA, lipids, and proteins. Food is a multicomponent system composed of a variety of biomolecules, and therefore, this definition describes well an antioxidant. However, regulatory bodies that overlook the food-supply categorize antioxidants under food additives and define them as “substances used to preserve food by retarding deterioration, rancidity, or discoloration due to oxidation” (Code of Federal Regulations, Food and Drug Administration). In foods, much of the work on antioxidants has emphasized retardation of lipid oxidation, which eventually triggers and transforms to the oxidation of other macromolecules such as proteins. It is the intention of this chapter to summarize the available information on the chemistry, technology, and regulatory aspects of compounds that can delay oxidation of unsaturated fats and lipids in food.

2 History of Antioxidants and Their Use

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References

Antioxidants may occur as natural constituents of foods, and may intentionally be added to products or formed during processing. Use of substances to enhance quality of food by means of delaying lipid oxidation has been in practice for centuries, although it was not chemically defined or understood. The first recorded scientific observation on oxidation inhibitors came from Berthollet in 1797 (2) and later from Davy (3). Their theory was described as “catalyst poisoning” in oxidative reactors, and this was well before the free radical theory of peroxidation had been proposed. Duclaux (4) first demonstrated participation of atmospheric oxygen in oxidation of free fatty acids. Later, it was found that oxidation of unsaturated acylglycerols can generate rancid odors in fish oils (5).

The earliest reported work on the use of antioxidants to retard lipid oxidation appeared in 1843, in which Deschamps showed that an ointment made of fresh lard containing gum benzoin (contains vanillin) or populin (from polar buds, contains saligenin and derivatives) did not become rancid as did the one with pure lard (2). Interestingly, the first reports on antioxidants employed for food lipids were about using natural sources; in 1852, Wright (6) reported that elm bark was effective in preserving butterfat and lard. Chevreul (7) showed that wood of oak, poplar, and pine (in the order of decreasing efficacy) retarded the drying of linseed oil films applied on them, and on all three, it took much longer time to dry than on glass. Moureu and Dufraise (8-11) first reported the possibility of using synthetic chemicals, especially phenolic compounds, to retard oxidative decomposition of food lipids. Their work provided the basic information leading to theories of lipid oxidation and antioxidants, which they referred to as “inverse catalysis.” Systematic investigation of antioxidant activity based on the chemistry of radical chain peroxidation of “model” chemicals was reported by Lowry and his collegues (12) and Bolland and tenHave (13) of the British Rubber Producers Research Association. Antioxidant synergism in food was first reported by Olcott and Mattill (14), and this was significant in achieving oxidative stability in food by using a combination of antioxidants found in the unsaponifiable fraction of oils. They described the antioxidants as inhibitors and grouped them into acid type, inhibitols, and hydroquinone and phenolics. Bailey (15) and Scott (16) have provided the history and a descriptive analysis of the development of antioxidants in their books, “The Retardation of Chemical Reactions” and “Antioxidants and Autoxidation”, respectively.

Since the early 1960s, the understanding of autoxidation of unsaturated lipids and antioxidative mechanisms have advanced significantly as a result of development of effective analytical tools. The last two decades have been very important to the antioxidant research. Around the world a revival is seen in studying the natural antioxidants in foods and the potential health benefits of natural antioxidants in relation to prevention and therapy of oxidative stress and related diseases. The emphasis has largely been on their implications on vital biological reactions that have a direct relationship to tissue injury and degenerative diseases. Enough scientific evidences have already been accumulated in relation to these conditions with free radicals and reactive oxygen species. Therefore, not only enhancing the shelf life stability of foods has been examined, but also control of lipid oxidation by suppressing free radical formation in foods to prevent their deleterious health effects has become important. The quest for understanding the oxidation of lipids and its prevention and control has continued since historical times and is still on.

3 Scope of Using Antioxidants in Food

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References

The function of an antioxidant is to retard the oxidation of an organic substance, thus increasing the useful life or shelf life of that material. In fats and oils, antioxidants delay the onset of oxidation or slow the rate of oxidizing reactions. Oxidation of lipids chemically produces compounds with different odors and taste and continues to affect other molecules in the food. The main purpose of using an antioxidant as a food additive is to maintain the quality of that food and to extend its shelf life rather than improving the quality of the food. Figure 1 illustrates how antioxidants can affect the quality maintenance of food in terms of oxidative rancidity development. Use of antioxidants reduces raw material wastage and nutrition loss and widens the range of fats that can be used in specific products. Thus, antioxidants are useful additives that allow food processors to use fats and oils economically in their product formulation.

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Figure 1. Typical curves for oxidation of lipids (a) No antioxidant added; (b) and (c) represent added or endogenous antioxidants. Antioxidant activity of (c) is higher than (b). IP1, IP2, and IP3 are induction period in hours or days.

4 Oxidation of Fats and Oils and Mechanism of Antioxidants

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References

In fats and oils, the process of oxidation is similar to that oxidation of any other unsaturated organic material and requires an initiation process, in order to generate free radicals from the substrate. As antioxidants inhibit oxidation or autoxidation process, the mechanism(s) involved need(s) to be discussed. Figure 1 explains the relationship of antioxidant activity and oxidation of a lipid as examined by a typical evaluation method.

Autoxidation is the oxidative deterioration of unsaturated fatty acids via an autocatalytic process consisting of a free radical chain mechanism. This chain includes initiation, propagation, and termination reactions that could be cyclical once started. The initiation process generates free radicals from the substrate. The α-methylenic H atom is abstracted from the unsaturated lipid molecule to form a lipid (alkyl) radical (R•) (Scheme 1, Equation [1]). The lipid radical is highly reactive and can react with atmospheric oxygen (3O2), a facile reaction resulting from the diradical nature of the oxygen molecule, and it produces a peroxy radical (ROO•) (Scheme 1, Equation [2]). In the propagation reactions, the peroxy radical reacts with another unsaturated lipid molecule to form a hydroperoxide and a new unstable lipid radical (Scheme 1, Equation [3]). As a new free radical is generated at each step, more oxygen is incorporated into the system. The newly propagated lipid radical will then react with oxygen to produce another peroxy radical, resulting in a self-catalyzed, cyclical mechanism (Scheme 1, Equation [4]).

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Scheme 1. Possible reactions of the autoxidation process. “R” is an alkyl group of an unsaturated lipid molecule. “H” is an α-methylenic hydrogen atom easily detachable because of the activating influence of the neighboring double bond or bonds. “ RO•” is alkoxy radical, “ROO•” is peroxy radical, and “I” is an initiator.

Hydroperoxides are unstable and may degrade to radicals that accelerate propagation reactions. These are branching steps of lipid autoxidation process (Scheme 1, Equations [5] and [6]). This chain reaction proceeds, and termination occurs only when two free radicals combine to form a nonradical product. Autoxidation can break down the substrate molecules as well as forming new molecules causing gross changes in the chemical and physical properties of the oxidizing substrate (17-19). Degradation of hydroperoxides may generate new molecules that have undesirable odors and flavors, associated with oxidative rancidity of unsaturated lipids. Such sensory perceivable changes are noted when oxidation of unsaturated lipids has been progressed to advanced stages. This is only a brief description of autoxidation process.

A lipid that contains double bonds undergoes autoxidation induced by various ways. It is now clear that metal-catalyzed decomposition of preformed hydroperoxides is the most likely cause for the initiation process. The direct oxidation of unsaturated lipids by triplet oxygen (3O2) is spin forbidden. This is because of the opposite spin direction of ground state lipid of single multiplicity and oxygen of triplet multiplicity, which does not match. When initiators are present, this spin barrier between lipids and oxygen can readily be overcome and produce radicals by different mechanisms. Ground state oxygen may be activated in the presence of metal or metal complexes and can initiate oxidation either by formation of free radicals or singlet oxygen. Exposure of lipids to light, metals, singlet oxygen and sensitizers (chlorophyll, hemoproteins, and riboflavin), or preformed hydroperoxide decomposition products causes generation of primary hydroperoxides. Photosensitized oxidation or lipoxygenase-catalyzed oxidation also produces hydroperoxides.

Thermal oxidation is also autocatalytic and considered as metal-catalyzed because it is very difficult to eliminate trace metals (from fats and oils or food) that act as catalysts and may occur as proposed in Equation 4. Redox metals of variable valency may also catalyze decomposition of hydroperoxides (Scheme 2, Equations [6] and [7]). Direct photooxidation is caused by free radicals produced by ultraviolet radiation that catalyzes the decomposition of hydroperoxides and peroxides. This oxidation proceeds as a free radical chain reaction. Although there should be direct irradiation from ultraviolet light for the lipid substrate, which is usually uncommon under normal practices, the presence of metals and metal complexes of oxygen can become activated and generate free radicals or singlet oxygen.

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Scheme 2. Possible reactions of generating hydroperoxides (Mn+ is the metal ion with transitional valency).

Photosensitized oxidation is a direct reaction of light-activated, singlet oxygen with unsaturated fatty acids, and subsequently hydroperoxides are formed. Photosensitized oxidation happens because of the presence of molecules that can absorb visible or near UV light to become electronically excited (sensitizers) (Equation 8). Pigments initiating photosensitized oxidation in foods include chlorophylls, hemoproteins, and riboflavin. The type I sensitizer serves as photochemically activated free radical initiator, and type II sensitizers in the triplet state interact with oxygen by energy transfer to form singlet oxygen (1O2) that reacts further with unsaturated lipid (Equation 9). Under photosensitized oxidation conditions, the reaction of unsaturated lipids with singlet oxygen (1O2) leads to rapid formation of hydroperoxides (Equation 10) (17, 19, 20).

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Scheme 3. Formation of hydroperoxides by photoxidation of a lipid with a sensitizer (hv is energy in the form of UV light, sensitizers that are naturally present in photosensitive pigments, their degradation products, or polycyclic aromatic hydrocarbons capable of transferring energy from light to chemical molecules).

5 Classification of Antioxidants

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References

Antioxidants may be broadly grouped according to their mechanism of action: primary or chain breaking antioxidants and secondary or preventive antioxidants. According to this classification, some antioxidants exhibit more than one mechanism of activity, therefore, referred to as multiple-function antioxidants. Another commonly used classification categorizes antioxidants into primary, oxygen scavenging, and secondary, enzymatic and chelating/sequestering antioxidants. However, synergistic antioxidants are not included in this classification. During the past two decades, several naturally occurring compounds have been added into the list of antioxidants that are effective against oxidation of unsaturated fats and oils and most of them fall into the multifunctional category. Classification of antioxidants according to the mode of activity as primary and secondary is preferred in this discussion.

5.1 Primary Antioxidants

Primary antioxidants are also referred to as type 1 or chain-breaking antioxidants. Because of the chemical nature of these molecules, they can act as free radical acceptors/scavengers and delay or inhibit the initiation step or interrupt the propagation step of autoxidation. Figure 2 illustrates possible events that primary antioxidants may interfere along the lipid autoxidation pathway. Primary antioxidants cannot inhibit photosensitized oxidation or scavenge singlet oxygen.

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Figure 2. Possible interactions of primary and secondary antioxidants with lipid oxidation pathway in foods.

The first kinetic study of antioxidant activity was conducted by Boland and tenHave (13) who postulated the Equations 11 and 12. The primary antioxidants (AH) react with lipid and peroxy radicals (ROO•) and convert them to more stable, nonradical products as shown in Scheme 4, Equations 13 and 14. These antioxidants are capable of donating a hydrogen atom to lipid radicals and produce lipid derivatives and antioxidant radicals (A•) that are more stable and less readily available to participate in propagation reactions (Equation 12). Primary antioxidants have higher affinities for peroxy radicals than lipids and react predominantly with peroxy radicals. The following reasons have been listed for their high affinity. Propagation is the slow step in lipid oxidation process; thus, peroxy radicals are found in comparatively larger quantities than other radicals. In addition, peroxy radicals have lower energies than alkoxy radicals; therefore, they react more readily with the low-energy hydrogen of primary antioxidants than unsaturated fatty acids. As the free radical scavengers are found in low concentration, they do not compete effectively with initiating radicals (e.g., hydroxyl radicals) (21, 22). Therefore, primary antioxidants inhibit lipid oxidation more effectively by competing with other compounds for peroxy radicals, and they are able to scavenge peroxy- and alkoxy-free radicals formed during propagation (Equation 3) and other reactions (Equations 4 and 5) in autoxidation.

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Scheme 4. Mechanism of primary antioxidant activity (AH is an antioxidant molecule).

The antioxidant radical produced because of donation of a hydrogen atom has a very low reactivity toward the unsaturated lipids or oxygen; therefore, the rate of propagation is very slow. The antioxidant radicals are relatively stable so that they do not initiate a chain or free radical propagating autoxidation reaction unless present in very large quantities. These free radical interceptors react with peroxy radicals (ROO•) to stop chain propagation; thus, they inhibit the formation of peroxides (Equation 13). Also, the reaction with alkoxy radicals (RO•) decreases the decomposition of hydroperoxides to harmful degradation products (Equation 14).

Most of the primary antioxidants that act as chain breakers or free radical interceptors are mono- or polyhydroxy phenols with various ring substitutions. The antioxidant effectiveness is influenced by the chemical properties of the compound including hydrogen bond energies, resonance delocalization, and susceptibility to autoxidation. The ability of the primary antioxidant molecule to donate a hydrogen atom to the free radical is the initial requirement. The ability of the free radical interceptor (scavenger) to donate a hydrogen atom to a free radical can be predicted from standard one-electron potentials (Table 1). According to Buettner (21), each oxidizing species is capable of stealing an electron (or H atom) from any reduced species listed below it. That means when the standard one-electron reduction potential is concerned, the free radical scavengers that have reduction potential below peroxy radicals are capable of donating an H atom to peroxy radical and form a peroxide. The resulting antioxidant radical should be of low energy, ensuring the lesser possibility of catalyzing the oxidation of other molecules. The formed antioxidant radical is stabilized by delocalization of the unpaired electron around the phenol ring to form a stable resonance hybrid (Figure 3) and as a result attained low-energy levels (18, 22, 23).

Table 1. Standard One-Electron Reduction Potential inline image at pH 7 for Selected Radical Couples (Adapted from Ref. 21)
Coupleinline image at pH 7.0
HO·, H+/H2O2310
RO·, H+/ROH (alkoxy)1600
ROO·, H+/ROOH (peroxyl)1000
PUFA·, H+/PUFA-H (polyunsaturated fatty acid) 600
α-Tocopheroxyl·, H+/α-tocopherol500
Ascorbate·, H+/ascorbate 282
Dehydroascorbic/ascorbate·−−174
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Figure 3. Stable resonance hybrids of phenoxy radical of phenolic antioxidant [adapted from (18)].

Antioxidant radicals are capable of participating in termination reactions with peroxy (Equation 13), alkoxy (Equations 14 and 15), or antioxidant (Equation 16) radicals removing reactive free radicals from the system. In fats and oils containing phenolic antioxidants, dimers of antioxidant molecules are usually found. This is a good indication that antioxidant radicals readily undergo termination reactions and form dimers as proposed in Equation 13. When considering all of these, the primary antioxidants or free radical scavengers can inactivate at least two free radicals, the first one during the interaction with peroxy radical and the second in the termination reaction with another peroxy radical.

The compounds that exhibit primary antioxidant activity include polyhydroxy phenolics as well as the hindered phenolics. There are several synthetic ring-substituted phenolics as well as naturally occurring phenolic compounds that may perform via the primary antioxidant mechanism, as discussed later in this chapter. The common feature of all of these antioxidants is that they are mono- or polyhydroxy phenols with various ring substitutes (Figure 4). Substitution with an electron-donating group/s ortho and/or para to the hydroxyl group of phenol increases the antioxidant activity of the compound by an inductive effect (e.g., 2,6-di-tert-butyl-4-methylphenol or BHA). Thus, the presence of a second hydroxyl group in the 2- (ortho) or the 4-position (para) of a phenol increases the antioxidant activity (e.g., TBHQ). In the dihydroxybenzene derivatives, the semiquinoid radical produced initially can be further oxidized to a quinone by reacting with another lipid radical (Figure 5). This semiquinoid radical may disproportionate into a quinone and a hydroquinone molecule, and the process of this conversion contributes to antioxidant activity as peroxy radical scavenging potential (18, 24). Table 2 summarizes most commonly used primary antioxidants in fats and oils and lipid- containing foods. Substitution with butyl or ethyl group/s para to the hydroxy groups also enhances the antioxidant activity. Substitution of branched alkyl groups at ortho positions enhance the ability of the molecule to form a stable resonance structure that reduces the antioxidant radical’s participation in propagation reactions (18, 23).

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Figure 4. Chemical structures of synthetic phenolic antioxidants commonly used in fats and oils.

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Figure 5. Possible mechanism of antioxidant activity of dihydroxybenzene derivative.

Table 2. Primary Antioxidants that are Commonly Used in Foods
Natural Synthetic
Carotenoids Butylated hydroxyanisole (BHA)
Flavonoids Butylated hydroxytoluene (BHT)
Phenolic acids Ethoxyquin
Tocopherols and tocotrienols Propyl gallate (PG)
  Tertiary-butylhydroquinone (TBHQ)

To be most effective, primary antioxidants should be added during the induction or initiation stage of the autoxidation reaction cascade. Antioxidants can scavenge the formed free radicals, as the cyclical propagation steps have not occurred at this stage. Addition of primary antioxidants to a lipid that already contains substantial amounts of lipid peroxides may result in loss of antioxidant activity (25).

5.2 Secondary Antioxidants

Secondary antioxidants are also classified as preventive or class II antioxidants. They offer their antioxidant activity through various mechanisms to slow the rate of oxidation reactions. The main difference with primary antioxidants is that the secondary antioxidants do not convert free radicals into stable molecules. They act as chelators for prooxidant or catalyst metal ions, provide H to primary antioxidants, decompose hydroperoxide to nonradical species, deactivate singlet oxygen, absorb ultraviolet radiation, or act as oxygen scavengers. They often enhance the antioxidant activity of primary antioxidants. Table 3 provides examples of some of these compounds that exhibit secondary antioxidant activity.

Table 3. Compounds that Exhibit Secondary Antioxidant Activity
Mode of ActivityCompounds in use
Metal chelationCirtic, Malic, Succinic and Tartaric acids
  Ethylenediaminetetraacetic acid, Phosphates
Oxygen scavenging and reducing agentsAscorbic acid, Ascorbyl palmitate, Erythorbic acid, Sodium erythorbate, Sulfites
Singlet oxygen quenchingCarotenoids (β-Carotene, Lycopene and Lutein)
5.2.1 Sequestering/Chelating Agents or Metal Deactivators

Heavy metals with two or more valency states with a suitable oxidation-reduction potential between them (e.g., Co, Cu, Fe, Mn, etc.) shorten the induction period and increase the maximum rate of oxidation of lipids. Trace amounts of these metal ions are present in the lipid-containing foods coming from naturally present compounds or included during processing operations. The effectiveness of copper as a catalyst for hydroperoxide decomposition has been reported (26-28). Transition metals such as iron exhibit low solubility at pH values near neutrality (29). That means in foods, transition metals may exist chelated to other compounds; many compounds form complexes with these metals and change their catalytic activity. Chelation can increase the prooxidant activity of transition metals by making them more nonpolar (increase solubility in lipids; 30), and some can increase oxidative reactions by increasing metal solubility or altering redox potential (31). According to Graf and Eaton (32), chelators may exert antioxidant activity by prevention of metal redox cycling, occupation of all metal coordination sites, and formation of insoluble metal complexes and steric hindrance of interactions between metals and lipids or oxidation intermediates (e.g., peroxides). Chelation of these metal ions or use of metal deactivators reduces the pro-oxidant activity by raising the energy of activation for intiation reactions. The most effective form of chelating agents as secondary antioxidants, which form σ-bonds with metal ions because they reduce the redox potential and stabilize the oxidized form of the metal ion. Chelating agents such as heterocyclic bases that form π-complexes raise the redox potential and may accelerate metal-catalyzed hydroperoxide decomposition (Equations 7 and 8) and act as prooxidants.

Multiple carboxylic acid compounds such as citric acid, ethylenediaminetetraacetic acid (EDTA), and phosphoric acid derivatives (polyphosphates and phytic acid) are commonly used in extending the shelf life of lipid-containing foods because of their metal chelating properties. Typically these chelators are water soluble, but citric acid exhibits solubility in lipids, which allows it to inactivate metals in the lipid phase (33). Chelator’s activity depends on pH and the presence of other chelatable ions (e.g., Ca). Most food grade chelators are unaffected by food-processing operations and storage; however, polyphosphates may decrease their antioxidant activity because of possible hydrolysis by endogeneous phosphatases in foods, especially in raw meat (22).

Several proteins that exist in food (e.g., lactoferrin, ferritin, transferritin, heme protein) possess strong binding sites for iron. Reducing agents (ascorbate, cysteine, superoxide anion) to low pH causes release of iron from proteins and accelerates lipid oxidation (34). Some amino acids and peptides found in muscle foods (e.g., carnosine) are capable of chelating metal ions and inhibit their prooxidant activity (35, 36).

5.2.2 Oxygen Scavengers and Reducing Agents

As oxygen is essential and is one of the reactants in the autoxidation process, scavenging of oxygen molecular species is one way of providing antioxidant activity. Ascorbic acid acts as a reducing agent and as an oxygen scavenger. The mechanism of antioxidant activity of ascorbic acid is discussed elsewhere in this chapter.

Singlet oxygen is the excited state oxygen, and its inactivation is an effective way of preventing initiation of lipid oxidation. Carotenoids are capable of inactivating photoactivated sensitizers by physically absorbing their energy to form the excited state of the carotenoid. Later, the excited state carotenoid returns to ground state by transferring energy to the surrounding solvent (37, 38). Other compounds found in food, including amino acids, peptides, proteins, phenolics, urates, and ascorbates also can quench singlet oxygen (20).

Compounds such as superoxide anion and peroxides do not directly interact with lipids to initiate oxidation; they interact with metals or oxygen to form reactive species. Superoxide anion is produced by the addition of an electron to the molecular oxygen. It participates in oxidative reactions because it can maintain transition metals in their active reduced state, can promote the release of metals that are bound to proteins, and can form the conjugated acid, perhydroxyl radical depending on pH, which is a catalyst of lipid oxidation (39). The enzyme superoxide dismutase that is found in tissues catalyzes the conversion of superoxide anion to hydrogen peroxide.

Catalase is capable of catalyzing the conversion of hydrogen peroxides to water and oxygen (40). Glucose oxidase coupled with catalase is well used commercially to remove oxygen from foods, especially fruit juices, mayonnaise, and salad dressings (19). Glutathione peroxidase that is found in many biological tissues also helps to control both lipid and hydrogen peroxides (41, 42). These enzymic reactions help to reduce various types of radicals that could be formed in lipid-containing biological systems.

5.3 Synergism and Synergists

Synergism is the cooperative effect of antioxidants or an antioxidant with other compounds to produce enhanced activity than the sum of activities of the individual component when used separately (43). Figure 6 illustrates how synergistic effect is expressed as antioxidant activity. Two types of synergism are observed, one involving primary antioxidants only and the other involving a combination of primary antioxidants with metal chelators or peroxy scavengers.

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Figure 6. Synergistic effect of antioxidants: (a) 0.32% (w/w) dipalmitoyl phosphatidylethanolamine, (b) 0.02% (w/w) propyl gallate, and (c) is (a)+(b), evaluated in lard at 120°C, and the induction period was used to compare antioxidant activity [modified and redrawn from (44)].

In a combination of two or more free radical scavengers, rapid reaction with free radicals occurs because of the differences in bond dissociation energies or steric hindrance of free radical scavenger/ROO• interactions (23). These differences result in one scavenger being used faster than the other. Also, it is possible to regenerate the primary antioxidant by transferring its radical to another scavenger. Ascorbic acid together with α-tocopherol also shows a good synergism, which is explained by the regeneration and recycling of the tocopheroxyl radical intermediate to the parent phenol, α-tocopherol (44, 45).

In the combination(s) of free radical scavenger and metal chelator, the chelator decreases the oxidation rates by inhibiting metal-catalyzed oxidation; thus, fewer free radicals are generated in the system. Inactivation of antioxidants via termination reaction or participation in autoxidation occurs to a lesser extent in such situations. This makes the concentration of antioxidant, which is available to scavenge free radicals, to be always greater at a given time than when no metal chelator is present. Therefore, the combination of chelator and radical scavenger decreases free radical generation and increases radical scavenging potential (22). Strong synergistic activity has been observed in the mixtures of natural tocopherols and citric acid. The synergistic effect of this mixture is caused by the chain-breaking ability of tocopherols and metal chelation of citric acid (19).

In an antioxidant combination that contains compounds exhibiting different mechanisms of action and physical properties, inhibition of oxidation occurs in many different phases. This suggests that food antioxidants should be carefully selected considering such factors as the type of oxidation catalyst, physical state of lipid (bulk, emulsified), pH, temperature, and the ability to interact with other components in the food.

6 Evaluation of Antioxidant Activity

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References

Antioxidant activity of a given compound is assessed as resistance to oxidation of lipids in the presence of that particular compound. Therefore, most of the methods described and used to assess antioxidant activity follow oxidation and the stages of oxidation of unsaturated lipid substrates. Many techniques have been developed to determine the antioxidant efficacy of the compounds of interest, but all of these have to be employed and interpreted carefully. Frankel (19, 46) has listed following parameters that are fairly important in choosing methods to evaluate antioxidants.

  • Substrate

    Should be relevant to foods. Triacylglycerols and phospholipids, in the bulk, emulsion, or liposome form represent the closest model in biological systems including foods.

  • Conditions

    Test under various conditions. Under different temperatures, with metal catalysts, with surface exposure, etc., select these conditions to mimic conditions in food.

  • Analysis

    Measure relatively low levels of oxidation (below 1%) and include measurement of initial or primary products of lipid oxidation (e.g., hydroperoxides, conjugated dienes) as well as secondary decomposition products of lipid oxidation (e.g., carbonyls, volatiles, dialdehydes).

  • Concentrations

    Compare antioxidants at the same mole concentration of active compound, and an appropriate reference compound should also be used, which may be a structurally related reference compound. With crude extracts (e.g., natural antioxidants), compositional data are needed to compare samples.

  • Calculations

    Use the induction period, percentage inhibition or rates of hydroperoxide formation or decomposition, or IC50 value (concentration required to achieve 50% inhibition) based quantification (this is discussed further in a later section).

Obviously attention should also be paid to the system under examination. Thus, bulk oil, water-in-oil, or oil-in water emulsions behave differently under similar oxidation conditions.

An updated review by Antolovich et al. (47) discusses the methods of determining antioxidant activity extensively. The methods used in measuring antioxidant activity may be categorized into three groups, which directly or indirectly measure the rate or extent of the following:

  1. Decay of substrate, probing compound, or oxygen consumption

  2. Formation of oxidation products by the oxidizing substrate

  3. Formation or decay of probing free radicals.

Methods that use approaches (1) and (2) measure antioxidant activity as an inhibitory effect exerted by the test compound on the extent or rate of consumption of reactants or the formation of oxidation products. The antioxidant activity (AA) of a compound or a component mixture that is a function of many parameters of the assay method employed may be defined using the following mathematical expressions (47; Schemes 5 and 6):

  • mathml alt image

For a fixed set of assay conditions, AA could be defined independent of the test method. Scheme 5 provides equations for a situation that measures time as the independent variable.

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Scheme 5. Proposed expressions to calculate antioxidant activity.

According to the equations in Scheme 5, if:

  • tCONTROL = tAH, no antioxidant activity is exerted

  • tCONTROL < tAH, an antioxidant activity is exhibited

  • tCONTROL > tAH, a prooxidant activity is observed and AA has a negative value

A similar expression can be formulated for rate of oxidation.

Another expression that can be used is relative antioxidant activity (RAA; Scheme 6).

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Scheme 6. Proposed expressions to calculate relative antioixidant activity.

RAA represents the activity equivalence of the test compound relative to the reference antioxidant, which is suitable for activity comparison.

Methods of category (3) tract the capacity of the test compound to capture radicals or to inhibit radical formation rather than monitoring the actual oxidation product formation or substrate oxidation. Several new methods are developed based on this concept, and a variety of new parameters for expressing results are used. It is expected that a high correlation exists between these two types of measurements. It should be noted here that there are no standard units for reporting the antioxidant activity because such activity (assay, capacity, efficiency, effectiveness, etc.) is independent of the test procedure. Table 4 summarizes the methods available for measuring antioxidant activity and how the results of such determinations are expressed.

Table 4. Methods, Entities Tested, and Units to Express Results in Determining Antioxidant Activity
Method/TestReference/sMeasurementResults and Units
Substrate oxidation or oxidation product formation   
Active oxygen method 49, 50Change of mass, peroxide value or hydroperoxides, conjugated dienes, 2-thiobarbituric acid reactive substances, anisidine value, formation of hexanal, ethane, or pentane

Induction period (h,d)

 Time to reach a set level of oxidation during preinduction period (h, d)

 Rate of oxidation during preinduction period (mol kg−1hr−1, g L−1d−1)

 Concentration required to produce equivalent effect to reference antioxidant during preinduction period (mol kg−1, g L−1)

 Concentration of a functional group after a set time period (mequiv. kg−1)

 Concentration of an oxidation product after a set time period (mg kg−1, ppm w/w)

 Scale reading after a set time period (absorbance, conductivity, etc.)

Oven storage test 51, 52
Shelf storage test 48
Free radical capturing or suppression of formation   
DPPH quenching assay 53, 54Ability to quench DPPH radical in solutionPercentage inhibition, EC50 (concentration of test compound required to decrease the concentration of test free radical by 50%), TEC50 (time to decrease concentration of test free radical by 50%).
Hydroxyl radical quenching assay 55Ability to quench hydroxyl radicals generated in a model system
Superoxide radical quenching assay 56-58Ability to quench superoxide radicals generated in a model system
Electron paramagnetic resonance (EPR) spectrometry/spin trap tests 59-61Detects free radical involved in autoxidation and related processIntensity or rate of change in EPR signal
Ferric Reducing Antioxidant Power 62-64Spectrophotometric measurement of Fe (II) complex formed due to reducing ability of the test compoundsChange of absorbance
Oxygen Radical Absorption Capacity 65-67Based on Phycoerythrin assayFluorencence intensity, μmol of Trolox equivalents
Total Radical-trapping Antioxidant Parameter 68, 69Measure oxygen consumption during controlled lipid oxidation induced by thermal decomposition of 2,2′Azobis(2-aminopropane) hydrochloride; AAPHμmol peroxy radical deactivated L−1
Trolox Equivalent Antioxidant Capacity 70, 71Based on inhibition of production of 2,2′Azinobis(3-ethylbenzthiazoline)-6-sulfonic acid; ABTS radical cation and mM concentration of a Trolox solution having antioxidant capacity equivalent to 1.0-mM solution of test substancemM L−1 Trolox equivalents

Another way of categorizing the methods of determining antioxidant activity is (1) accelerated stability tests, and (2) free radical-based methods. Most of the studies that are currently used tend to employ accelerated test systems and try to relate them to real food systems.

6.1 Methods Based on Lipid/Substrate Oxidation (Stability Tests)

These methods are based on lipid (substrate) oxidation and specific to the analysis of oxidation that occurs in food lipids. The tests employed strongly correlate to the conditions that oils and fats are subjected to during processing, food preparation, and storage. The substrate is a model compound that could be a pure triacylglycerol, fatty acid methyl ester, or an actual edible oil/lipid. Favorable conditions for substrate oxidation (e.g., high temperature) are provided to facilitate increased rate of oxidation reactions in a controlled environment. The end point is determined by measuring chemical (e.g., primary or secondary oxidation products) or physical changes (e.g., change of mass or energy) of the oxidizing substrate. Table 5 provides a summary of methods commonly used for stability testing of edible oils.

Table 5. Commonly Used Accelerated Stability Tests for Oils in Evaluating Antioxidants 72
Method/TestConditions and characteristics
Ambient storageAtmospheric pressure and room temperature, too slow and time consuming
Active oxygen method (AOM)Bubbling air in a closed environment, 98°C, do not represent normal storage
 Rancimat is the automated version, also OSI instrument
LightAtmospheric pressure and room temperature, rapid screening test, photo-oxidation occurs
Metal catalystsAtmospheric pressure and room temperature, rapid screening test, more decomposition occurs
Oxygen uptakeAtmospheric pressure, 80–100°C, do not represent normal storage
Oxygen bomb65–115 psi, O2, 99°C, do not represent normal storage
Schaal ovenAtmospheric pressure, 60–70°C, generally correlates well with actual storage
Weight gainAtmospheric pressure, 30–80°C, not always very sensitive
6.1.1 Shelf Storage Test

The test material is stored under similar conditions as in retail and is evaluated for the effectiveness of antioxidants in prolonging the premium quality of the product. Periodic evaluation of the lipid oxidation products (primary or secondary) by chemical tests (e.g., peroxide value, conjugated diene value, 2-thiobarbituric acid reactive substances, hexanal content) or sensory evaluation will be used to find out the onset of oxidation. The main drawback of this kind of evaluation is the time taken; therefore, rapid evaluation or accelerated methods are often preferred (19, 51).

6.1.2 Active Oxygen Method (AOM)

This is one of the widely used methods for evaluating antioxidant activity. This test involves bubbling air through the heated lipid sample to accelerate its oxidation. Periodic analysis of peroxide value is carried out to determine the time required for the fat to oxidize under the conditions provided by AOM. This method has also been referred to as the Swift stability test. The fully automated version of this method is available as Rancimat apparatus (Metrohm Ltd, Herisau, Switzerland) and is accepted as a standard method by ISO (ISO6886) and American Oil Chemists’ Society (AOCS Cd 12b-92) (47-49, 73). Similarly, the Oxidative Stability Instrument (OSI, Omnion, Inc., Rockland, MA) uses a similar principle as AOM. This instrument is sensitive to the change of conductivity of water, which receives the air passed through the oxidizing lipid. OSI uses induction period or oxidative stability index as the measure of stability of the antioxidant-containing lipid, and it is accepted by the AOCS (73) [AOCS method Cd-126-92 (97)].

6.1.3 Oven Storage Test

The lipid with or without antioxidants is allowed to oxidize in an electrically heated convection oven (60–70°C). The oil is periodically assessed for change of its mass and its formation of primary oxidation products (hydroperoxides; peroxide value, conjugated dienes; conjugated diene value) or secondary products of oxidation (aldehydes; hexanal, dialdehydes, 2-thiobarbitiuric acid reactive substances; TBARS) or off-odor formation (50-52) . This method is commonly referred to as the Schaal oven method and is widely used for bulk oil substrates. Conditions provided in this process are suitable for a low degree of oxidation; thus, the results correlate well with actual shelf stability of the antioxidant-containing lipids.

6.1.4 Multiphase Systems

Antioxidant activity depends very much on the lipid substrate used for evaluation and the hydrophilic/lipophilic nature of the antioxidative compound. Solubility and partition properties of the compound in the medium affect the activity of antioxidants in the bulk lipid systems. As most foods cannot be related to bulk oil systems (e.g., meat, fish, eggs, mayonnaise, salad dressings, etc.), evaluation of antioxidants in multiphase systems is more relevant to their physical and chemical nature. Because of the very same reasons, several studies have found that compounds exhibiting strong activity against oxidation of lipids in bulk systems are often inefficient in colloidal and emulsion systems.

Three systems are generally used for such evaluations: emulsions (water-in-oil, oil-in-water), liposomes (uni- or multilamellar vesicles formed with aqueous phase and phospholipids), and micelles (emulsions formed with free fatty acids and aqueous phase). A lengthy discussion about how to use these multiphase systems in evaluating antioxidant activity is provided by Frankel (19).

Porter (74) has proposed a theory for distinguishing the effectiveness and behavior of antioxidants in bulk oils and emulsions and membranes. The “polar paradoxical behavior” (75, 76) describes the anomalous effect of antioxidants on lipids when they are in different physical systems. Work carried out by Frankel et al. (77) used the interfacial partitioning phenomenon to explain the reciprocal effect of antioxidants in bulk oil versus multiphase/colloidal systems. This phenomenon recognizes the discrete phases of the oxidative stability and antioxidative mechanism; however, exact details are not yet fully understood. The oxidative stability of food lipids varies according to their colloidal location because of the exposure of the lipid to the antioxidant/proxidant is different in such an environment. The partitioning of the antioxidant between the aqueous and nonaqueous phases depends on their solvent properties. Also, the partitioning of antioxidants into the nonaqueous phase can exert an important effect on activity by protecting the lipid oxidizable substrate. Figure 7 explains probable interfacial distribution of antioxidative compounds based on their hydrophobicity and hydrophilicity in monophasic and bipahsic systems. Surfactants can improve the solubility of lipophilic antioxidants in the interface and exert a significant effect on activity (77, 78). Thus, it is important to use several methods to measure different products of oxidation under various conditions, including multiphase systems, especially in evaluating natural compounds as antioxidants in foods or biological systems (46).

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Figure 7. A schematic of probable distribution of hydrophilic and hydrophobic antioxidants in bulk oil (oil–air interface) and oil-in-water emulsion interface [adapted from (46)].

6.2 Methods Based on Radical Scavenging Ability

Several methods have been described and used based on the fact that antioxidants are radical scavengers in aqueous and lipid phases. The radicals employed in these methods do not necessarily originate from lipid oxidation. In general, two approaches are used in these methods. One involves the generation of a free radical species and direct measurement of its inhibition caused by the test compound; such methods do not require an actual substrate to be oxidized. The other approach uses assay systems that involve oxidation of a substrate that is coupled with generated free radicals, which is actually an indirect measurement. The ability of a compound to inactivate radicals that are generated in a model system is extrapolated to its potential as an antioxidant in lipid and lipid-containing foods. These methods are widely and effectively used as screening and comparison tests in search for naturally occurring antioxidants. They are simple and easy to use but should be carefully interpreted. An extensive discussion of the methods of antioxidant activity determination that are based on free radical scavenging is found in the review by Antolovich et al. (47).

6.2.1 DPPH Free Radical

Blois (53) showed that α,α-diphenyl-β-picrylhydrazyl radical (DPPH•) can be used for determining antioxidant activity of ascorbic acid, tocopherol, and quinones (Figure 8a and b). DPPH in ethanol shows a strong absorption band at 517 nm (independent of pH from 5.0 to 6.5), and the solution appears to be deep violet in color. As the DPPH radical is scavenged by the donated hydrogen from the antioxidant, the absorbance is diminished according to the stoichiometry.

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Figure 8. Proposed reaction between α,α-diphenyl-β-picrylhydroxyl (DPPH) radical and Oxidizable groups. (a) oxidation of conjugated group of ascorbic acid to the dehydro form and (b) oxidation of hydroquinone [adapted from (53)].

As DPPH radical is paramagnetic and can become a stable diamagnetic molecule by accepting an electron or hydrogen radical, it can exhibit a change of its spin resonance using electron paramagnetic resonance (EPR). If the compound in question is able to scavenge DPPH radicals, the EPR signal of DPPH is attenuated (Figure 9a), and this can be quantified by integration into an appropriate calculation program. Use of DPPH radical in combination with monitoring EPR signal has been commonly used to assess natural antioxidants (54, 79-81). Although DPPH is a comparatively stable free radical at room temperature, it is not water soluble and the reaction mechanism between the antioxidant and DPPH radical depends on the structural conformation of the antioxidant (82).

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Figure 9. Electron paramagnetic resonance of (a) α,α-diphenyl-β-picrylhydroxyl (DPPH) radical and (b) hydroxyl radical as spin adduct of DMPO-OH. (A test solution composed of 50 μl of 1:10 (v/v) diluted methanolic extract of Elusine coracana was added as a radical quencher, and splitting constant of N and H was 14.791 G.) [From (79), with permission.]

6.2.2 Oxygen Radicals

The oxygen radical absorbing capacity (ORAC) method (66, 67) is developed based on the ability of antioxidant compounds to scavenge oxygen (e.g., peroxy) radicals. The peroxy free radical generated using 2,2′- azobis(2-amidinopropane) dihydrochloride (AAPH; as the generator) in a buffered system is targeted to damage β-phycoerythrin (β-PE, a phycobilliprotein containing a red photoreceptor pigment) molecule. The fluorescent signal of β-PE is recorded and interpreted as ORAC (as micromole Trolox equivalents per weight of material). An automated system of ORAC coupled with chromatographic systems is available for measuring total antioxidant capacity of natural products (67).

6.2.3 Hydroxyl Radicals

Hydroxyl radical generated from Fenton reaction (Equation 17) in a buffered system can be used to evaluate hydroxyl radical scavenging ability of an antioxidant. Shi et al. (59) have shown that EPR may be used to assess the hydroxyl radical scavenging ability of compounds when an appropriate spin trapping agent (i.e., 5,5-dimethyl-1-pyrroline-N-oxide; DMPO) is used (see Figure 9b).

Halliwell et al. (55) have described a model that uses hydroxyl radicals generated from Fenton reaction to degrade 2-deoxy-D-ribose. The decomposed products of deoxyribose are 2-thiobarbituric acid-reactive substances (TBARS). If the antioxidant present in the system scavenges hydroxyl radicals generated, deoxyribose is protected and the amount of TBARS produced is less.

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Scheme 7. Hydroxyl radical generation via Fenton reaction.

6.2.4 Superoxide Radical

The scavenging ability of antioxidants for superoxide radical anions generated in a model reaction is also employed to assess antioxidant activity. Superoxide radical anions can be generated in vitro by enzymatic (Equation 18) or nonenzymatic reactions using a xanthine–xanthine oxidase system, which requires oxygen for the reaction to generate superoxide radicals. Quantification of superoxide radicals can be achieved using chemiluminescence (58) with an appropriate agent (e.g., lucigenin) or by using the oxygen consumption kinetics (83). The nonenzymic system uses reduction of tetrazolium salt induced by superoxide radicals generated in a phenzine methosulfate/NADH mixture (56, 57). Meyer et al. (84) showed that scavenging of superoxide anion radical is not necessarily effective in preventing lipid oxidation by phenolic compounds in natural extracts. Also, no equilibrium can be achieved when superoxide radicals are generated continuously during the test, which is seen as a shortcoming when using O2•−-scavenging ability to assess antioxidant activity.

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Scheme 8. Generation of superoxide radical by an enzymatic reaction.

7 Commonly Used Antioxidants in Foods

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References

This discussion is carried out based on the origin of the antioxidative compound: synthetic (manufactured chemical molecules) and natural (originated from food related material), which is widely used by the food industry. Although compounds such as α-tocopherol and D-ascorbic acid are synthesized, they are considered as naturally existing compounds; thus, they are considered as “natural” and are discussed under natural antioxidants.

7.1 Synthetic Antioxidants

Synthetic antioxidants are manmade and are used to stabilize fats, oils, and lipid-containing foods and are mostly phenolic-based. Many compounds are active as antioxidants, but only a few are incorporated into food because of strict safety regulations. These phenolic derivatives usually contain more than one hydroxyl or methoxy group. Ethoxyquin is the only heterocyclic, N-containing compound that is allowed for use in animal feeds.

Synthetic phenolic antioxidants are p-substituted, whereas the natural phenolic compounds are mostly o-substituted. The p-substituted substances are preferred because of their lower toxicity. The m-substituted compounds are inactive. Synthetic phenolic antioxidants are always substituted with alkyl groups to improve their solubility in fats and oils and to reduce their toxicity (24, 85, 86). The primary mechanism of activity of these antioxidants is similar to those of primary antioxidants. An antioxidant molecule reacts with a peroxy radical produced by the oxidizing lipid, thus forming a hydroperoxide molecule and an antioxidant free radical. A similar path of reaction may occur with the alkoxy free radicals formed during the decomposition of hydroperoxides. The antioxidant free radical so formed may be deactivated by a lipid peroxy or an alkoxy radical or with another antioxidant radical. Dimers and even trimers of antioxidant molecules are formed because of the reaction of antioxidant radicals, and these may have a modest antioxidant activity of their own. With the help of synergists such as ascorbic acid, some of these original antioxidant molecules may be regenerated. Quinones are formed from phenolic antioxidants by reaction with peroxy radicals. When antioxidants are present in excess, the reaction of antioxidant free radicals with oxygen may become important; even their reaction with polyunsaturated fatty acids has some impact on the course of oxidation. Therefore, at high concentrations, phenolic antioxidants may act as pro-oxidants.

In most countries, use of synthetic antioxidants is regulated and the safety of the compounds involved has been tested based on long-term toxicity studies. The ability of an antioxidant to withstand thermal treatment (e.g., frying or baking) and to retain sufficient stabilizing activity for the food (fried or baked) is termed as “carry through property.” Table 6 provides a summary of physical properties of commonly used synthetic antioxidants. Several researchers have studied the effectiveness of these compounds in suppressing lipid oxidation in fats and oil, and Tables 7 and 8 provide comparative effects of synthetic antioxidants (82).

Table 6. Physical Properties of Synthetic Antioxidants Used in Foods 18, 88, 89
   Gallates 
Property/CharacteristicBHABHTDodecylPropylTBHQ
AppearanceWaxy solidWhite crystalsWhite crystalsWhite crystalsWhite-tan crystals
Carry through propertiesVery goodFair–GoodFair–GoodPoorGood
Boiling point (°C)264–270265Decompose above 148300
Melting point (°C)50–5269–70146–148146–148126–128
Solubility (%, w/w) in
 Corn oil3040005–10
 Glycerol1025<1
 Lard30–405015–10
 Methyl linoleatevery solublevery soluble11>10
 Propylene glycol5006.5430
 Water00<1<1<1
SynergismBHT & gallatesBHABHABHA
Table 7. Effect of Different Antioxidants on Oxidation of Soybean Oil [Adapted from 90]
 Antioxidant Activity (as time in hours taken to reach peroxide value of 70)
Antioxidant (at 0.02% level)45°Ca98°Cb
  • a

    Oxidation at 45°C as a thin layer of oil.

  • b

    Oxidation at 98°C at AOM conditions.

Control (no added antioxidant)168 5
Ascorbyl acid28843
Ascorbyl palmitate45613
BHA216 9
BHT24011
PG36014
TBHQ54428
Table 8. Effect of Antioxidants and Metal Inactivators on the Oxidation of Soybean Oil [Adapted from 87]
 Antioxidant Activity
Antioxidant or Combination(hours based on peroxide value of soy bean oil)
  • a

    Naturally present mixed tocopherol.

  • b

    Carbon black treated oil to remove natural tocopherol partially.

  • c

    Antioxidant concentration is 0.57 mmol/kg.

Controla (contain 1500 ppm tocopherol)26
C-treatedb (contain 45 ppm tocopherol)17
C-treated + ascorbic acidc43
C-treated + BHAc18
C-treated + BHA + 0.01% citric acidc56
C-treated + BHTc23
C-treated + BHT + 0.01% citric acidc53
C-treated + citric acidc50
C-treated + propyl gallatec26
C-treated + α-tocopherolc21
C-treated + α-tocopherol + 0.01% citric acidc53
7.1.1 Butylated Hydroxyanisole (BHA)

This monophenolic compound exists as a mixture of two isomers (Figure 4), 3-tertiary-butyl-4-hydroxyanisole (90%) and 2-tertiary-butyl-4-hydroxyanisole (10%). The 3-isomer shows a higher antioxidant activity than the 2-isomer. BHA is commercially available as a white, waxy flakes that is lipid soluble. BHA exhibits good antioxidant activity in animal fats as compared to vegetable oils. It has good carry through properties but is volatile at frying temperatures. When BHA is included into packaging materials it easily migrates to the containing food and delays lipid oxidation (18, 74, 91).

7.1.2 Butylated Hydroxytoluene (BHT)

BHT is also a monohydroxyphenol (Figure 4) and is widely used in foods. This fat-soluble antioxidant is available as a white crystalline compound. BHA is less stable than BHT at high temperatures and has lower carry-through properties. BHA and BHT act synergistically, and several commercial antioxidant formulations contain both of these antioxidants. BHT is effectively used in oxidation retardation of animal fats. It is postulated that BHA interacts with peroxy radicals to produce a BHA phenoxy radical. This BHA phenoxy radical may abstract a hydrogen atom from the hydroxyl group of BHT. BHA is regenerated by the H radical provided by BHT. The BHT radicals so formed can react with a peroxy radical and act as a chain terminator (92, 53).

7.1.3 Ethoxyquin

Ethoxyquin, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (Figure 4), is used as an antioxidant in animal feeds primarily to protect carotenoid oxidation. It may also be used in fish products, fish oil, poultry fats, potatoes, apples, and pears during storage and especially to protect pigment oxidation in ground chili and paprika (93). Ethoxyquin may act as a free radical terminater. Dimerization of the radical may inactivate the antioxidant (94).

7.1.4 Gallates

Esters of gallic acid (Figure 4), namely, n-propyl, n-octyl, and n-dodecyl gallates, are approved antioxidants for food use. Propyl gallate (PG), the most commonly used gallate, is slightly water soluble and is available as a white crystalline powder. PG is not suitable for use in frying oils because it is volatile at the high temperatures of frying (18, 92). Octyl and dodecyl gallates are more lipid soluble and heat stable and have better carry-through properties. Gallates can chelate metal ions effectively, thus retarding lipid oxidation catalyzed by metal ions. However, this may negatively effect the esthetic appeal of the food, because of the dark color of the metal-gallate complexes. Therefore, gallate formulations are always available with a metal chelator such as citric acid to prevent any discoloration in the incorporated food. Gallates show synergistic activity with both primary and some of the secondary antioxidants (94). Propyl gallate works well with BHA and BHT because of a synergistic action, however, its use together with TBHQ is not permitted in the United States (25).

7.1.5 Tertiary-Butylhydroquinone (TBHQ)

This is a diphenolic antioxidant (Figure 4) and is widely used in a variety of fats and oils. TBHQ has excellent carry-through properties and is a very effective antioxidant for use in frying oils. It is available as a beige color powder that is used alone or in combination with BHA or BHT. TBHQ can be used in a variety of lipid-containing foods and fats and oils. Chelating agents such as monoacyglycerols and citrates enhance the activity of TBHQ, mainly in vegetable oils and shortenings.

TBHQ reacts with peroxy radicals to form a semiquinone resonance hybrid. The semiquinone radical intermediate may undergo different reactions to form more stable products; they can react with one another to form dimers, dismutate, and regenerate as semiquinones; and they can react with another peroxy radical (Figure 5; 18, 24). A possible mutagenic effect of TBHQ has been the subject of extensive studies, and few countries in the world including Japan and the European Union countries do not yet approve its use in foods. Since 1999, TBHQ is included in the class IV preservative list in Canada, with a maximum usage level of 0.02% (Canada Food and Drug Act).

7.2 Natural Antioxidants

Use of plant parts (bark, leaves, seeds, etc.) and their extracts to preserve food from developing a rancid taste is a practice that has continued since prehistoric time. There is evidence that even for the industrial materials, plant-based components were used as antidrying agents to prevent oxidation and polymerization of polyunsaturated fatty acid-rich plant oils (2, 5, 48). During the past two decades, intensive research has been carried out on naturally occurring antioxidative compounds from different sources. The main drive behind this search was to reduce the use of synthetic compounds as food additives because of their potential negative health effects and as a result of consumer demand.

Plant-based components have increasingly been advocated as “safe and natural” antioxidants considering their existence in regular foods that are consumed. Much of the interest on naturally occurring antioxidants is developed because of the trend to minimize or avoid the use of synthetic food additives. Continuous effort in searching for naturally occurring antioxidative compounds during the past 20 years has helped to develop efficient models for activity screening, structure function relationship assessment, categorizing sources of antioxidant groups, developing methods of isolating purified antioxidative compounds from natural sources, and developing branded foods (e.g., claims for marketing purpose). Again, one has to keep in mind that the “safe it is natural” is based on the fact that these secondary metabolites are present in small concentrations in regular foods. If these are to be added to foods that are largely consumed, they should undergo all safety clearances. There are many naturally occuring compounds that act as antioxidants in fats and lipid-containing foods. Among these, only a few are currently approved and employed in foods. Groups of compounds that are found naturally and exhibit strong antioxidant activity are discussed here.

7.2.1 Ascorbic Acid and Ascorbic Acid Esters and Salts

Vitamin C or ascorbic acid is widespread in nature but sparingly associated with fats of oils because of its hydrophilic nature (95). Ascorbic acid in the free form, salts of sodium and calcium, and esters of stearic and palmitic are commonly used as antioxidants in foods. Erythorbic acid is the D-isomer of naturally present L-ascorbic acid (Figure 10) and is often used as an antioxidant in dried fruits and a cure accelerator in cured meat. Unlike ascorbic acid, erythorbic acid is not a natural constituent of foods and has minimal vitamin C activity. Similar to ascorbic acid, erythorbic acid is highly water-soluble but remains insoluble in oils (97).

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Figure 10. Chemical structures of L-ascorbic acid, erythorbic acid, and ascorbyl palmitate.

In foods, water-soluble ascorbic acid acts as a secondary antioxidant and participates in various antioxidative and related functions. Ascorbic acid is capable of quenching various forms of oxygen (singlet oxygen, hydroxyl radicals, and superoxide). When ascorbic acid acts as a hydrogen donor, ascorbyl radical so produced may reduce or terminate radical reactions; hydroperoxides may then be converted into stable products. Ascorbic acid can reduce primary antioxidant radicals and thus act as a synergist. A very good example is donating a hydrogen atom to tocopheryl radical and then regeneration of tocopherol (Figure 11), which is commonly observed in the biological systems. In addition to that, ascorbic acid can shift the redox potential of food systems to the reducing range and can act synergistically with chelators and regenerate primary antioxidants other than tocopherols (98).

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Figure 11. Regeneration path of tocopherol by ascorbic acid and during participation in radical scavenging in biological systems [adapted from (46)].

In vivo ascorbic acid acts as a primary antioxidant and in tissues it is essential for the prevention of oxidative cellular damage by hydrogen peroxide (99). In a solution, ascorbic acid readily oxidizes to dehydroascorbic acid. This formation occurs through one- or two-electron transfer that is due to its enediol structure. It is a reductone and has a very high affinity for oxygen. The 2- and 3-positions of ascorbic acid are unsubstituted. Oxidation happens via the intermediate semidehydroascorbic acid or monodehydroascorbic acid or ascorbate free radical. The semidehydroascorbic acid is either reduced to give ascorbic acid again or oxidized to give dehydroascorbic acid. In nature, these compounds complete a redox system (Figure 12). The redox cycle is completed in living tissues by enzymatic reduction of dehydroascorbic acid to ascorbic acid. Seib (101) has reviewed the oxidation and other reactions of ascorbic acid.

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Figure 12. Ascorbic acid-dehydroascorbic acid redox system (a) oxidation of ascorbate to semidehydroascorbic acid, (b) disproportionation of semidehydroascorbic acid, and (c) reduction of dehydroascorbic acid [From (100), with permission].

Ascorbyl palmitate and ascorbyl stearate are synthetic derivatives of ascorbic acid. Ascorbic palmitate is soluble in lipid-containing foods because of its relatively good hydrophobicity (88). Ascorbyl palmitate is hydrolyzed by the digestive system to provide nutritionally available ascorbic acid and palmitic acid, but health claims cannot be made for its vitamin C contribution.

As an antioxidant, ascorbic acid is very attractive as it carries GRAS (generally recognized as safe) status with no usage limits; it is a natural or nature-identical product and is highly recognized as an antioxidant among the nutrient category. Ascorbic acid is also used as an acidulant and flavorant. Heat treatment makes ascorbic acid unstable, and it participates in nonenzymic browning reactions and degrades through reductone formation. Ascorbic acid and its salts (Na- and Ca-ascorbate) are water soluble and are not applicable as antioxidants in oils and fats. These salts are used extensively for stabilizing beverages that contain oxidizable substrates. In fats, ascorbyl palmitate is used mostly because of its superior solubility. Ascorbyl palmitate also has GRAS status, and there are no restrictions for its usage level.

7.2.2 Carotenoids

Carotenoids are ubiquitously found lipid-soluble-colored compounds, mainly from green plants, fruits, and vegetables. The two classes of carotenoids, carotenes and xanthophylls, are composed of 40-carbon isoprenoid or tetraterpenes with varying structural characteristics. Carotenes are polyene hydrocarbons and vary in their degree of unsaturation (e.g., β-carotene, lycopene; Figure 13). Xanthophylls are derived from carotenes by hydroxylation and epoxidation, thus containing oxygen (e.g., astaxanthene, canthaxanthin). Some carotenoids exhibit biological activity of vitamin A and hence are categorized as provitamin A. β-Carotene is the most abundantly found provitamin A. Many fats and oils, especially those from plant sources, contain β-carotene, and it contributes to the deep intense orange red color of many oils (94, 102).

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Figure 13. Chemical structures of some carotenoids.

Carotenoids can act as primary antioxidants by trapping free radicals or as secondary antioxidants by quenching singlet oxygen. In foods, carotenoids usually act as a secondary antioxidant; however, at low oxygen partial pressure (<150 mm Hg, in the absence of singlet oxygen), carotenoids may trap free radicals and act as a chain-breaking antioxidant (95, 104). At high oxygen concentrations, the antioxidant activity of β-carotene is diminished. Increased oxygen concentration leads to the formation of carotenoid peroxy radicals because the conjugated double bonds of carotenoid molecules are very susceptible to the attack by peroxide radicals. This reaction favors autoxidation of β-carotene over inactivation of lipid peroxy radicals (103). At low oxygen concentrations, the lifetime of the carotenoid radical is long enough to permit its reaction with another peroxy radical and to form nonradical species. The unsaturated structure of β-carotene allows the molecule to delocalize electrons in the radical and to produce a resonance-stabilized product with the peroxy radical. This carotene radical participates in termination reactions and converts peroxy radicals to less damaging products. Lieber (104) has provided details of antioxidative reactions of carotenoids. The combination of carotenoids and tocopherols results in synergistic action (105, 106). The stability of carotenoids is affected by oxygen, heat, pH, light, and metals; therefore, care should be taken in their handling as antioxidants.

Singlet oxygen, which is unstable, preferentially transfers energy to β-carotene to produce triplet state β-carotene. This occurs through an exchange electron transfer mechanism. Triplet state β-carotene releases energy in the form of heat, and the carotenoid is returned to its normal energy state. This mechanism allows the carotenoid molecule to be an effective quencher of numerous molecules of singlet oxygen (102). The ability of carotenoids to quench singlet oxygen is related to the number of carbon double bonds in their chemical structures. Carotenoids with nine or more conjugated double bonds are very effective antioxidants. Because of the presence of additional functional groups in the hydrocarbon structure, xanthophylls cannot perform as effectively as antioxidants (94).

7.2.3 Tocopherols and Tocotrienols

Tocopherols and tocotrienols are the natural antioxidative compounds found widely in different tissue, even if it is in trace amounts. Tocopherols are found abundantly in vegetable oil-derived foods. These monophenolic compounds possess varying antioxidant activities. Tocopherols and tocotrienols comprise the group of chromanol homologs that exert vitamin E activity in the diet. These different homologs vary in the extent of methylation of the chromane ring. The α-, β-, γ-, and δ-tocopherols contain a saturated phytyl (trimethyltridecyl) side chain (Figure 14). The corresponding tocotrienols have unsaturated phytyl chain at the 3-, 7-, and 11-positions. Only RRR isomers are found naturally. Synthetic α-tocopherol (all rac-α-tocopherol) is a combination of eight sterioisomers that are found in equal amounts in the mixture. Biologically, RRR-α-tocopherol is the most active vitamin E homolog. The antioxidant activity of α-, β-, γ-, and δ-tocopherols and tocotrienols decreases in the order of α > β > γ > δ in vivo (107, 108) and α > β > γ > α in bulk oils and fats (108). Vitamin E activity of tocopherols decreases in the order of α > β > γ > δ (107).

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Figure 14. Chemical structures of tocopherols and tocotrienols.

Antioxidant activity of tocopherols is mainly by scavenging peroxy radicals thus interrupting chain propagation (95), which is based on the tocopherol–tocopherylquinone redox system. The active configuration is the phenolic group in the benzene ring, located at the para position to the oxygen atom bound next to the dihydropyrone cycle. Alpha-tocopherol donates a hydrogen atom to a peroxy radical resulting in an α-tocopheryl semiquinone radical (Figure 15). This radical may further donate another hydrogen to produce methyltocopherylquinone or react with another tocopheryl semiquinone radical to produce an α-tocopherol dimer. The methyltocopherylquinone is unstable and will yield α-tocopherylquinone (100). The α-tocopheryl dimer continues to possess antioxidant activity. Also, two tocopheryl semiquinone radicals can form one tocopheryl quinone molecule and regenerate one tocopherol molecule. The decomposition products of tocopherols (during thermal oxidation) can slowly oxidize and release tocopherol that can act as an antioxidant (100).

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Figure 15. Possible mechanism of participation of α-tocopherol in free radical scavenging.

Commercially, tocopherol is available as a pure all-rac-α-tocopherol, mixed tocopherols having various contents of α-, β-, γ-, or δ-tocopherols (diluted in vegetable oil) and synergistic mixtures containing tocopherols, ascorbyl palmitate or other antioxidants, and synergists such as lecithin, citric acid, and carriers. Extraction of tocopherols from natural sources and chemical synthesis of tocopherols are well described by Schuler (100).

Tocopherols are considered as natural antioxidants for lipid-containing foods and marketed as “all natural.” They are permitted in food application according to GMP regulations (21CFR 182.3890). Natural tocopherols are limited to 0.03% (300 ppm) in animal fats (9 CFR 318.7). As most vegetable oils naturally contain tocopherols, the addition of this antioxidant may pose prooxidant effects.

7.2.4 Other Phenolic Antioxidative Compounds from Plants

Higher plants are rich in a myriad of phenolic compounds in their secondary metabolite pool. Among these, phenolic acids and polyphenolic derivatives are found to be the most important series of hydrophilic–hydrophobic antioxidative compounds naturally present. In foods, these polyphenolic compounds act as radical scavengers or metal chelators, and some may play a multifunctional role. Numerous plants and their parts have been identified as sources of phenolic acids, flavonoids, and related compounds. Antioxidant activity of phenolic compounds from various plant sources has been reported in several peer-reviewed papers, reviews, and books. Interested readers are referred to reviews by Shahidi et al. (18, 109-112) about the sources of natural phenolics possessing antioxidative activity that are obtained from plants and have potential applications in oils and fats (as discused below).

7.2.4.1 Antioxidants from Cereals, Oilseeds, and Related Sources

Seeds rich in oils are also abundant sources of various types of antioxidative compounds. Among these carotenoids, phenolic acids, and their derivatives, flavonoids, phytic acid, lignans, and tocopherols are predominantly found depending on the plant genera and species. Reviews by Wanasundara et al. (110) and Shukla et al. (111) discuss antioxidants of oilseeds and their products in detail.

7.2.4.1.1 Phenolic Acids and Their Derivatives

Phenolic acids are found in plants and have the basic chemical structure of C6-C1 (benzoic acids) and C6-C3 (cinnamic acids) (Figure 16). A range of substituted benzoic acid or cinnamic acid derivatives comprise these two major families of phenolic acids that are found in plants. Both of these families occur as free, conjugated, or esterified form, and sometimes as depsides such as chlorogenic acid (3-caffeoyl-quinic acid). Phenolic acids serve as free radical acceptors and chain breakers. The presence of a phenolic ring in the molecular structure and side chains facilitates the radical accepting ability of phenolic acids. According to Chimi et al. (113) and Pokorný (114), monohydroxy phenolic acids are less efficient as antioxidants than polyhydroxy phenolic acids. The presence of the CH[DOUBLE BOND]CH[BOND]COOH group in cinnamic acids ensures a greater antioxidative activity than a [BOND]COOH group as in benzoic acids. The participation of the C[DOUBLE BOND]C bond is important in stabilizing the antioxidant radical by resonance.

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Figure 16. Chemical structures of antioxidative phenolic acids.

The antioxidant activity of phenolic acids and their esters depends on the number of hydroxyl groups in the molecule, and this would be strengthened by steric hindrance. Hydroxylated cinnamic acids are more effective antioxidants than their benzoic acid counterparts. When the acid group is esterified with a bulky group such as a sugar, the antioxidant potency of the molecule is further enhanced (115). Introduction of a second hydroxyl group in the ortho or para position increases the antioxidant activity of hydroxylated phenolic acids. Therefore, acids with ortho diphenolic groups (caffeic and protocatechuic acids) are more efficient antioxidants than their respective monophenolic acids (p-hydrobenzoic and p-coumaric acids). Gallic acid that has three hydroxyl groups is more active than proteocatechuic acid, but more than three hydroxyl groups in the structure does not appear to improve the antioxidant efficiency in oil systems (114). The aromatic ring substituted with two or three phenolic groups in the ortho position are particularly important; some hydroxyl groups may be methoxylated. Substitution of one or two methoxy groups at the ortho position relative to the hydroxyl group markedly increases the antioxidant activity of phenolic acids. Therefore, sinapic acid is a more efficient antioxidant than ferulic acid, which is more efficient than p-coumaric acid. For the same reason, syringic acid is more active than vanillic acid and p-hydroxybenzoic acid (115). Ortho substitution of the phenolic acid with electron donor alkyl or methoxy groups increases the stability of aryloxy radical and thus the antioxidant activity. Methoxy substitution strengthens the antioxidant activity than the addition of a hydroxyl group to the molecule (113, 114).

7.2.4.1.2 Lignans

Lignans are compounds with great chemical diversity and found in all parts of the plants. They are dimers of phenyl proponoid (C6-C3) units linked by the central carbons of their side chains (116). Among these, bisepoxy lignans and cyclolignans that occur in oilseeds (sesame and flax) exhibit strong antioxidative activity in aqueous and lipid media. Lignans of sesame (Sesamum indicum L.) seed include sesamin, sesamolin, sesaminol, and sesamol, which act as endogenous antioxidants for the oils (117-119). Sesamolin may undergo chemical changes during thermal treatment and under processing conditions (e.g., bleaching) and forms sesaminol and sesamolinol (120-122). High oxidative stability of sesame oil obtained from roasted seed may be largely attributed to the presence of lignan compounds (123-125).

Lignans of flaxseed exist as secoisolariciresinol diglucoside (SDG; 126). SDG is a potent antioxidant in biological systems because of its tendency to associate in the aqueous phase. Much of the work on antioxidant activity of SDG is related to its radical mediated disease prevention (127, 128). Lignans of both sesame (121, 122) and flax (129, 130) have shown hydrogen-donating ability and scavenging activity for various free radicals.

7.2.4.1.3 Sterols

Phytosterols are mostly associated with unrefined vegetable oils and exist as derivatives of phenolic acids (e.g., ferulic acid). Several studies are available on antioxidant activity of sterols and their derivatives from sources such as corn fiber, oats, and rice. These compounds can be obtained from the unsaponifiable fraction that is removed during vegetable oil refining. Triterpene alcohols and hydrocarbons (131), or sterols (Figure 20) from oats (132, 133), rice (134, 135), and corn fiber (135, 136), were able to exert the antioxidative effect on frying oils and as a result displayed antipolymerizing effects. It has been suggested that donation of a hydrogen atom from the allylic methyl group in the side chain of sterols followed by isomerization to a relatively stable tertiary allylic free radical may be the mechanism for sterol antioxidant activity (132). The ethylidine group (CH3[BOND]CH[DOUBLE BOND]) in the side chain of the sterol molecule seems essential for performing the antipolymerizing effect on the frying oils at high temperatures (137, 138). The γ-oryzanol of rice bran performed good antioxidant activity in a linoleic acid model system at 37°C as opposed to frying temperatures. γ-Oryzanol of rice is composed of at least ten compounds that are mainly ferulic acid derivatives of triterpene alcohols, stigmasterol, campesterol, sitosterol, and cycloartinol (Figure 20; 137). Plant sterol-based compounds are available as physiological antioxidants to prevent certain disease conditions. Commercial preparations of sterol–based natural antioxidants for high-temperature food applications are not abundantly available yet.

7.2.4.2 Antioxidants from Labiatae Herbs

Among phenolic diterpenes, carnosic acid and carnosine are the very active antioxidants that are commercially available as natural antioxidants for lipid-containing foods. Chemically, carnosic acid has a structure consisting of three six-membered rings, including a dihydric polyphenolic ring and a free carboxylic acid, and carnosol is a derivative of carnosic acid containing a lactone ring (Figure 17). These active antioxidants are found especially in plants of the Labiatae family (oregano, rosemary, sage, and thyme). Commercial extracts of these plants are produced by organic solvent extraction of the plant parts and subsequent deodorization and bleaching. Commercially these antioxidants are available as powder, paste, or liquid and formulations in propylene glycol, medium-chain triacylglycerols, or vegetable oils. Schuler (100) has discussed preparation of rosemary antioxidants and their effectiveness in oils compared with other antioxidants.

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Figure 17. Chemical structures of carnosine, canosic acid, and rosemarinic acid.

Extensive studies (139, 140) on rosemary extracts containing carnosol, carnosic acid, and rosmarinic acid have shown that the activities of these natural antioxidants are system-dependent and that their effectiveness in different food systems is difficult to predict. In bulk vegetable oils (corn, soybean and peanut) and fish oils, carnosol and carnosic acid are effective antioxidants. It has been hypothesized that this behavior of rosemary antioxidants is attributed to the partitioning differences in the biphasic system. These hydrophilic antioxidants are oriented in the oil–air interface and protect the bulk oil phase from oxidation. However, in oil-in-water emulsions, they are less effective in the oxidation of the oil–water interface, where most of the oxidation reactions take place (see illustration in Figure 7). It has been hypothesized that the hydrophilic antioxidants (e.g., rosmarininc acid, gallic acid, catechins, propyl gallate) partitioned more (>90%) into the aqueous phase, thus allowing a considerably low concentration of antioxidants in the oil phase, causing less antioxidative protection (141). Carnosic acid and carnosol are oxidized during oxidation at 60°C and higher temperatures; however, antioxidant activities are maintained; apparently the oxidation products are active antioxidants at high temperatures. These compounds have good carry-through properties and protect frying oils and fried foods.

7.2.4.3 Flavonoids from Green Tea

Among the natural flavonoids studied as antioxidants for lipid-containing foods, polyphenolic catechins from green tea (Camellia sinensis L) have been extensively scrutinized (142, 143). Extracts of immature leaves of the plant (green tea) are rich in flavan-3-ols and their gallic acid derivatives, namely, (+)-catechin, (−)-epicatechin, (+)-gallocatechin, (−)-epicatechin gallate, (−)-epigallocatechin, and epigallocatechin gallate (Figure 18).

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Figure 18. Chemical structures of flavonoids according to their hydroxylation.

Flavonoids are a heterogeneous group of phenolic compounds having a benzo-γ-pyrone structure (Figure 18) in the molecule and occur ubiquitously in plants. Approximately 90% of flavonoids occur in plants in the glycosidic form (143, 144). Antioxidant activity of flavonoids is bimodal, and they are very effective in counteracting lipid oxidation; however, this is very much dependent on the chemical and physical properties of the system. Flavonoids function as primary antioxidants in systems when metal catalyzed oxidation is not present. Because of their lower redox potentials (230<E0<750 mV), flavonoids (145) are thermodynamically able to reduce highly oxidizing free radicals with redox potential in the range of 2310–1000 mV, such as alkoxy, hydroxyl, peroxy, and superoxide (See Table 1, 21) radicals by hydrogen donation. Flavonids can form resonance-stabilized radicals while scavenging oxidative free radicals (18). For a molecule that has 3′, 4′-dihydroxylation donation of one H atom to a free radical may produce a flavonoid aryloxy radical. This flavonoid aryloxy radical may react with a second radical and acquire a stable quinone structure (Figure 19a; 145, 146). At the same time, flavonoids are good metal chelators that can be used for inhibition of metal-catalyzed oxidation initiation. Metal chelation ability of flavonoids is caused by the ortho-diphenol structure in rings A and B (3-hydroxy-4-keto group or the 5-hydroxy-4-keto) and ketol structure in ring C (Figure 19b). An ortho quinol group at the B ring has also demonstrated metal ion chelating activity (18, 147).

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Figure 19. (a) Free radical scavenging by flavonoids. (b) Binding sites of flavonoids for metal ions.

The position and the degree of hydroxylation (especially at the A and B rings) are of primary importance in determining the antioxidant activity of flavonoids. Dihydroxylation at ortho position of the B ring contributes to antioxidant activity; however, para and meta hydroxylation of the B ring do not occur naturally. All flavonoids with 3′,4′-dihydroxy configuration possess antioxidant activity (18, 147). Other important features include carbonyl group at position 4 and a free hydroxyl group at position 3 or 5 (91). Among aglycones, the presence of a free 3-hydroxyl group in the C ring is a requirement for maximal radical scavenging activity of flavonoids (148, 149). When a disaccharide is glycosylated to a flavonoid (e.g., rutin), the substituent at position 3 becomes a poorer leaving group; thus, the molecule becomes less oxidizable and exhibits a lower antioxidant activity in free fatty acid systems than monosaccharide glycosides (e.g., quercetin; 149). Many of the flavonoids and related substances display a significant antioxidant behavior in lipid-aqueous and lipid–lipid food systems (147). Most of these compounds have very low solubility in the lipid phase, and it is a serious disadvantage if the aqueous phase is present to a in considerable extent in the food.

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Figure 20. Chemical structure of antioxidative sterols identified from (a) oats, and (b) rice and corn fiber.

The antioxidant activity of these catechins and the derivatives showed a marked difference depending on the substrate used for evaluation. In bulk corn oil that was oxidized at 50°C epigallocatechin, epigallocatechin gallate and epicatechin gallate exhibited better antioxidant activity than epicatechin or catechin. These catechins have been very effective in retarding oxidation of polyunsturated fatty acids-rich vegetable, and marine oils (150-152). In the oil-in-water emulsions, all catechins tested were pro-oxidants; however, in the liposomes comprising lecithin, epigallocatechin gallate was the best antioxidant, followed by epicatechin, epigallocatechin, epicatechin gallate, and catechin (78, 152). When tea catechins were added to noodles and to the frying oils, they were able to improve the oxidative stability of the fried product and the oil used for frying (153). In addition to that, tea polyphenols exhibited protecting ability against β-carotene oxidation; i.e., tea catechins were able to exert an antidiscoloring effect on beverages containing β-carotene that were UV-light irradiated (153).

The antioxidant activity of individual tea polyphenols in different model assays showed a proportional relationship to the number of hydrogen radical donors of catechins. A synergistic effect was observed between tea catechins and caffeine, ascorbic, citric, malic, and tartaric acids and tocopherols (153). Formation of oxidation products of (+)-catechin during the antioxidative process has been observed in oxidation model studies. According to the proposed mechanism, (+)-catechin can scavenge four radicals per molecule (154, 155). Yamamoto et al. (156) have summarized the chemistry and application aspects of green tea, especially in relation to using their catechins.

8 Estimation and Analysis of Antioxidants in Foods

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References

Qualitative and quantitative detection of antioxidants and potential antioxidative compounds is of utmost importance to researchers and industry and regulatory agencies. Numerous methods based on colorimetry, spectrometry, fluorometry, voltammetry, polarography, thin-layer chromatography, paper chromatography, gel permeation chromatography, gas chromatography, and high-performance liquid chromatography have been described for both natural and sysnthetic compounds for their antioxidant activity in foods (157). Almost all of these procedures require considerable sample preparation and estimation of individual antioxidants. A considerable number of procedures have been developed and tested in collaborative studies for determination of commonly used antioxidants, which are strictly regulated for their use. Kochhar and Rossell (157) have provided an elaborative discussion about the methods used to determine BHA, BHT, gallates, tocopherols, and TBHQ. Official methods have been developed to determine phenolic antioxidants under the category of food additives (AOAC method 983.15; 158).

9 Technological Considerations in Using Antioxidants

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References

The type of food to which antioxidants may be added is variable and ranges from baked goods, biscuits, chewing gum, dry snacks, fruit drinks, mayonnaise, meat products, nuts, and oils and fats, among others. For food applications, the antioxidants must be effective at low concentrations (below 0.02%, w/w) because at high concentrations, they may act as pro-oxidants. The antioxidants should also be nontoxic.

Usually the antioxidant is directly added to the food as a concentrate in lipid/oil, dissolved in a food grade solvent, or in an emulsified form that may be sprayed onto the food product. Antioxidants must be thoroughly blended with the lipid to obtain their maximum potency. To be effective, antioxidant(s) should partition between oil–air interfaces in bulk oil systems or between oil–water interfaces in the emulsion systems. Antioxidants must be added, as soon as possible, to the fresh product as they cannot reverse any oxidation reactions that have already occurred (157). Metal deactivators such as citric acid are added to vegetable oils after heating, during the cooling stage of deodorization step of vegetable oils (19). An effective antioxidant should be stable under processing conditions, especially at high temperatures, and possess a good “carry through property.”

Antioxidants that are added to fats and oils are usually in the form of liquid formulations. The major considerations of devising antioxidant formulations include the following:

  1. Antioxidants with different degrees of potency are formulated in an antioxidant combination/mixture.

  2. Better control and accuracy in applying of antioxidants should be achieved with the mixture or formulation.

  3. Ability to use synergistic effect of antioxidants to enhance their activity.

  4. Complete distribution or solvation of antioxidants in fats and oils.

  5. Prevent or minimize discoloration associated with specific antioxidants.

  6. Ease of handling.

Most synthetic antioxidants are formulated with polypropylene glycol, glyceryl monooleate, mono- and diacylglycerols, or vegetable oils as carriers to enhance their solubility or dispersibility in foods. Several synergistic mixtures are available commercially, especially citric acid with synthetic antioxidants. Commercial preparations of natural antioxidants are predominantly tocopherol- and ascorbic acid ester-based. Few formulations are available with rosemary, sage, and tea chatechin-based antioxidative ingredients. In these formulations, vegetable oils and starches are used as carriers and citric acid is also included. Elliot (159) has extensively discussed the technological considerations when using ascorbic acid, β-carotene, and tocopherols as antioxidants in lipid-containing foods. These antioxidants should be handled differently than the synthetic antioxidants because of their reactive nature.

10 Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References

All synthetic antioxidants are generally categorized under direct food additives. They are subjected to careful scrutiny and complex toxicological studies for approval. However, the usage and approval of an antioxidant may differ from one country to another. Many countries have adopted regulations similar to the United States regarding the usage of these antioxidants; however, significant differences exist among different countries on their type, application, and usage levels. Table 9 provides a summary of regulations governing the use of synthetic antioxidants in Canada and the United States.

Table 9. Regulations Governing the Use of Synthetic Antioxidants in Canada and the United States
 CanadaaUnited Statesb
CompoundItem Number and Permitted in or uponMaximum level of useCitation and Permitted inMaximum level of use
  • a

    From Canada Food and Drugs Act.

  • b

    From U.S. Code of Federal Regulations.

Ascorbic acidClass IV, A.1 21 CFR 182.3013GRAS with GMP
 In fats and oils, monoglycerides and diglyceride, shortenings, Unstandardized foodsGMP  
Ascorbyl palmitateClass IV, A.2 21 CFR 182.3149GRAS with GMP
 In fats and oils, lard, monoglycerides and diglyceride, ShorteningsGMP  
 Unstandardized foods except meat and meat byproducts, fish, poultry meat and its byproductsGMP  
 MargarineNot to exceed 0.02% of the fat content, alone or in combination with ascorbyl stearate  
Ascorbic stearateClass IV, A.3   
 In fats and oils, monoglycerides and diglyceride, ShorteningsGMP  
 MargarineNot to exceed 0.02% of the fat content, alone or in combination with ascorbyl palmitate  
BHAClass IV, B.1 21 CFR 172.110Alone or in combination with BHT
 In fats and oils, shortenings, margarineNot to exceed 0.02%, alone or in combination with BHT, PG or TBHQDehydrated potato shreds50 ppm
 Dried breakfast cereals, dehydrated potato productsNot to exceed 0.005%, alone or in combination with BHT or PGActive dry yeast1,000 ppm
 Chewing gumNot to exceed 0.02%, alone or in combination with BHT or PGBeverages and desserts prepared from dry mixes2 ppm
 Essential oils, citrus oil flavors, dry flavoursNot to exceed 0.125%, alone or in combination with BHT or PGChewing gum base1,000 ppm
 Citrus oilsNot to exceed 0.5%, alone or in combination with BHT or PGDry breakfast cereals50 ppm
 Partially defatted pork or beef fatty tissuesNot to exceed 0.065%, alone or in combination with BHTDry diced glazed fruit32 ppm
 Vitamin A liquids for foods5 mg/1,000,000 international unitsDry mixes for beverages and desserts90 ppm
 Dry beverage mixes, dry dessert and confection mixes0.009%Edible fats and oils excluding butterfat and margarine200 ppm
 Active dry yeast0.1%Emulsion stabilizers for shortenings200 ppm
 Unstandardized foods except preparations of meat and meat by products, fish, poultry meat and its by productsNot to exceed 0.02% of total fat content of food, alone or in combination with BHT or PGEssential oils1,000 ppm
 Dry vitamin D preparations for food10 mg/1,000,000 international unitsMargarine200 ppm
 MargarineNot to exceed 0.01% of the fat content, alone or in combination with BHT or PG or bothPotato flakes50 ppm
 Dried cooked poultry meat0.015% of the fat content alone or in combination with PG or citric acid or bothPotato granules10 ppm
  Sweet potato flakes 50 ppm
BHTClass IV, B.2 21 CFR 172.115Alone or in combination with BHA
 Fats and oils, lard, shorteningNot to exceed 0.02%, alone or in combination with BHA, PG or TBHQDehydrated potato shreds50 ppm
 Dried breakfast cereals, dehydrated potato productsNot to exceed 0.005%, alone or in combination with BHA or PGDry breakfast cereals50 ppm
 Chewing gumNot to exceed 0.02%, alone or in combination with BHA or PGChewing gum base1,000 ppm
 Essential oils, Citrus oil flavors, Dry flavoursNot to exceed 0.125%, alone or in combination with BHA or PGEdible fats and oils excluding butter fat and margarine200 ppm
 Citrus oilsNot to exceed 0.5%, alone or in combination with BHA or PGEmulsion stabilizers for shortenings200 ppm
   Essential oils1,000 ppm
 Partially defatted pork or beef fatty tissuesNot to exceed 0.065%, alone or in combination with BHAMargarine200 ppm
 Vitamin A liquids for foods5 mg/1,000,000 international unitsPotato flakes50 ppm
 Parboiled rice0.0035%Potato granules10 ppm
 Unstandardized foods except preparations of meat and meat byproducts, fish, poultry meat and its byproductsNot to exceed 0.02% of total fat content of food, alone or in combination with BHA or PGSweet potato flakes50 ppm
 Dry vitamin D preparations for food10 mg/1,000,000 international units  
 MargarineNot to exceed 0.01% of the fat content, alone or in combination with BHA or PG or both  
Citric acidClass IV, C.3   
 In fats and oils, monoglycerides and diglyceride, shortenings,GMP  
 Unstandardized foods except preparations of meat and meat byproducts, fish, poultry meat and its byproductsGMP  
 Dried cooked poultry meatNot to exceed 0.015% of fat content, alone or in combination of BHA or PG or both  
Monoacylglycerol citrateClass IV, M.1 21 CFR 172.832Not to exceed 200 ppm of total fat
 In fats and oils, monoglycerides and diglyceride, shorteningsGMP  
 Unstandardized foods except preparations of meat and meat byproducts, fish, poultry meat and its byproductsGMP  
 MargarineNot to exceed 0.01% of fat content, alone or in combination of mono i sopropyl citrate or stearyl citrate or both  
Mono isopropyl citrateClass IV, M.2 21 CFR 582.6511GRAS with GMP
 In fats and oils, monoglycerides and diglyceride, shorteningsGMP Use as a sequestrant
 Unstandardized foods except preparations of meat and meat byproducts, fish, poultry meat and its byproductsGMP  
 MargarineNot to exceed 0.01% of fat content, alone or in combination of mono glyceride citrate or stearyl citrate or both  
Propyl gallateClass IV, P.1 In fats and oils, lard, shorteningsNot to exceed 0.02%, alone or in combination with BHA, BHT or TBHQ21 CFR 184.1660Not to exceed 0.02% of fat content including essential (volatile) oil content
   Chewing gum base100 ppm
 Dried breakfast cereals, dehydrated potato productsNot to exceed 0.005%, alone or in combination with BHA or BHTEdible oils and fats excluding butterfat and margarine100 ppm
 Chewing gumNot to exceed 0.02%, alone or in combination with BHA or BHT  
   Margarine100 ppm
 Essential oils, dry flavorsNot to exceed 0.125%, alone or in combination with BHA or BHT  
 Citrus oilsNot to exceed 0.5%, alone or in combination with BHA or BHT  
 Unstandardized foods except preparations of meat and meat byproducts, fish, poultry meat and its byproductsNot to exceed 0.02% of the fat content alone or in combination with BHA or BHT  
 MargarineNot to exceed 0.01% of the fat content, alone or in combination with BHA or BHT or both  
 Dried cooked poultry meat0.015% of the fat content alone or in combination with BHA or citric acid or both  
TBHQClass IV, T.1A In fats and oils, lard, shorteningsNot to exceed 0.02% alone or in combination with BHA or BHT or PG21 CFR 172.185Not to exceed 0.02% of fat content including essential (volatile) oil, Alone or in combination with BHA and/or BHT
Tocopherols (α-,concentrate or mixed)Class IV, T.2 In fats and oils, lard, monoglycerides and diglyceride, shorteningsGMP21 CFR 182.3890GRAS with GMP
 Unstandardized foods except preparations of meat and meat by products, fish, poultry meat and its by productsGMP  

In Canada, the use of antioxidants is regulated under the Food and Drug Act (Heath Canada), and in United States, it is regulated by the Federal Drug Administration and the U.S. Department of Agriculture. When it comes to the European Economic Community, directives regulate the use of antioxidants; however, individual member countries still have the control of usage levels. In Japan, the Food Sanitation Law specifies the use of antioxidants (48).

Antioxidative compounds naturally present in food are not covered under present regulations; obviously, it is not a controlled substance as it is part of the raw material of food processing. However, if an antioxidative compound isolated from a natural source is to be added to food, the compound should comply with the appropriate regulations and safety clearances.

11 Safety Considerations of Antioxidants Used in Foods

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References

The safety of food additives is always a controversial discussion because of their possible toxic effects during long-term intake. It has become clear that antioxidants may share a number of toxic properties at high doses. However, it is logical to consider using antioxidants at low levels to reduce the deleterious effects of consuming lipid oxidation products that may be produced if no antioxidants are used. The intake amount of each antioxidant is different with the food and the dietary habits. The use of antioxidants in different countries is limited by specific regulations, established on the basis of their safety for use and technological need. Many countries follow the recommendations of the Joint FAO/WHO Expert Committee on Food Additives and Contaminants (JECFA) on the safe use of food additives. The safety evaluations produced by international bodies such as the Joint FAO/WHO expert committee on food additives (JECFA) and the European Commission (EC) scientific committee for food additive (SCF) are used to establish acceptable daily intake (ADI). ADI is defined as the average amount of the substance that can be consumed daily for a lifetime without health hazards and expressed on the basis of bodyweight. In determining ADI, a range of toxicity tests is carried out. From these tests, the effect that is most sensitive is studied to ascertain the maximum dose at which that effect is no longer observed (no-effect level). A reduction or a safety factor is used to refine the no-effect level, considering the possible difference in sensitivity between species (animals and human) and individuals. To ensure there is an adequate margin of safety for consuming groups, an arbitrary safety factor of 100 is normally used (159). A factor lower than 100 is used if ADI is based on human toxicity study data. For some compounds, ADI is “not specified.” According to the Joint FAO/WHO Expert committee recommendations, this is “specified on the basis of the available data (biochemical, chemical, toxicological and other), the total daily intake of the substance, from its use at levels necessary to achieve the desired effect and from its acceptable background in food, and does not, in the opinion of the committee, represent a hazard to health. For that reason and for the reasons stated in the individual evaluations, the establishment of an ADI expressed in numerical form is not deemed necessary” (160, 161). For some of the compounds, because of the insufficient information available, an ADI level is not specified or “No ADI allocated.” Table 10 provides information on ADI available for food antioxidants in use. Barlow (160) has provided a very descriptive examination on the toxicological studies on antioxidants used as food additives.

Table 10. Acceptable Daily Intake (ADI) Levels of Antioxidants Commonly Used as Food Additives
AntioxidantADIReference
Ascorbic acid and DerivatesNo ADI specified for salts of Na, K, Ca162
 0–1.25 mg/kg bw for ascorbyl palmitate or stearate or sum of both if used together163
BHA0–0.5 mg/kg bw (JECFA)164
 0–0.5 mg/kg bw temperory (SFA) 
BHT0–0.125 mg/kg bw (JECFA)161
 0–0.05 mg/kg bw (SFA)165
TBHQ0–0.2 mg/kg bw temperory allowed161
Tocopherols0.15–2.0 mg/kg bw for dl-α-tocopherol and d-α-tocopherol concentrate161

Toxicological risks may develop when the daily doses of a compound rise above a certain threshhold limit; therefore, toxicity is a matter of dose as well. Natural antioxidants, especially carotenoids, phenolic acids, flavonoids, and sterols, may also exert in vivo pro-oxidative activity. Rietjens et al. (166) have provided an elaborate discussion on the pro-oxidative chemistry and toxicity of well-known natural antioxidants, including ascorbic acid, tocopherols, carotenoids, and flavonoids.

References

  1. Top of page
  2. An Antioxidant—Definition
  3. History of Antioxidants and Their Use
  4. Scope of Using Antioxidants in Food
  5. Oxidation of Fats and Oils and Mechanism of Antioxidants
  6. Classification of Antioxidants
  7. Evaluation of Antioxidant Activity
  8. Commonly Used Antioxidants in Foods
  9. Estimation and Analysis of Antioxidants in Foods
  10. Technological Considerations in Using Antioxidants
  11. Regulatory Status and Safety Issues of Synthetic and Natural Antioxidants
  12. Safety Considerations of Antioxidants Used in Foods
  13. References