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Food Additives and Contaminants, Vol. 21, No. 2 (February 2004), pp.
93124
Chemical interactions between additives in foodstus: a review

M. J. Scotter* and L. Castle
Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK
(Received 3 March 2003; revised 15 September 2003; accepted 30 September 2003)
This paper critically reviews the key literature on food additiveadditive chemical interactions published over the last 30 years together with appropriate relevant information on food additivefood component interactions. Five main classes of food additive are included, reecting the research eort to date: the sulfur (IV) species of preservatives, synthetic food colouring materials, nitrate and nitrite, ascorbic acid, and sorbic acid. Within each class, aspects of the chemistry (reactivity), functionality, stability, use and reactions with other specic food additives are reviewed. Where appropriate, the importance of interactions of food additives with other components of food (i.e. nutrients and non-nutrients) has been assessed and certain aspects of toxicology included. The practical outcome of this review is presented as a set of recommendations for future research in this area. The use of the data in this review is proposed as a training set to develop the framework into a diagnostic tool. This might be used ultimately for the development of a multilevel framework, operating systematically, to understand the important parameters that dictate the outcome of additive interactions. Keywords: food additives, contaminants, chemistry, stability, interactions, research, processing
food chemistry is complex. The subject area of food additives is similarly complex and includes both natural and synthetic substances, covering a wide range of chemical entities. The food additive industry is growing rapidly. Consumers have created a demand for processed foods requiring one or more additives as ingredients and low-calorie foods requiring substitutes for (principally) fats and sugars. There are some 2500 chemicals that function as food additives that give rise to some 5000 trade name products on a world-wide basis (Ash and Ash 1995). For regulatory compliance, not only must each additive have a demonstrated technological need, but also it must be subjected to extensive toxicity testing before approval. This is to establish a level of exposure, i.e. the acceptable daily intake (ADI), which is associated with acceptable risk (see below). The interaction (i.e. target function) of a food additive with food components, whilst being important for the appearance, nutritional value, taste, and safety of food, may have dierent implications in vivo. Food safety issues are of increasing concern to consumers. For food additives, concern has focussed mainly on adverse reactions to certain additives such as the articial food colouring tartrazine or the sulte group of preservatives. The consequences of chemical interactions between food additives permitted for use in the European Union (EU) are the main consideration in this review. The use of a threshold of no concern approach (i.e. threshold of regulation, linked to a threshold of no toxicological signicance) might permit certain interaction products to be disregarded. For instance, for an additive that constitutes (say) 0.1 mg kg1 in the diet and reacts to the extent of 1% to form unidentied products, these will be present at only 0.001 mg kg1 in the diet and may be considered to be below the need for specic identication and toxicological assessment. Moreover, it may be that in a few cases, the nature of the interaction allows the conclusion that the products of an additivefood interaction (or an additive decomposition) was covered by the toxicological tests upon which the ADI for that additive was established.
Introduction
Food is a heterogeneous mixture of chemicals, both natural and man-made; hence, the science of food and
*To whom correspondence should be addressed. e-mail: m.scotter@csl.gov.uk
Food Additives and Contaminants ISSN 0265203X print/ISSN 14645122 online # 2004 Taylor & Francis Ltd http://www.tandf.co.uk/journals DOI: 10.1080/02652030310001636912
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M. J. Scotter and L. Castle
The term interaction may be interpreted dierently, depending upon the particular scientic point of view. In toxicological terms, for instance, interaction may be used as a general term to describe an altered outcome arising from the presence of two compounds and could be antagonistic, additive or synergistic. The work done by Groten et al. (2000), from which such descriptions arise, is a comprehensive and detailed analysis of the possible health implications of such joint actions and interactions of food additives in relation to their potential to increase or decrease the risk of a toxic eect. Risk assessment was based on the potential for additives to share common sites and mechanisms of action or common pathways of elimination. All food additives approved for use in the EU allocated with numerical ADIs were studied, initially based on reports by Food and Agricultural Organization (FAO)World Health Organization (WHO) Joint Expert Committee for Food Additives (JECFA). In all but a very few cases, the possibility of joint actions or interactions could be excluded on scientic grounds. It was concluded that those additives that could not be excluded did not represent a signicant health concern. Toxicokinetic interactions were considered unlikely because of the low dosages involved, the diverse nature of routes of metabolism and elimination, and the fact that enzyme induction or inhibition would have inuenced selection of the no observable adverse eect level (NOAEL). Many of those additives that could not be excluded from showing joint actions or interactions would have low intakes; in some cases, they were alternatives for the same application, thereby lowering the combined intake. Over the last 30 years, research in the area of food additiveadditive interactions has been wide-ranging. Several workers have reviewed specic areas of food additive interactions (Wedzicha 1984, 1995, Dav dek et al. 1990, Massey and Lees 1992, Adams and Langley 1995, Adams 1997), whilst others have focussed on the interactions between additives and other food components, especially nutrients (Cort 1987, Vanderveen 1988, Culik and Kellner 1995). The rst clue to an additives reactivity is its function. Many additives are intentionally reactive and exert their function by an interaction with biological material. Examples are the preservatives sulfur dioxide and nitrite, and the phenolic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene. Their reactivity is well recognized and evidenced, for example, by disappearance data.
Other additives are incidentally reactive. Examples are food colours that confer their eect cosmetically, but may incidentally be photosensitive giving reactive species. Lastly, other additives can be considered chemically relatively inert (e.g. gums and bulk sweeteners), but even these may have important nonspecic interactions, for example, by reducing the bioavailability of essential food components. The boundary between undesirable interactions and desirable interactions (such as those associated with functional foods for example) in many instances is not clear and therefore merits further work. Additivefood interactions are more problematic than additiveadditive interactions. This is because knowledge of the detailed chemical composition of food is still surprisingly limited, beyond the major food constituents and trace nutritional components. A whole food approach, whereby not only the toxic factors present in a food are taken into account, but also the eects of other benecial chemicals, such as nutrients, protective factors and of substances that modify toxicity. While this approach does not attempt to provide a complete chemical description of foods, it could be used to assess the balance between benecial and detrimental eects of natural food constituents, individually and in combination. Such an approach may prove useful in assessing the (arithmetical) additive and synergistic combination of eects of all food additives that may be present in any given foodstu. The identication and quantication of degradation products may also be important in discussions of potential toxicity of food additives, as these products may react further with other food components including other additives, to form undesirable compounds. So, how does one dene the term interaction within the context of food additives? In the simplest case, for two additives to interact, it can be presumed to a rst approximation that encounter at the molecular level the pre-exponential term A (the frequency factor) in an Arrhenius rate equation is second-order as the product of the concentration of the two species under consideration (Moore 1972). Therefore, two species each at low concentration have a low rate of encounter, and vice versa. Within the context of food additives and foodstus, the most important of the thermodynamic and kinetic factors to consider are (arguably) time, temperature, pH, water activity, oxygen, light and catalysis (including enzymes). Wedzicha et al. (1993) have discussed the importance of a correct approach to the design of model systems
Chemical interactions between additives in foodstus
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for additivefood component interactions. They noted that many factors aect the rates of reaction between food additives and food components and presented a diverse set of examples to show the need for a critical approach to model system design. The eects of concentration, ionic strength, non-electrolytes, pH, surfactants and solute transport were addressed. It was concluded that whilst identication of the kinetically signicant food components was the main problem, they need not necessarily feature in reaction stoichiometry. Furthermore, the solution lies in the principles concerned with solutesolute and solute solvent interactions that could be appropriately extended to solutesolid interactions. This review focuses on ve classes of food additive (and their reaction products) that reect published literature to date: . Nitrates and nitrites. . Synthetic food dyes and other food colouring materials. . Sulfur (IV) species. . Sorbic acid and sorbates. . Ascorbic acid. Caramel food colours are chemically heterogeneous hence their chemistry is complex and constitutes a large subject area, which is covered elsewhere (Finot et al. 1990, OBrien et al. 1998). For this reason, this important group of food additives and Maillard-type reactions are not described here, except where other additives are involved too. Similarly, natural and synthetic avourings are also complex substances (and in some cases mixtures) used in foods, but they do not appear on the EU list of permitted food additives and have separate regulations governing their use. Where appropriate, the importance of interactions of food additives with other components of food (i.e. nutrients and non-nutrients) is discussed, as well as certain aspects of toxicology.
100 and 175 mg kg1 (EC 1995a). The antibacterial eect of nitrite and the formation of the so-called Perigo-type factor (PTF) in certain heat-treated meat products has been discussed elsewhere (Christiansen et al. 1973, Skovgaard 1992). Walters (1992) has reviewed the reactions of nitrate and nitrite in foods, with special reference to the determination of N-nitroso compounds.
Reactions of nitrate and nitrite with specic additives

Sorbic acid. The reaction between nitrite ions and sorbic acid analogues is discussed separately and in more detail below. Sulfur (IV) oxo-anions (see also below). Nitrate reacts with bisulte to form sulfonates of ammonia or hydroxylamine (Wedzicha 1984). Such reactions are reported to destroy the preservative activity of the individual additives and so they are rarely used in combination (Adams 1997). The destruction of excess nitrite using bisulte has been suggested in a patented process for curing meats, where the reaction was reported not to aect the nitrosyl pigments (Coleman et al. 1974). Lecithin. Lecithin preparations are used commercially as emulsifying agents, as dietary supplements and as release agents. Lecithin is a source of choline, a quaternary ammonium compound that can decompose on heating to yield trimethylamine. Demethylation can then produce dimethylamine, which in turn can react with nitrite to form dimethylnitrosamine, a known carcinogen. The formation of dimethylnitrosamine occurs on heating sodium nitrite and lecithin at pH 5.6 in a model system (Pensabene et al. 1975).
Nitrates and nitrites

The nitrate and nitrite salts of sodium and potassium are permitted preservatives that may be added to foods, such as cured and dried meats, canned meat products, certain cheeses, and pickled herring and sprat. Nitrate can be used up to a maximum level of between 50 and 250 mg kg1, and nitrite between
Nitroso compounds
The occurrence of nitrosamines in the diet has warranted much concern over the last 20 years or so, hence most of the data generated on the reactions of nitrate and nitrite with other food components have focused on this area. Within the context of food toxicity, the reactions of nitroso compounds arising from the interactions of food constituents with
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OH + OH OH + HNO 2 NO NOH + HONO O O (C 2H5 ) NH 2 NOH + O (C 2H5 ) NNO 2 NONO + H 2O 2 HNO 2 O OH + H2O O + 2 NO + 2 H 2O
food additives have been reviewed (Culik and Kellner 1995). Inhibition of the formation of N-nitroso compounds. Nitrite scavengers react over a wide range of pHs to produce predominantly nitric oxide, a poor nitrosating reagent (Walters and Taylor 1964). Nitric oxide is oxidized in air to nitrogen dioxide (NO2, in equilibrium with dinitrogen tetroxide, N2O4), which reacts readily with secondary amines to form N-nitrosamines. However, the oxidation of nitric oxide in air is trimolecular and is very slow at low concentrations. Thus, the inhibitory eect of ascorbic acid on the conversion of amines, amides, etc. into their N-nitroso derivatives is markedly dependent on the products of its reaction with nitrite under the prevailing conditions. Where quantities of lipid are present, ascorbyl palmitate is particularly useful in inhibiting N-nitrosation. Ascorbic acid and tocopherols inhibit nitrosation eectively at concentrations of 5001000 mg kg1 (ascorbic acid) and 100500 mg kg1 (tocopherols). Mixtures of ascorbic acid and tocopherols have a synergistic eect. The inhibiting inuence of ascorbic acid has been discussed in detail (Douglass et al. 1978) and the inhibiting eect of tocopherols on nitrosation has been reviewed (Lathia and Blum 1989). Other inhibitors include sulfur dioxide, sulfamate, cysteine and glutathione (Gray and Dugan 1975). The ammonium ion, hydroxylamine and retinol have been reported as nitrosation inhibitors (Douglass et al. 1978). The nitrosation of N-alkylureas can also be inhibited strongly by lowering the dielectric constant of the reaction medium (Garcia-Prieto et al. 1998). Wedzicha and Wei (1989) postulated that 3-deoxyhexosulose, and compounds derived from it, are important intermediates in the formation of colour in the non-enzymic Maillard browning reaction. The reaction kinetics were consistent with C-nitrosation (equation 1): H NO 2 DH CH3 COOH
Figure 1. Examples of the role of phenols in the formation of nitrosamines (Walker et al. 1982).
Davies et al. (1978) found that the smoking of meats prepared using nitrite can deposit up to 300 mg kg1 phenols in the matrix. This type of contaminant can be of great importance in relation to nitrite breakdown. The initial product of the nitrosation of phenol is a C-nitrosophenol, but oxidation of this to C-nitrophenol can take place readily. Phenols thus play an interesting role, since they can inhibit or enhance the formation of nitrosamines, depending upon the relative concentrations of nitrite and phenol. Alcohols in high concentration inhibit the formation of N-nitrosodimethylamine and N-nitrosodiethylamine from sodium nitrite at pH 3.0, but enhance the reaction at pH 5.0 (Kurechi et al. 1980). The reaction between nitrite and ferulic acid (an abundant plant phenolic cinnamic acid) under acidic conditions, gives complex mixtures of products (Rousseau and Rosazza 1998), including vanillin, 2-methoxy-4,6-dinitrophenol and a new benzoxazin4-one. It was suggested that the nature of these products might give a basis to the role of ferulic acid as an eective antioxidant or chemoprotective agent in the quenching of nitrosating species. Kalus et al. (1990) isolated and characterized some products of the reaction between BHA and nitrite and examined their mutagenicity. Among the products separable by thin-layer chromatography (TLC) from
DHNO CH3 COO H2 O:
Nitroso compound formation from phenolic compounds. The formation of C-nitroso products from phenolic compounds has been reported by Knowles et al. (1974, 1975) and Douglass et al. (1978), as has the role of phenols in the catalysis of nitrosamine formation from secondary amines (gure 1) (Walker et al. 1982).

OH NO+ BHA OCH 3 N O or NO 2 3 2 O + O t-BuQ OCH3 t-Bu-O-Q O O
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(Walters and Taylor 1964). Lee and Cassens (1976) have demonstrated that the nitrosylhaemochrome obtained on heating contains two nitric oxide moieties coordinated to the iron atom. Reaction with amines. Primary amines, such as certain amino acids, react with nitrous acid, the principal products being the corresponding hydroxy compounds. Secondary amines are converted into their N-nitroso derivatives, along with those from substituted amides and guanidines, etc. (Walters 1992). Walters also reported that despite many reports to the contrary, nitrous acid reacts with tertiary amines and even with quaternary ammonium compounds. The principal products formed are a carbonyl compound, resulting from oxidation of a substituent group, and the N-nitroso derivative of the secondary amine arising from its cleavage. An example of this is the reaction between the alkaloid hordenine and oxides of nitrogen during the direct red drying of malt, which is considered to be responsible for the formation of N-nitrosodimethylamine. Nitrite may undergo a number of reactions with various components of food matrices, including C-, S-, O- and N-nitrosation reactions. Isotope (15N) labelling studies have indicated that the majority of nitrite nitrogen is still present in cured meat, but no longer as nitrite (Massey and Lees 1992). Reaction with thiocyanates. Thiocyanates are widely distributed in vegetables of the Brassica type and are known to catalyse the formation of N-nitroso compounds. Catalytic activity is greatest at low pH, such as that found in the human stomach (Boyland and Walker 1974).
OH NO2 BHA-NO2 OCH 3
Figure 2. Dominant products from the reaction between BHA and nitrite (Kalus et al. 1990).
the reaction in aerobic aqueous solution, 1-hydroxyl2-tert-butyl-4-methoxy-6-nitrobenzene (BHA-NO2), tert-butyl substituted p-quinone and 3-tert-butyl-5methoxy-1,2-benzoquinone (t-Bu-O-Q) were dominant (gure 2). One further compound with nitroso character was also isolated along with traces of tert-butylhydroquinone. No evidence of mutagenicity in the Ames test was found and some of the products were unstable. The authors concluded that only a one-electron reduction and/or a reaction with amino acids may be involved in the cytotoxicity of the quinones, and that BHA appeared to exert a protective eect against nitrosating agents in the media containing it. Reaction with thiols. Thiols such as glutathione are readily converted by nitrite into their S-nitroso derivatives (see below), although most such compounds are relatively unstable. In the presence of sources of Fe and nitrite, thiols can give rise to iron thionitrosyls, such as the salts of Roussin, which exert a bactericidal eect. Reaction with haems. The primary action of nitrite in relation to the haem proteins, myoglobin and haemoglobin in preserved meats is to oxidize the natural oxygenated ferrous to the ferric met- forms, whilst itself being oxidized to nitrate. Subsequently, reduction occurs both of nitrite to nitric oxide and of ferric met-haem to the ferrous forms of haem protein, nitrosylmyoglobin and nitrosohaemoglobin. These compounds give cured meat its characteristic pink colour. These reductions can be eected by residual respiratory enzymes in uncooked meat products and by sulfhydryl systems in heat-processed products
Synthetic food dyes

The most common articial food colours are azo dyes that include mono-, dis- and tris-azo types. Triphenylmethyl (triarylmethyl) compounds, as well as a small number of dyes based on quinoline, xanthene and indigoid structures, constitute the remainder. Table 1 shows the EU maximum permitted levels of usage for those synthetic dyes with numerical ADIs. All of the synthetic colouring materials permitted for food use in the EU and USA (except lithol rubine BK and citrus red, which have restricted use, and the lake colours) are soluble in water to a greater or lesser
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I O I COO O I O I
Table 1. EU-permitted synthetic food colouring materials with numerical ADIs.

Colour Allura red AC Amaranth Black PN Brilliant blue FCF Brown FK Brown HT Carmoisine Green S Indigo carmine Patent blue V Ponceau 4R Quinoline yellow Sunset yellow FCF Tartrazine
a
ADI Maximum permitted (mg kg1 bw day1) level (mg kg1) 7 0.8 5 10 0.15 3 4 5 5 15 4 10 2.5 7.5 500 100 500a 500a 20b 500a 500a 500a 500a 500a 500a 500a 500a 500a
a
(I) O 1[O ] 2 N H (II) N . O (III) O
Use at this level in sauces, seasonings, salmon substitutes, surimi, decorations and coatings; main range of application 50200 mg kg1 and Quantum satis permitted in, for example, edible casings; b use restricted to kippers.
Figure 3. Food colour erythrosine (I), a xanthine-type dye, and the scheme for oxidation of 2,2,6,6-tetramethyl4-piperidone (II) to the corresponding N-oxide radical (III) (Rosenthal et al. 1988).
extent and insoluble in oils and fats. The chemistry and stability of synthetic food dyes has been covered in other texts (Scotter 2003), but many factors can and do contribute to colorant stability, such as heat, light, pH, redox systems, other food ingredients (especially preservatives) and trace metals. Rosenthal et al. (1988) screened 43 certied US Food, Drug and Cosmetic (FD&C) colours for photosensitizing activity using photogeneration of singlet oxygen (1(O2)) as an index. Only xanthine-type dyes substituted with Br or I (e.g. erythrosine) (gure 3 (I)) generated 1(O2). This was monitored by oxidation of 2,2,6,6-tetramethyl-4-piperidone (gure 3 (II)) to the corresponding N-oxide radical (gure 3 (III)) measured by electron spin resonance (ESR) spectrometry. The formation of reactive species of oxygen such as 1(O2) implied potential for dye phototoxicity as a result of photodynamic activity, but this was considered a low risk due to the deactivation of such species in food matrices, especially those which contain antioxidants. Indigo carmine showed mutagenic activity and growth inhibition of Bacillus subtilis following ultraviolet light (UV) irradiation (Ozaki et al. 1998). Mutagenic activity reached a maximum after 6 days of irradiation and DNA-damaging activity after 14 days. No toxicity or mutagenicity was observed after 30 days, indicating that the photodegradation
products became inactive on further degradation. Six intermediate products of photodegradation were observed after 6 days using high-performance liquid chromatography (HPLC) analysis, which was reduced to 5 after 14 days. No diagnostic peaks could be characterized. Superoxide radicals and hydrogen peroxide were postulated as intermediates in the photo-induced generation of hydroxy radicals under certain conditions (Ishimitsu et al. 1995, Ozaki et al. 1998). Heat can cause loss of colour during food processing and cooking. For example, during the thermal degradation of indigo carmine (Taru and Takoka 1982), the heterocyclic ring was decomposed at 340470 C, leading to loss of colour. The majority of colour additives are unstable in combination with oxidizing and reducing agents. Since colour depends on the existence of a conjugated unsaturated system within the dye molecule, any substance which modies this system (e.g. oxidizing or reducing agents, sugars, acids, and salts) will aect the colour. All dyes, those with an azo group in particular, will exhibit accelerated fading under both acid and alkaline conditions in the presence of metals, including zinc, tin, aluminium, iron and copper, especially at higher temperatures. This is mostly due to the reducing eect of liberated hydrogen. Dyes will often react with the metal in food cans.
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Interaction of synthetic food dyes with other food additives

Preservatives. The added colour in canned products may degrade in the presence of tartaric and citric acids, because the acids can react with the metal of the container to liberate hydrogen. A study of nine red colours in comminuted meat products found that most of the dyes were destroyed to some extent, but, with nitrite added, more of the colour survived (Knowles et al. 1974). Subsidiary dye components and colourless uorescent products were formed as a result of heat processing and in certain cases additional products were observed in the presence of nitrite. Nitrite can also cause rapid de-tinning to produce Sn2, a strong reducing agent. Sulfur dioxide is known to cause rapid decolourization of dye solutions (see below). Model food systems and foods. McCormick (1971) studied the degradation of allura red AC in 10% solutions of acetic acid, citric acid, malic acid and tartaric acid, as well as solutions of sucrose, glucose, SO2, sodium benzoate and buers, ranging from pH 3 to 8. No colour fading was detected, but increases in the blue hue were observed in 10% solutions of various alkalis after 1 week. The fading of colour at temperatures above 135 C has been reported (Stange Co. 1970, Lancaster and Lawrence 1986). Several factors have been listed as possible causes, including excessive heat and reaction with protein, reducing ions and reducing sugars (Krukar 1980). When heated in aqueous solution in the presence of fructose, amaranth decomposes slowly (Ross 1975, Bibeau and Clydesdale 1978a), whereas sunset yellow FCF is reported to be much more resistant to fructosemediated decomposition (Bibeau and Clydesdale 1978b). Synthetic azo colour additives are often used in foods processed at 100170 C or even higher, especially sugar-based confectionery commodities, which account for a signicant proportion of the consumption of azo food colours (MAFF 1987). Within this temperature range, reducing sugars are degraded to highly reactive species (Fleming et al. 1968, Ross 1975), which are capable of reducing azo dyes, demonstrated by heating aqueous solutions under reux for several hours. Amino R-salt (1-amino-2hydroxynaphthalene-2,7-disulfonic acid) is a reactive degradation product of amaranth and has been the subject of several studies. Singh (1970) reported
that amino R-salt completely decomposed when heated with an aqueous mixture of sucrose or dextrose. Baking soda was also reported to accelerate the decomposition. In a separate study, the compound was degraded by 60% when heated at 103 C for 15 min in a 10% sucrose solution. In a candy mixture, it was reported to degrade faster than naphthionic acid (4-aminonaphthalene-1-sulfonic acid). Simulated candy making at 120162 C showed azo dye degradation by 1025%. Amaranth degraded to naphthionic acid and amino R-salt, whereas tartrazine and sunset yellow FCF showed no increase in intermediates (Lancaster and Lawrence 1986). No signicant increases in the levels of intermediates (above regulatory limits) were found in commercial candies. The same authors also reported that reactive intermediate compounds arising from the manufacture of food colouring materials may be present at low levels (< 1%) in food colouring preparations and thus may also be aected by heat during production of confectionery commodities. Candies containing the amaranth intermediates naphthionic acid and R-salt at levels of 419 mg kg1 in the absence of food colour were prepared at 162 C. Naphthionic acid was reported to decompose by an average of 40% whilst R-salt degraded by about 20%. Similar studies with sunset yellow FCF at 120162 C using Schaeers salt showed no signicant decomposition whereas sulfanilic acid (4-aminobenzene-1-sulfonic acid) showed 46% decomposition at 126 C. The tartrazine intermediate pyrazolone-T (PyT or 1-(4-sulfophenyl)-3carboxy-5-hydroxypyrazolone) was shown to degrade substantially (8090%) over the temperature range 120162 C. The stability of azo dyes in food systems depends upon a number of parameters, the most important of which is related to the presence of reducing species. These result in the cleavage of the azo double bond, ultimately to form primary amines such as aniline, sulfanilic acid and naphthionic acid. These products can degrade further to ammonia (Nursten and Williams 1969, Fogg and Summan 1983, 1984) (see below). The current EU regulations limit the amounts of unsulfonated aromatic amines allowed to 0.01% (EC 1995b), so the degradation of azo dyes could produce a signicant contribution to this risk. Nursten and Williams (1969) reported that carmoisine decolourized more rapidly at pH 7 than at pH 3 at 121 C in the presence of ascorbic acid (AA). Ponceau 4R, however, was stable at this pH. The results suggested that the sulfonate group peri to
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c a NaO S 3 N N N SO Na 3 SO Na 3 SO Na 3 OH HN b N COCH 3
the azo bond was the source of the increased stability of ponceau 4R. The disazo dye black PN was also found to be particularly unstable in the presence of AA. This was attributed to the reductive cleavage of both azo groups to yield 1,4-diaminonaphthalene-6-sulfonic acid, a compound that had previously been shown to be capable of catalysing the breakdown of the original dye (after Eisenbrandt and Lang 1966). AA-mediated degradation of azo dyes was reduced by the presence of ethylenediaminetetracetic acid (EDTA), but enhanced by tungsten light at pH 5.5 (Fogg and Summan 1983, 1984). A major source of synthetic dyes in the diet is from soft drinks, to which AA is added as an antioxidant/ vitamin supplement. This provides a chemically reducing environment in which azo dyes have the potential to be degraded. Marovatsanga and Macrae (1987) studied the stability of amaranth under a range of conditions similar to those found in soft drinks. The dye was reduced under reference conditions to provide authentic degradation products as reference substances. Soft drink model solutions containing amaranth were made in citrate buer preserved with sodium benzoate and analysed for dye degradation in the presence of AA during storage. The eects of other components such as sugar were investigated and also the eect of removing oxygen by ushing with nitrogen. These ndings were rationalized in terms of the amount of AA available. Sugars have been reported as having a protective eect on AA in solution by virtue of their ability to chelate metal ions, which would otherwise catalyse AA oxidation. AA is also oxidized by atmospheric oxygen and, in this instance, particularly by dissolved oxygen (Birch and Parker 1974, Birch and Pepper 1983). In a study on the fate of black PN used to stain meat (Scotter 1983), TLC was used to identify degradation products both with and without autoclaving of the dyed meat samples. The expected products of azo-bond cleavage, sulfanilic acid, 1,7-cleves acid, and acetyl-K acid (4-acetamido-5-hydroxynaphthalene-1,7-disulfonic acid), were identied along with coloured monoazo dyes, corresponding to the residual coupled products sulfanilic acid ! 1,7-cleves acid and 1,7-cleves acid ! acetyl-K-acid (gure 4). The deacetylated analogues were also identied in the autoclaved samples. Indigo carmine is known to be unstable in solution to heat, acids and alkalis (Nursten and Williams 1969). Investigations into the losses incurred during indigo
Figure 4. Structure of black PN showing the points of azo cleavage (a, b) and deacetylation (c) (Scotter 1983).
O NaO S 3 N H (I) hv O2 O NaO S 3 O N H (II) N H O SO3 Na
Figure 5. Degradation of indigo carmine (I) to sodium 2,3-dioxo-5-indolinesulfonate (II) (Boley et al. 1981).
carmine analysis of boiled sweets revealed that oxygen was essential for photochemical degradation of the dye to sodium 2,3-dioxo-5-indolinesulfonate (Boley et al. 1981). Whilst this is not an additive additive interaction per se (gure 5), it could be postulated that mediation by other oxidizing agents could occur and any products such as indoline derivatives formed as a result may be reactive towards other food additives. Sulfur (IV ) species. The stability of mono-azo dyes in the presence of S(IV) oxo-species is reported to be variable (Wedzicha 1984). Reduction to hydrazo products would be expected to be reversible, but that to the amino compound would not. Irreversible formation of the amine is proposed if one of the
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OH- groups present in the aromatic ring is in the 2-position. Where the OH- group occurs in the 4-position, relatively stable hydrazo compounds may be formed. Carmoisine is reduced by S(IV) oxo-anions with the transfer of two electrons thus facilitating the conversion of S(IV) to sulfate. The stabilities of the triarylmethane dyes permitted in foods (green S, brilliant blue FCF and patent blue V) have not been studied in any depth. However, the triarymethyl cation may be decolourized by combination with sulte ion, e.g. in the use of pararosaniline as a Schi reagent for the histochemical determination of S(IV) oxoanions. Malachite green and crystal violet are triarylmethane dyes that are not permitted for use in foodstus, but their reactivities towards S(IV) oxoanions have been studied. At the pH of acetic acid/acetate buer (3.75.7), both dyes react with bisulte to give colourless sulfonates (Wedzicha 1984):
Ar3 C HSO 3 Ar3 CSO3 H
suggested a 1:1 reaction of sulte with the azo compound to form a complex and it was suggested that a hydrazo compound could then be formed via hydrolysis. Saxby and Stephen (1981) reported that sunset yellow in the presence of SO2 forms a secondary dye. Further work by Damant et al. (1989) and Damant (1990) using nuclear magnetic resonance (spectroscopy) (NMR) and fast atom bombardmentmass spectrometry (FAB-MS) revealed the structure of this secondary dye to be 1-(40 -sulfo-1-phenylhydrazo)-2-keto-3,3,4-trihydronaphthalene-4,6-disulfonic acid and elucidated the mechanism of formation. Because these dyes exist predominantly as hydrazone tautomers, the addition of bisulte to the para position is facilitated, the observed secondary rate constants being proportional to pH and hence bisulte availability. Studies of interactions between amaranth and SO2 were also undertaken, where amaranth was found to degrade to an orange-yellow substance which could not be identied due to its instability. Adams and Langley (1995) repeated aspects of this work in their studies on the reactivity of six food colours selected on the basis of their frequent use in combination with sulte, ascorbate and nitrite, and reported similar results, but were unable to identify the structure of the secondary dyes. Kinetic studies of the food colours, amaranth, black PN, ponceau 4R, sunset yellow FCF and tartrazine, were carried out to determine the conditions necessary to obtain degradation products that could be identied by mass spectrometry. The rate of degradation of sunset yellow FCF at pH 3 in the presence of AA and bisulte to a lemon yellow compound was found to be proportional to bisulte concentration and temperature (Damant et al. 1989, Damant 1990). In the absence of bisulte, AA caused oxidative degradation of sunset yellow FCF. Conversely, black PN, tartrazine and carmoisine degraded without an observed shift in their corresponding lmax values. Disazo and other more complex azo dyes are reported to have fair to good stability to sulfur dioxide, but the degradation products do not appear to have been studied in depth. The most likely products of degradation in this case are thought to be sulfonate derivatives. Indigo carmine has poor stability to SO2; even so, the products are unknown. Quinoline yellow, the only permitted quinoline food colouring material, is reported to have excellent stability to SO2 (Adams and Langley 1995).
The stability of permitted articial food colouring agents with respect to S(IV) oxospecies is shown in table 2. Wedzicha and Rumbelow (1981) investigated the kinetics and mechanisms of the interaction between sulte species and carmoisine. The kinetic data
Table 2. Reactivity of articial food colour additives towards S(IV) oxospecies (Wedzicha 1984).
Common name Tartrazine Quinoline yellow Sunset yellow FCF Carmoisine Amaranth Ponceau 4R Erythrosine Red 2G Allura red AC Patent blue V Indigo carmine Brilliant blue FCF Green S Black PN Brown FK Brown HT Chemical class monoazo quinoline monoazo monoazo monoazo monoazo xanthene monoazo monoazo triarylmethane indigoid triarylmethane triarylmethane disazo azoa disazo Stability towards S(IV) excellent excellent fair fair fair fair good good not listed not listed poor not listed good fair good fair
a Mixture of six main components; only used for the colouring of kippers.
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R1
3' 4' 5'
Other food colours

Within the EU, only the natural colouring materials annatto, carmines, carotenes and the Cu complexes of chlorophyll have been allocated numerical ADIs (0.065, 5, 5 and 15 mg kg1 bw day1, respectively). Annatto is permitted for use in a restricted range of foodstus up to 50 mg kg1, whereas the carmines and carotenes can be used up to a level of 500 mg kg1. The Cu complexes of chlorophylls may be used quantum satis.
HO
7
OH R2
O
5
OG
OH
(a)
RO H R HO N
+
COO-
Anthocyanins
The chemistry of anthocycanins is complex and several detailed reviews have been published (Markakis 1982, Jackman et al. 1987, Harborne et al. 1988, Francis 1989, Hendry and Haughton 1992, Mazza 1997). Anthocyanins are derivatives of 2-phenylbenzopyrylium salts with the substitution pattern such as those shown in gure 6(a). Where these substances occur in fruit juices and other plant cell materials, and as puried food colouring materials in foodstus, they will interact readily with other naturally occurring materials, such as avonols. Such interactions depend upon intermolecular association through, for instance, hydrogen bonds, and may therefore aect anthocyanin stability. The avylium nucleus is electron decient and therefore highly reactive. Its reactions usually involve decolourization. The rate of anthocyanin degradation depends on pH, temperature, O2 partial pressure, metal ion (i.e. Mn), ascorbate concentration, and other components (Dav dek et al. 1990). In most food processing operations, the anthocyanins are relatively stable, especially when low pH conditions are maintained. Naturally present ascorbic acid can be problematic. In the presence of iron or copper ions and oxygen, the oxidation of ascorbic acid to dehydroascorbic acid is accompanied by the formation of hydrogen peroxide, which in turn can oxidize anthocyanins to colourless malvones, a reaction implicated in the loss of colour from canned strawberries (Coultate 2002). The mechanism, however, is not clear. Under anaerobic conditions, anthocyanins and ascorbic acid may react (in the presence of amino acids, phenols, sugar derivatives, etc.) via condensation-type mechanisms, but the products
COOH H COOH COOH H COOH
N H (b)
N H (c)
Figure 6. (a)2-Phenylbenzopyrylium(avylium)nucleus; G, glycoside; R1, R2 H, OH or OCH3; (b) betalains (R, glucose betacyanin) and (R H betanidin); (c) vulgaxanthin-I (R, glutamine), vulgaxanthin-II (R, glutamic acid).
of these interactions, some of which are complex polymers, have not been well characterized (Jurd 1964, Harborne et al. 1975). Mazza (1997) has reviewed the structure, stability and analysis of anthocyanins and concludes that in addition to pH, structure, concentration, co-pigmentation and metal complexing, the intensity and stability of anthocyanin colours are inuenced by other factors. These include temperature, light, oxygen, acetaldehyde, ascorbic acid, sugars and their degradation products sulfur dioxide, amino acids and catechins. Oxygen is an important factor inuencing the rate of anthocyanin degradation, due to the partial open-ring structure. Anthocyanins are oxidized by peroxides, depending upon the substructure at the C3 hydroxyl. The hydrogen peroxide oxidation of avylium salts produces a number of dierent compounds 1985). (Chichester 1972, Havlikova Anthocyanins form adducts with S2O 5 as a result of its common use as an antimicrobial preservative in fruit juices and wines, as well as in other food commodities, such as dried fruits, comminuted meat products and compound foodstus (Hendry and
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HO
Ph :N OG or
HO
Ph OG
OH
OH
:N
Figure 7. Sites for nucleophilic attack on the avylium cation (Wedzicha 1984).
Houghton 1992). The bisulte ion adds to position 2 or 4 of the avylium nucleus, discolouring the pigment and simultaneously conferring high heat stability on the glycosidic bond (Adams 1973, Adams and Woodman 1973). According to Wedzicha (1984), the reaction involves the reversible formation of a 1:1 adduct between S(IV) and the avylium cation, which is pH dependent. Nucleophilic attack on the cation leads to the formation of Meisenheimer-type sigma-complex in which positions 2 and 4 are reactive (gure 7). (A Meisenheimer complex, or adduct, is a cyclohexadienyl derivative formed as a Lewis adduct from a nucleophile (Lewis base) and an aromatic or heteroaromatic compound.) According to Timberlake and Bridle (1968), attack at the 4-position is preferred. However, polymeric anthocyanins are resistant to S(IV) reaction, since the 4-position is already occupied. Anthocyanin reactivity and the status of the avylium 4-substituent have been reported to play an important role in the colour stability of anthocyanins (Garcia-Viguera and Bridle 1999).
Lipoprotein-bound chlorophylls are somewhat protected against acids, but can be aected by cooking and processing. In alkaline media, the decomposition of chlorophylls is very rapid and they are not stable against the action of free-radicals, e.g. during lipoxygenase-catalysed oxidation of lipids, probably due to the eect of hydroperoxides. The allomerization reaction of chlorophylls takes place spontaneously in a polar medium and is metal-ion catalysed; allomeric 10-hydroxychlorophylls and 10-methoxylactones have been detected (Schaber et al. 1984). Chlorophylls also form Schi bases, the colour maximum of which is pH dependent (Maggiora et al. 1985). The major organic acids involved in the degradation of chlorophylls are acetic acid and 5-oxopyrrolidinecarboxylic acid (Lin et al. 1971).
Betalains
Betalains are the red and yellow plant pigments obtained from members of the order Centrospermae, the most food-signicant of these being the beetroot (Beta vulgaris L.). The betalains are divided into two groups, the betacyanins which are purplish-red in colour and the less common yellow-coloured betaxanthins (gure 6 (b, c)). The betacyanins comprise about 90% of beetroot betalains. They are all either the glycoside or the aglycon of betanidin or its C-15 isomer. Iso forms account for only about 5% of the total betacyanins and some 95% of betanidin and isobetanidin carry a glucose residue at C-5, and a very small proportion of these glucose residues, esteried with sulfate. In beetroot, the betaxanthins are characterized by the vulgaxanthins (types I and II in roughly equal proportions), which are characterized by the lack of an aromatic ring system attached to N-1 and by the absence of sugar residues (Coultate 2002). Betacyanins dier from other naturally occurring water-soluble plant pigments, especially anthocyanins, in that their colour is hardly aected by pH changes in the range normally encountered in foodstus. Betacyanins are fairly stable under food-processing conditions, although heating in the presence of air at neutral pH causes breakdown to brown compounds. According to Dav dek et al. (1990), the most important betalain is betanin (or phytolaccanin), the b-D-glucopyranoside of betanidin, which may be enzymically hydrolysed to the corresponding aglycon. In the presence of acids, it is transformed into its isomer, and further, to yellow betalamic acid products, containing an open ring system, and nally to
Chlorophylls
Chlorophyll degradation during food processing has been reviewed by Dav dek et al. (1990), who cite the work carried out by White et al. (1963). Chlorophyll destruction can proceed as an acid-, base- or enzymecatalysed reaction. Weak acids liberate the Mg atom bound to the porphyrin ring to form pheophytins by substitution with hydrogen. This results in a colour change from green to dull brown. Magnesium may be replaced by copper (as in copper chlorophyll additives, i.e. chemically modied chlorophylls) and by tin and zinc. Alkaline salts may also be produced, but these are very unstable. Chlorophylls may also undergo photo-oxidation accompanied by the loss of desirable colour. The rate of oxidation has been shown to be dependent upon water activity and the temperature and duration of blanching in dehydrated products.
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brown products (Saguy et al. 1978). In alkaline medium, the red-violet pigment is decomposed into colourless products (Mabry 1980). Vulgaxanthin I, the most important betaxanthin, and its amide vulgaxanthin II, are hydrolysed in acid medium to amines or amino acids bound to the dihydropyridine moiety. The stability of betalains during food processing is inuenced by many factors, most signicantly by temperature, pH, aw, Mn, O2, and hn (Singer and Von Elbe 1979).
Carminic acid
Carminic acid (7-a-D-glucopyranosyl-9,10-dihydro3, 5,6,8-tetrahydroxy-1-methyl-9,10-dioxoanthracene2-carboxylic acid) from cochineal is a pale yellow compound which complexes readily with aluminium (as in cochineal carmines), tin and other metals to give brilliant red pigments. The cochineal complexes are relatively stable under food processing conditions, but are easily bleached by SO2 (Dav dek et al. 1990). Carminic acid has close structural and chromogenic similarities to the highly toxic anthracycline antitumour drug doxorubicin and is reported to redox cycle to produce free radicals (Gutteridge and Quinlan 1986). These radicals, in the presence of trace amounts of iron salts, readily damage membrane lipid and degrade the carbohydrate deoxyribose. Damage to membrane lipid appears to involve mainly organic oxygen radicals, such as alkoxy and peroxy radicals, whereas that to deoxyribose implicates the hydroxyl radical formed in a Fentontype reaction. a-Tocopherol, the phenolic antioxidants BHA and butylated hydroxytoluene (BHT), the hydroxyl radical scavengers mannitol, benzoate, and thiourea, and iron chelators, such as EDTA, were all reported to prevent such damage. The product of the reaction between carmine and hot ammonia solution has been reported to be the acid-stable 4-amino analogue, which is permitted for food use in the USA (Sugimoto et al. 2002).
principles comprise one major and two minor structurally related species, characterized by the presence of a dione system with two substituted aromatic moieties, each conjugated to a keto group. These are curcumin itself (major), demethoxycurcumin and bisdemethoxycurcumin. Curcumin is relatively stable to heat processing except in alkaline medium, where it degrades to vanillin and diferuloylmethane. Curcumin is sensitive to light, which limits its applications in food. Curcumin gives a lemon-yellow with a distinct green shade in solution. Cations will tend to induce a more orange-brown shade and SO2 reduces the colour intensity. Majeed et al. (2000) have reviewed the chemistry and pharmacology of curcumin. Bisdemethoxycurcumin was observed to be less susceptible to degradation at pH 10.2 than curcumin or demethoxycurcumin. In buer solutions, curcumin was observed to decompose in a pH-dependent manner, with faster reactions at neutral-basic conditions, trans-6-(40 -hydroxy-30 methoxyphenyl)-2,4-dioxo-5-hexanal being predicted to be the major degradation product. Vanillin, ferulic acid and feruloylmethane were the minor degradation products, with vanillin increasing with the time of incubation. The dierential physical properties of the naturally occurring curcuminoids and selected derivatives were explained in a study that investigated intra- and intermolecular hydrogen-bond formation in these compounds using absorption and emission properties (Tonnesen et al. 1995). Carotenoids (see also below). The carotenoids include the carotenes, which are hydrocarbons, and the xanthophylls, which are oxygen-containing analogues (Britton et al. 1995). Within the context of food additives, their reactivities and general properties may be considered to be similar. Carotenoids may exist in the free state, in solution in the lipid phase, or associated with proteins or polysaccharides. They can also exist as esters and glycosides, etc. In food, the main causes of carotenoid degradation are various oxidative reactions mainly involving oxygen (principally 1(O2)), hydroperoxides and peroxy-radicals. The presence of a polar (e.g. hydroxyl) group in a carotenoid molecule may eect the reactivity of neighbouring carbon atoms. Carotenoids act as very eective 1(O2) scavengers in the photosensitized oxidation of unsaturated fatty acids, where chlorophylls may act as sensitizers. The oxidative stability of carotenoids is improved by the addition of antioxidants, such as BHA, BHT, tocopherols and ascorbyl palmitate. The addition of ascorbic
Curcumin
Curcumin is the collective term given to the yellowcoloured compounds obtained from rhizomes of Curcuma domestica Val. (syn. Curcuma Longa Koenig non L.), which themselves are used as turmeric spice (Hendry and Haughton 1992). The colouring
105
acid is reported to improve the colour stability (Bunnell 1968). Flour bleaching agents used to decolourize carotenoids, include benzoyl peroxide, nitrosyl chloride and chlorine dioxide (Fortmann and Joiner 1971). Degradation of carotenoids. Several studies have reported on the formation of volatile aromatic hydrocarbons as anaerobic thermal degradation products of (principally) b-carotene (Kuhn and Winterstein 1932, 1933a, b, Day and Erdmann 1963, Mulik and Erdmann 1963, McKeown 1965). These and other studies were reviewed by Onyewu et al. (1982), who also reported on the formation of two nonvolatile colourless carotene-like compounds in the thermal degradation of b-carotene. In a later study on b-carotene degradation (Onyewu et al. 1986), over 70 non-volatile compounds were observed. Marty and Berset (1988) have reported 15 coloured degradation products arising from the degradation of trans-b-carotene during extrusion cooking. Factors aecting the thermal degradation of b-carotene were discussed in a subsequent report (Marty and Berset 1990). This study showed that prolonged heating in a model extrusion-cooking system at 180 C caused only limited breakdown, but the presence of common food constituents, such as water or starch, combined with mechanical mixing favouring the diusion of oxygen, led to much higher losses. Several hypotheses concerning the sequence of reactions involved in oxidative degradation of b-carotene were proposed, in which reversible stereoisomerization was important in the formation of both non-oxidized volatiles and oxidation products. A mechanism for the formation of volatile compounds by thermal degradation of carotenoids in aqueous medium has been proposed (Kanasawud and Crouzet 1990). The formation of a- and bionones by the thermal decomposition of b-carotene present in distilled alcoholic beverages in addition to these aromatic hydrocarbons, has also been reported (LaRoe and Shipley 1970). The eects of dierent types of oxidation-autoxidation at 20 and 80 C (photosensitized and chemical) on the degradation of b-carotene have been reported (Gloria et al. 1993). Although similar volatile degradation products were found for the dierent types of oxidation, their relative concentrations varied. The oxidation of capsanthin is reported to involve primarily the oxidation of hydroxyl groups, followed by scission of the chain at the carboncarbon bond a- to the
in-chain carbonyl group (Philip and Francis 1971). Since many of the non-volatile degradation products of b-carotene possess chemically reactive groups such as carbonyls, they are likely to react with amino acids and/or their degradation products during the thermal processing of foods. Four major (yellow) coloured products were isolated by HPLC from methanol extracts of model systems comprising mixtures of b-carotene and phenylalanine, following heating in liquid paran at 210 C (Papadopoulou and Ames 1993). Following on from the degradation studies on bixin reported by McKeown (1965), characterization of the coloured thermal degradation products of bixin from annatto food colouring material, and a revised mechanism for their formation has been reported (Scotter 1995a). The main product was conrmed as 4,8-dimethyltetradecahexaenedioic acid and its production was shown to be accompanied by the formation of m-xylene and (to a lesser extent) toluene. m-Xylene has been shown to be present in annatto food colour preparations up to a level of 200 mg kg1 (Scotter et al. 2000) and low levels of both m-xylene and toluene have been detected in the headspace of model systems and foods containing annatto, heated in situ (Scotter 1995b).
Sulfur (IV) species SO2 and sultes

Wedzicha (1984, 1991, 1992) has comprehensively reviewed the chemistry of sulfur dioxide in foods. Detailed accounts of the safety aspects of SO2 and sultes have been given by Taylor et al. (1986) and by Rose and Pilkington (1989), who reported a mechanism for the antimicrobial action of this group of preservatives. Most of what follows has been taken from these works. The terms sulfur dioxide and sultes are used commonly to describe the oxo-species of sulfur oxidation state (IV). The chemical nature of each chemical species is well documented. In the normal pH range of food (about pH 36), the principal S(IV) species is HSO 3 in equilibrium with small but pH-sensitive amounts of SO2H2O and SO2 3 . The minor species are responsible for the preservative eect and the chemical reactivity of the additive. However, the pK
106
values of SO2H2O are sensitive to other compositional factors, such as salt concentration. This is in accordance with theoretical predictions based on the variation of solute activity coecients with high ionic strength. Certain non-electrolytes such as ethanol have a very marked eect, increasing pK values by up to 2 units, whereas high concentrations of sucrose have very little eect (Wedzicha and Goddard 1988, 1991); hence, there are no simple rules.
Chemical reactivity of sulfur (IV)

The main cause of reactions between S(IV) species and food components is the nucleophilicity of sulte, which can act as both a carbon and sulfur nucleophile (Davis 1968, Wedzicha 1984, 1987). Sulte is the more nucleophilic, metabisulte showing no signicant nucleophilic activity because its action involves S-S bond breaking. The pKa of bisulte is similar to that of H2PO 4 , so it is reasonable to expect that S(IV) species can act as general acidbase catalysts as do phosphate species. The potential for such activity is illustrated by the eect of S(IV) on the rate of mutarotation of glucose, the hydrolysis of gluconolactone and, most particularly, in the hydrolysis of p-nitrophenyl acetate, for which it is some 1600 times more eective than, for example, phosphate another commonly used food additives. This is attributed to the ability of sulte to initiate the hydrolysis by nucleophilic attack at the carboxyl carbon atom. Thus, whilst the implications of this reactivity are not fully appreciated, it is possible that esters that are either naturally present or added (as avours for instance) could be destabilized by S(IV). It is also possible that the rate of degradation of ascorbic acid, which proceeds by hydrolysis of the lactone moiety, is increased by S(IV) (Davies and Wedzicha 1992). This may therefore be relevant to preserved fruit beverages. The metabisulte ion is potentially a better acidbase catalyst, although the tendency for it to catalyse reactions between food components has not been conrmed. The most signicant homolytic reactions of S(IV) are the result of oxidation by molecular oxygen catalysed by transition metal ions (Yang 1984). Complexing agents such as EDTA or citrate may act as antioxidants. Free-radical scavengers such as alcohols are eective antioxidants. Caramel colour can act as a pro-oxidant at low concentration, presumably by providing initiating free radicals, but acts as an
antioxidant at high concentrations (Wedzicha and Clayton 1994). It is generally recognized that antioxidants can become pro-oxidants under certain conditions. High (>30%) concentrations of ethanol for instance allow rapid oxidation of S(IV), provided that transition metal ions and amino compounds (including EDTA) are also present. These form redoxactive complexes in solution that are oxidized by molecular oxygen and in turn oxidize S(IV). Despite the fact that many foods contain numerous known antioxidants to the oxidation of S(IV), it is well recognized that some (520%) of the additive becomes oxidized in food (Wedzicha and Herrera-Viloria 1991). The nature of the oxidizing agent, the catalyst and the type of oxidation taking place have not been established and so no hard and fast rules exist.
Oxidation of sulfur (IV)

There are several implications of S(IV) oxidation for food quality. The depletion of S(IV) after exposure to air can lead to an increased risk of microbial spoilage, so benzoate is often used in combination with sulte. Free-radical intermediates may be formed which are capable of initiating oxidation of other food components in the same way that OH or O2 do. The accepted role of S(IV) in foods with respect to oxidation is that of an antioxidant, despite evidence that the species can easily mediate the oxidation of unsaturated organic compounds in model systems. Such implications may be speculative and it is interesting to note that Til and Feron (1992) observed that an oxidized lipid fraction, which is peculiar to sulted animal diets, has some toxicological eect against rats. Low levels of sodium sulte have been implicated in causing oxidative-reductive depolymerization of polysaccharides (i.e. cassava starch), which can be prevented by the addition of the antioxidant propyl gallate (Mat Hashim et al. 1992).
Free and bound sulfur (IV)

The reversible binding of S(IV) is the reaction between carbonyl components of the food and S(IV) to form hydroxysulfonates (HS). The rate of decomposition of the HS depends upon the ease of proton removal, which in turn increases with pH. Furthermore, the ease with which the initial sulte attack takes place also increases with pH. The two eects are almost in
107
balance over the pH range of foods. However, this is only representative of a simple model and the case in foodstus is far more complex; here reactivity is dependent upon the nature of other functional groups in close proximity to the carbonyl moiety.
. Use of S(IV) as an additive for biscuit our. . In the non-reversible hydrolysis of S-sulfonates:
RSSO 3 H2 O ! RSH HSO4 :
Inhibition of non-enzymic browning (NEB)

It is generally observed that the levels of free and bound S(IV) in food decrease with time. Thus, the additive is converted by reaction to products that do not release S(IV) under conditions of analysis. Apart from sulfate, these products are organic sulfonates and can arise from the inhibition of NEB (i.e. Maillard and ascorbic acid browning). S(IV) is the only permitted additive known to be eective against NEB and consequently this property is of considerable technological value.
. Interaction with hydroperoxides via nucleophilic 2 displacement, i.e. SO2 3 ROOH SO4 ROH. At lower pH, the situation is more complex with the formation of ketones, olens and ethers in which there is evidence for the involvement of free radicals in solution. . Addition to Schi bases. The most likely reaction between a Schi base and S(IV) oxoanions is addition to the CN bond:
0 RCH NR0 HSO 3 ! RCHSO3 NHR
RCHSO 3 OH RNH2 ,
H2 O
Inhibition of enzymic browning (EB)

EB is the result of oxidation of phenolic and o-diphenolic compounds of plant material to o-quinones, catalysed by polyphenol oxidase. Whilst the enzyme is not inhibited by S(IV), the latter reacts with the quinone intermediates to give substituted dihydroxybenzenesulfonic acids. In foods, the substrates for polyphenol oxidase are often 4-substituted catechols, e.g. dihydroxyphenylalanine, which forms 3- or 5-sulfo derivatives.
where the reaction product of the corresponding aromatic amine appears to be stable to hydrolysis and exists as an amine salt. . Addition to olenic groups. The reaction of S(IV) oxoanions with carboncarbon double bonds may proceed by both heterolytic and homolytic routes, depending upon the nature of the unsaturated compound. The reaction is reported to proceed over a wide range of pH. For example, the sulfonation of the sodium salt of crotonic acid takes place in weakly acidic media and is unaected by the presence of oxygen or antioxidants. RCH CHCOOH
Other sulfur (IV)food component interactions of interest

The nucleophilic behaviour of sulte renders it able to react with any electron-decient chemical present in food such as in the following areas: . Cleavage of thiamine (see also below). . Cleavage of protein disulde bonds, which has been suggested as a reason for the observed inhibition of a large number of enzymes (and thence microorganisms) by sulte, which is reversible and subject to steric eects of P and P0 , and charged groups in the vicinity of SS:
0 PSSP0 SO2 3 P SSO3 PSH:
! RCSO 3 CH2 COH O:
SIV
. Interaction with isothiocyanates, e.g. as found in Brassicas and in some food avourings, where subsequent oxidation of the intermediate species produces a carbylamine and thiosulfate: RNCS KHSO3 ! RNHCS:SO3 K: 6
. Formation of anionic g-adducts with aromatic compounds such as anthocyanins. . Addition to pyridine compounds, e.g. NADH readily undergoes nucleophilic addition of S(IV) oxoanion at position 4. NADH also reacts with S(IV) by both free radical and ionic mechanisms to give a variety of products. Other reactions of
108
O Vitamin K 1 free radical mechanism
O Vitamin K 3
sulte (at least 10) are not well-dened, but are thought to be highly oxygenated, i.e. they have a relatively low sulfur content. The nal degree of S(IV)-mediated oxidation of b-carotene is similar to that resulting from its autoxidation (Neto et al. 1981). Catalytic amounts of Mn2 and glycine promote oxidation of S(IV) species in media high in alcohol, but at a slower rate than comparable aqueous systems. The S(IV)-mediated oxidation of food components is possible both in aqueous solution and in dispersions. However, the probability of such reactions taking place must be considered. It is likely that the success of the S(IV) oxoanion as an antioxidant (in foods) arises from the physical state of the substrate in contact with the aqueous medium and the presence of natural or added antioxidants in the aqueous and non-aqueous phases. The second possibility is demonstrated by the observation that when b-carotene is solvent-extracted from carrots, the crude product is less easily oxidized in the presence of S(IV) oxoanion than is pure b-carotene (B. L. Wedzicha and O. Lamikanra, unpublished results, 1982).
OH or SO 3 Na OH Thermodynamic product
SO 3 Na O Kinetic product
Figure 8. Reactions of S(IV) with K vitamins.
S(IV) that have been studied are those with pyrimidines and disuldes. . Quinines, such as vitamins K1 (natural product) and K3 (synthetic) (gure 8).
Uses of sulfur (IV) in food: general considerations sulte-mediated oxidations (Wedzicha 1984)
The pro-oxidant action of the sulte ion in systems is usually discussed in the context of lipid browning, which can be described basically as the interaction of carbonylic oxidation products of unsaturated fats with amines. An important step is the autoxidation of the sulte ion and interaction between oxidizing radical intermediates and the unsaturated compound. Other organic compounds aected in this way include methionine, which in the presence of Mn2 forms methionine sulfoxide (Yang 1970), tryptophan, which forms four dierent products (Yang 1973), and chlorophyll (Peiser and Yang 1977, 1978). Chlorophyll was rapidly destroyed in the presence of bisulte and linoleic acid hydroperoxide, both of which were necessary, but there was no oxygen requirement. Chlorophyll destruction occurred most readily at acidic pH, with little destruction occurring above pH 8. However, chlorophyll loss was monitored spectrophotometrically at 665 nm, thus subtle changes in porphyrin structure caused by the action of acid (loss of magnesium and phytyl side chain) and alkali (E-ring cleavage) were not taken into account. b-Carotene has been similarly studied (Peiser and Yang 1977, 1979, Wedzicha and Lamikanra 1983). The products of the reaction between b-carotene and
Eects of sulfur (IV) on avourings

The most important eects of S(IV) on avour are the results of interactions with the volatile components. A wide range of functional groups is encountered with these substances; an indication of the known and possible reactivity of avour compounds towards S(IV) oxospecies is given in table 3. For instance, the reaction between bisulte ion and carbonyls is reported to suppress the formation of oxidized avour in beer (Hashimoto 1972) and allyl isothiocyanate (from mustard) forms a non-volatile odourless sulfonate (Griths et al. 1980, Frijters et al. 1981). The interactions of S(IV) species and anthocyanins have already been discussed (see above). More specically, in wines, bisulte combines with anthocyanins, with pyruvic acid, and with 2-ketoglutaric acid. The product of the reaction with acetaldehyde is practically undissociable and the combined SO2 is totally ineectual as a preservative. A similar eect is observed when SO2 is combined with pyruvic acid when the acid concentration is high, because in this case the rate of dissociation is low. The reaction between SO2 and 2-ketoglutaric acid is more dissociable and can be perceived as a reservoir of free and active SO2 in wine (Usseglio-Tomasett 1992).
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Table 3. Reactivity of avour components of foods towards S(IV) oxospecies (Wedzicha 1984).
Reactivity towards S(IV) free radical? free radical? observed free radical? expected expected expected expected expected unlikely
N OCH3
Food
Compound
Group C C C C C C CHO C C CHO CHO CHO C C CHO

N R
Citrus fruits terpenes terpenoid alcohols and esters citral citronellal Apple aliphatic aldehydes hexanal hex-2-enal
reactivity of these two substances are dierent on account of the ionic environment with respect to the reactivity of SO2 3 and the non-ionic/micellar environment of sorbic acid. These observations allow the proposal of a general protocol for food additivefood component (and potentially for additiveadditive?) interactions in model systems, with particular emphasis on the presence of the appropriate ionic medium, surfactants, dispersed phases (e.g. oil), and dispersed proteins. Other, less studied components did not aect the stoichiometry of reactions, but may have a profound eect on the rate and yield of reaction product(s). These studies on the chemistry of S(IV) and sorbic acid led the authors to appreciate the diverse range of reaction products that are known to arise or that are possible, when these additives are used in food. Reaction with nitrite. The reaction between S(IV) oxo-anions and nitrite is discussed above.
Carrot Potato Cabbage
Onion Garlic
2-alkyl-3methoxypyrazines isothiocyanates suldes disuldes hydrogen sulde aldehydes thiols disuldes trisuldes hydrogen sulde allylisothiocyanate
RNCS R2S RSSR0 H2S CHO RSH RSSR0 RSSSR0 H2S RNCS
expected free radical? expected expected expected free radical? expected expected expected observed
Sorbates and other carboxylic acid preservatives

Thakur et al. (1994) have given a basic perspective on the chemistry of sorbates with respect to their use in foods. For example, the autoxidation products of sorbic acid in solution and in foods are reported to be associated with non-enzymic browning processes. The authors considered the many factors that aect sorbic acid stability, notably pH, temperature, aw, food composition and food packaging. The reactions of sorbic acid as an electrophile have been reviewed (Khandelwal and Wedzicha 1990a). The conjugated dienoic structure of sorbic acid renders it susceptible to nucleophilic attack. Nucleophiles known to react with sorbic acid include sulte ion and amines. These attack the sorbic acid molecule at position 5 and, in the case of amines, cyclization to form substituted dihydropyridones may follow. Recent investigations show that thiols in general can also add to sorbic acid (Khandelwal and Wedzicha 1990b). Cysteine, for example, reacts slowly with sorbic acid at 80 C and pH 5.5, leading to the formation of a 5-substituted 3-hexenoic acid. Reaction products are susceptible to acid- and base-catalysed hydrolysis. Alkylthiols give di-adducts with sorbate esters, whilst low molecular mass thiols containing an oxygen atom give a mono-adduct. Among the mechanisms that have been discussed is the reaction of sorbic acid with nitrite (see below).
Reaction with ascorbic acid (AA) (see also below)

In the presence of oxygen, AA is oxidized to dehydroascorbic acid (DHAA), which can react with S2O 5 to give the monohydroxysulfonate adduct. Under anaerobic conditions, dehydration and decarboxylation of AA can lead to the formation of 3,4-dideoxypentulos-3-ene, which can react with bisulte to give the corresponding 4-sulfopentosulose (Wedzicha 1984). Reaction with sorbate and benzoate. The interactions between SO2 and these two preservatives that can form ions, but which also dissolve in non-aqueous systems, have been used to illustrate the diversity of chemical interactivity possible between food preservatives and other food components (Wedzicha 1995) in the context of the relationship of such reactivity to food quality and safety. S(IV) and sorbic acid are the two most commonly used food preservatives world-wide and show considerable reactivity in the food environment. The factors that aect the
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3

OH O or Nu:
5
OH O
NHR O
OH and N R I II O N R III O
Nu:
Figure 9. Sites of nucleophilic attack on sorbic acid (Khandelwal and Wedzicha 1990a).
The C-3 site of sorbic acid has the lowest electron density (using NMR) and is thus the more likely site for nucleophilic attack by HNu, where Nu represents the nucleophile. Strong nuclophiles, such as amines and thiols (represented by Nu:), will attack at C-5 also and without the need for the initial protonation (gure 9). In this case, the addition of H takes place as the nal stage of the mechanism. Simple 1,2- or 1,4-addition of a strong nucleophile is generally under thermodynamic control, i.e. due to the relative stability of carbonium ion intermediates. The much greater extent to which the charge on the intermediate arising from attack on position 5 is delocalized, suggests that this should be preferred despite the lower electron density at position 3 of sorbic acid. The reaction is completed by addition of H to positions 2 or 4. The products thus formed depend upon the relative rates of H addition to dierent resonance forms of the carbonium ion intermediates (and somewhat on solvent polarity). The consequence of 1,2-addition is the possibility of adding a second nucleophile. Foods contain numerous nucleophiles; those most studied in reaction with sorbic acid are sulte and nitrite. The reaction between sulte ion and sorbic acid is well documented (e.g. Ha gglund and Ringbom 1926, Heintze 1976). Only the 1,4-addition product is formed as a labile 1:1 adduct.
Figure 10. Products of the high-temperature reaction of sorbic acid with ammonia and other amines (Khandelwal and Wedzicha 1990a).
with simple amines, such as butylamine or glycine, even after prolonged heating at 100 C in water at pH 57.
Reactions with thiols

Sorbic acid reacts slowly with thiols in aqueous solution at 80 C and pH 3.75.7 (Wedzicha and Brook 1989). The reaction of sorbic acid with mercaptoethanol, mercaptoacetic acid, cysteine, and glutathione are all second order. The adducts were dicult to isolate due to decomposition. Structural analysis was achieved using synthesized methyl and ethyl esters of the adducts. Under similar conditions, methyl 2-mercaptoacetate, ethyl 2-mercaptoacetate, and mercaptoethanol, all give the corresponding monoadducts. Alkylthiols (e.g. ethanethiol, 2-methyl-2propanethiol, butanethiol) react with sorbic acid esters to give only di-adducts, the proposed mechanisms being somewhat speculative. The situation is complicated by the competing thermodynamic, kinetic and steric factors implying a range of potential products.
Reaction with amines

Sorbic acid reacts with ammonia, alkylamines, aromatic amines, and benzylamine at high temperature (about 150200 C) to form (reportedly) the amino acids (I) and dihydropyridones (i.e. lactams II and III) as shown in gure 10, lactam II being thermodynamically favoured. There are important processing and toxicological considerations in relation to the use of sorbic acid in foods. For instance, in the grilling of cheese, sorbic acid may be exposed to high temperatures. Moreover, a large number of 3,4-unsaturated lactones have been reported to be physiologically active (Haynes 1948). There is no evidence to suggest that sorbic acid reacts
pH eects
Undissociated sorbic acid is more susceptible to autoxidation than dissociated sorbic acid and the process is thus inuenced by pH. The nature of other acids present is also important; HCl, HOAc, TFA and H2SO4 all exert a strong catalytic eect on the degradation of sorbic acid. This may be due to the reaction of the terminal hydroxyl group of sorbic acid with acid to form peroxy esters, which decompose to yield radicals (Pekkarinen and Rissanen 1966). However, there is no appreciable eect of various acids on sorbic acid stability in aqueous solution at 60 C (Petropavlovski and Usinova 1967). Note that dierent pH and temperature conditions
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were used in these studies. Vidyasagar and Arya (1983) investigated the role of various acids found in foodstus, where all the acids studied were reported to exhibit a catalytic eect, except citric and malic acids. It was suggested that acid specicity had a greater eect than overall pH.
tion. Dierential rates of sorbic acid degradation have been observed in sucrose and glycerol solutions; a protective eect may be due to the fact that in viscous solutions, diusion of oxygen through the aqueous layer becomes the rate-determining step in autoxidation.
Antioxidants
The degradation of sorbic acid is aected by antioxidants such as BHA, dodecyl gallate and nordihydroguaiaetic acid, which act by inhibiting free-radical mechanisms.
Ascorbic acid
AA has been observed to exert an inhibitory eect on sorbic acid degradation in fruit preserves. In aqueous systems, AA alone did not exert any inhibitory eect, but, in the presence of Fe3/Cu2, degradation was accelerated. In complex food systems, AA may compete with sorbic acid for available oxygen.
Transition metal ions

Those transition metal ions that possess two or more valency states with suitable redox potential between them are found generally to accelerate the rate of autoxidation. However, with sorbic acid, the eect is concentration dependent. At low concentration, autoxidation is enhanced, since it proceeds via the decomposition of peroxides to form radicals (or via the formation of free radicals following olenic bond cleavage). Conversely, at high concentration, autoxidation is retarded, due to the ability of the metal ions to complex with the peroxy radicals.
Amino acids and proteins

Amino acids and proteins, especially those containing free sulfhydryl groups, are found to interact with sorbic acid.
Reaction of sorbic acid and analogous food components with nitrite

Sorbic acid has been proposed as a partial replacement for nitrite in curing, since it inhibits Clostridium botulinum and also reduces the formation of nitrosamines (Ivey 1977). However, this practice may lead to other toxicological problems, since sorbic acid reacts with nitrite to yield mutagenic products (see below). According to Walker (1990), however, the conditions under which these compounds were shown to be formed (6090 C and high equimolar concentration) do not represent those which obtain in curing brines, and negative results have been recorded in mutagenic assays on curing brines containing sorbic acid. Nevertheless, the following investigations are worth noting. Osawa and Namiki (1982) examined the formation of mutagens by the reaction of sodium nitrite with some sorbic acid analogues using microbial mutagenicity tests (rec-assay and Ames test), together with chemical analysis using TLC. The conjugated dienoic carbonyl structure was reported to be essential for mutagen formation by the reaction with nitrite. For instance, the reaction of 2-furanacrylic acid
Other salts
The stability of sorbic acid in aqueous solution is enhanced by the presence of NaCl and KCl, particularly as their concentration increases. Conversely, stability is reduced in the presence of LiCl. Sulfates exhibit a strong pro-oxidant eect, but the eects of phosphate are more complex. The addition of Na2HPO4 increases the pH and hence the stability of sorbic acid, but, when the pH falls below the natural pH of sorbic acid (3.3), phosphate has a prooxidant eect. Bisultes and sulfur dioxide accelerate sorbic acid degradation.
Sugars and glycerol

At low concentration (< 5%), sucrose does not exert any signicant eect on sorbic acid stability, but, at high concentration, the rate of sorbic acid degradation is signicantly reduced in aqueous solu-
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O O (a) O N
and b-carotene with nitrite, both showed less than 20% of the starting material reacted, whereas piperine showed 2050% and methyl sorbate over 50% reacted. From a large-scale reaction of NaNO2 with methyl sorbate, 5-nitro-2,4-hexadienoic acid methyl ester and ethylnitrolic acid were isolated and identied as the main mutagens. The results implied that a nitro group adjacent to a double bond is an important factor in the development of mutagenic character. In a similar study, the development of mutagenicity by the reaction between sorbic acid and nitrite was reported to be highly dependent upon the reaction conditions, especially pH, reaching a maximum at pH 3.5, but being negative at pH 5 6 (Namiki et al. 1983). Most of the isolated mutagens were C-nitro and C-nitroso compounds. It was also concluded that the conjugated dienoic carbonyl structure is essential for mutagen formation, though details of the mechanism of formation were not clear. Among the food constituents tested was piperine, which formed strong mutagens. Ascorbic acid and cysteine were shown to inactivate the eective mutagenicities of the C-nitro compounds, which were reported to be more susceptible to being inactivated than N-nitroso mutagens. Osawa et al. (1982) proposed a mechanism for mutagen formation from the reaction between piperine (a) and nitrite. Initially, piperic acid (b) and piperidine (c) are formed, which both react with nitrite to give hitherto unknown C-nitro mutagens, including (d), along with N-nitrosopiperidine (e) (gure 11). The products of sorbate-nitrite interactions are among the putative mutagens and carcinogens in foods reviewed by Hartman (1983). Mutagen formation was reported to be blocked by ascorbate at low pH. Ascorbate at eightfold molar led to inactivation of 1,4-dinitro-2-methylpyrrole (DNMP) near neutral pH, but did not destroy the mutagenic nitro compound at low pH. It was concluded that the combination of sorbate with nitrite represents a potential health risk in the absence of adequate levels of ascorbate (cf. the comments made by Walker 1990). Osawa et al. (1986) reported further on the desmutagenic action of certain food components on DNMP. Whilst the results were somewhat inconclusive, it was proposed that (1) ascorbic acid specically reduces the 3-nitro or 4-nitro groups on pyrrole or furan rings to corresponding amino groups, (2) the mechanism for inactivation by cysteine involves direct attack on
OH O O NO/NO 2 (b) O + (c) HN
NO/NO 2
O O (d) + unknowns
CHO NO 2
ON (e)
Figure 11. Proposed mechanism for mutagen formation from the reaction between piperine (a) and nitrite (Osawa et al. 1982).
the aromatic ring of DNMP, followed by elimination of the C-nitro group and (3) reducing substances responsible for desmutagenic action against the products of the sorbic acid/nitrite reaction are present in vegetable juices. The principal nitrite-derived reactant is reported to be N2O3, since it is well established that it may be polarized as NO2 NO for electrophilic nitration. Such species are known to add across double bonds (Park and Williams 1976). Evidence for the initial product (a) and its subsequent isomerization to the oxime (b) is the isolation of the furoxan (e). The formation of structures, which may eventually degrade to nitrolic acid-type moieties, may only be conceived via a Beckmann rearrangement of (b). Product (c) is ethylnitrolic acid and product (d) is DNMP (gure 12).
Ascorbic acid
The interaction between AA and azo dyes, natural colours, sulfur (IV) species and sorbic acid have been discussed. Reaction with nitrite has also been discussed briey.

O NO2 O
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OR ON
OR
(a)
NO2
NO2
OR H3C
N
NO2
(d)
HO
(b)
O H3C NO2 H3C OH N HO
(c)
N (e)
Figure 12. Products formed from the interaction of nitrite and sorbate (Osawa et al. 1986).
Reaction with nitrite during curing

According to Fox et al. (1981) and Sebranek and Fox (1985), during the curing of meat, nitrite can react with AA under certain pH conditions to form dinitrosylascorbate. Under anaerobic conditions, the decarboxylation of AA can lead to the formation of 4,5-dihydroxy-2-oxopentanal, which can potentially react with nitrite (Wedzicha et al. 1982).
alone. However, no information was given on the degradation of aspartame under the given conditions.
Reaction with benzoate

Following the reports in 1990 of bottled water contaminated with benzene, test laboratories stumbled over the nding of benzene in other beverages. McNeal et al. (1993) examined the potential for benzoate and AA to react to produce benzene. Less than 2 mg kg1 benzene was found in beverages without added benzoate, whereas with added benzoate the levels were up to 38 mg kg1. A model beverage system containing 0.04% potassium benzoate and 0.025% AA was investigated. Exposure to UV light for 20 h at 45 C produced 300 mg kg1 benzene, which was constant after 8 days. At room temperature in the dark, 4 mg kg1 benzene were produced after 20 h, which increased to 266 mg kg1 after 8 days. In a separate series of experiments, 0.025% AA was combined with 0.04% of various compounds to produce the yields of benzene given in table 4. The decarboxylation of benzoate is a well-known probe for hydroxyl radicals (Winston and Cedarbaum 1982) and it seems likely that this is the mechanism for benzene formation in the beverages (Gardner and
Reaction with intense sweeteners

Since articial sweeteners are commonly used in beverages containing AA, the potential for interaction is of interest. The degradation of aspartame and acesulfam K in acidic aqueous solution at high temperatures is accelerated by the presence of AA (Kroyer et al. 1993). Under such high temperature conditions, AA degradation was reported to take place rst. Hsieh and Harris (1991) reported that the cupric ion-catalysed oxidation of AA occurs more rapidly in the presence of aspartame and concluded that this was presumably due to the formation of a complex between cupric ion and aspartame, which is a more eective oxidation catalyst than cupric ion
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Table 4. Summary of benzene yields resulting from the reaction between ascorbic acid and various compounds (McNeal et al. 1993).
Compound Benzaldehyde Benzoic anhydride Acetophenone Toluene Benzyl alcohol BHT BHA
a
Benzene yielda 74 ng g1 4 ng g1 5 ng g1 not detected not detected not detected not detected
Experimental conditions given in the text.
Lawrence 1993). The role of AA is probably as a source of reducing equivalents in the Udenfriendtype generation of hydroxyl radicals from molecular oxygen and a transition metal-chelate catalyst (Castle 1980). Even traces of metals in potable water, or in other ingredients used to make beverages, are sucient when combined with AA, to produce the levels of hydroxyl radicals responsible for formation of such low levels of benzene.
Upon heating, aspartame cyclizes to 5-benzyl-3,6dioxo-2-piperazineacetic acid (DKP) with one molecule of methanol liberated in the process. Slow formation of DKP may also be observed when aspartame is dissolved in aqueous solution. Up to 2% DKP was reported to be formed from an aqueous solution of aspartame after standing at 22 C for 4 h (Tsang et al. 1985). Other degradation pathways involve the formation of phenylalanine methyl ester and aspartylphenylalanine, which may undergo further hydrolysis to aspartic acid and phenylalanine. According to Vetsch (1985), the stability of aspartame is aected by time, temperature, and pH the latter being of particular importance, optimum stability being at pH 4.3. At elevated temperatures, such as those found in short-time pasteurization (80 C), aspartame becomes less stable, notably in the pH range 67. It was suggested that the decomposition of aspartame by ultrahigh temperature (UHT) processing, as used in aseptic processing, can be ignored since the time involved is too short for substantial degradation to take place. This view may or may not be correct, depending on the activation energy for the degradation process(es). Other intense sweeteners. Acesulfam K is reported to be highly stable in aqueous solution and at temperatures encountered during food processing, and no indications of undesired reactions with food ingredients have been reported (Von Rymon Lipinski 1985, 1995, Mayer and Kemper 1991). Neohesperidin dihydrochalcone (NHDC) has been reported (Borrego 1995) to show similarly good stability, no degradation being detected by HPLC in unavoured yoghurt fermentation (42 C for 6 h), blackcurrant jam manufacture (pH 3.0, 102106 C for 3540 min), and pasteurization of soft drinks, based on apple, pineapple, orange, or lemon juice (90 C for 30 min). HPLC data from stability studies on NHDC in aqueous buers from pH 1 to 7, with storage for up to 20 weeks at temperatures of between 30 and 60 C, as determined by Canales et al. (1993), have been examined by Lindley (1996). He showed that the breakdown of NHDC can be represented as a pseudo-rst-order reaction at all temperatures and pHs studied. Maximum stability was observed at a pH of about 4.0. Along with other stability data derived from model food systems, this led to NHDC being considered to pose no signicant stability problems for food manufacturers, even though there is a theoretical possibility of hydrolytic cleavage of the glycosidic linkage.
Other additives Intense sweeteners (see also above)

Information on the chemical interactions between intense sweeteners and other food additives/food components is sparse. Much of the published works has focused on stability and degradation studies in foods and model systems, including identication of reaction products. Here, these additives are discussed mostly from the point of view of the degradation products produced and their potential reactivity. Aspartame. Aspartame is the methyl ester of the dipeptide, aspartylphenylalanine, which is susceptible to degradation under a wide range of conditions (heat, moisture and pH). The stability of aspartame has been examined by Homler (1984), who showed that a cola-based soft drink, containing aspartame and stored at 20 C for 6 months, remained acceptably sweet. The levels of aspartame and its condensation and degradation products were not reported. Hussein et al. (1984) showed that aspartame is quite reactive towards aldehydes, which are among the principal avour compounds used in chewing gums and certain soft drinks.
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Saccharin and cyclamate have been reported to be highly stable in both bulk form and in solution. Stability has been proven over a wide range of pHs and temperatures (Beck 1983, Salminen and Hallikainen 1990, Mikuschka 1995). According to Beck (1983), the low solids content of some food products sweetened with non-nutritive sweeteners may render them susceptible to microbial spoilage. Consequently, it becomes especially important to use preservatives, such as sorbate or benzoate, or to employ thermal processes to achieve adequate shelf life. However, there is some controversy over the stability of saccharin and cyclamate. Kroyer and Washuettl (1982) have reported on the possible interactions of these sweeteners with food ingredients, particularly water-soluble vitamins and essential amino acids. Stability studies in model aqueous systems at ambient temperature and at 80 C showed no signicant losses of sweeteners over 28 days, except for systems containing phenylalanine and tryptophan, where reductions of up to 13 and 47% were observed for tryptophan in the presence of saccharin and cyclamate, respectively, at ambient temperature. In the same experiment, corresponding reductions in phenylalanine of 10 and 17% were observed in saccharin and cyclamate systems, respectively. Control systems containing sucrose showed up to 3% reduction of the amino acids. Mixtures of vitamins B2, B6 and B12 with sucrose, saccharin and cyclamate in solid form indicated no signicant losses after heating for 1 h at temperatures of 120 and 150 C, but distinct changes of the vitamins B1 and C were observed, particularly in the presence of saccharin (33% reduction of B1 and 93% reduction of C at 150 C). Under extreme conditions, cyclamate is known to break down to cyclohexylamine, small quantities of which were detected in model systems heated between 150 and 200 C. Further studies on saccharin and cyclamate stability during the preparation of foods, such as cake, jam, punch, coee and vinegar dressing, again showed various amounts of sweetener loss. These ranged between 0% (punch) and 64% (cake) for saccharin, and between 1% (punch) and 17% (vinegar dressing) for cyclamate. However, there was no direct evidence for the interaction of the sweeteners with other food components.
with iron and particularly with ferric iron. Kearsley and Birch (1985), considered it likely that the pHdependent chelation of inorganic species is not restricted to iron, but that all metal ions will be complexed to a greater or lesser extent. Whilst the complexing of metal ions with carbohydrate may ameliorate the deleterious eects of certain metal species, it is also possible that the bioavailability of certain trace elements such as iron may be compromised. Moreover, addition of carbohydrate (hydrogenated glucose) has been implicated in the inhibition of copper-catalysed oxidation of AA (Cross et al. 1984).
Interactions between micronutrients and food additives

Cort (1987), reviewing the eects of food additives on nutrition, reported the following points.
Sultes
Sultes cause a nucleophilic displacement on the methylene bridge of thiamine, which is thus cleaved to form the free thiazole and the pyrimidylmethanesulfuric acid. The reaction occurs faster at neutral pH. According to DeRitter (1969), SO2 will also cleave folic acid to pteridine and p-aminobenzoylglutamic acid moieties, where the amino group of the latter undergoes further destruction.
Acids and alkalis

In liquid or semiliquid foods adjusted to pH 4 or below, vitamin A esters will de-esterify and isomerize to the less bioactive cis-forms of retinol. Folic acid, pantothenic acid, and threonine would be expected to decompose under acidic conditions. Cort (1987) cites the work of Moore and Folkers (1968), who showed that dilute acids cleaved vitamin B12 to the inactive corrin nucleus. Aluminium sodium sulfate and the di- and tri-sodium potassium phosphates aid in the destruction of thiamine. Several breakdown products have been identied from alkaline oxidation of thiamine (Dwivedi and Arnold 1973). These include the ring-opened thiamine disulde, which has thiamine activity, along with thiochrome and thiothiazolone,
Starch-based sweeteners
It has been shown that all common carbohydrates, including glucose syrups, have the ability to complex
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which do not. In addition, thiamine may be cleaved under these conditions to the pyrimidine and thiazole moieties, where the latter may further degrade to a number of compounds, including 3-mercaptopropanol, hydrogen sulde, 2-methylfuran, 2-methylthiophene, 2-methyl-4,5-dihydrothiophene, and 2-methylthio-5-methylfuran.
Smoking and curing

Modern mild curing and smoking methods employ small amounts of salt, sugar, nitrite, ascorbates, phosphates, and liquid smoke condensates in processed meat, poultry and sh products. Losses of specic nutrients due to curing appear to be very small. Thiamine is reported to suer up to about 20% losses during heat treatment in the curing process, whereas riboavin and niacin are very stable (Schweigert and Lushbough 1960).
subsequent measurement of Fe2, has been the basis of the colorimetric assay for tocopherol for many years. Tocopherol will react to form the p-tocopherolquinone under most conditions. However, when EDTA is present, the dimeric keto-ether is formed (Cort et al. 1974). Fe2, Cu and Cu0 are reported not to react with tocopherol. Cu2, as shown by Dwivedi and Arnold (1973), also destroys thiamine (see above). In a separate review article, Borenstein (1971) reported that copper catalyses folic acid destruction. Methionine and linolenic acid, as well as a number of other substrates, have been reported to be degraded to ethylene by AA and copper (Liebermann and Kunishi 1967).
Other reactions detrimental to nutrients

Feller and Macek (1955) have shown that thiazole, produced as a product of thiamine decomposition, promotes the breakdown of vitamin B12. This is a problem in pharmaceutical preparations and, in fact, according to DeRitter (1969), most of the information on vitamin interactions is to be found in pharmaceutical research publications. Fortunately, the thiamine levels in most foods are so low that this is not a major pathway for vitamin B12 destruction. Gambier and Rahn (1957) have shown that riboavin aids the oxidation of thiamine to thiochrome in concentrated solutions. Niacinamide and AA react together to form a yellow-coloured nicotinamide-AA complex. Amen (1973) reported that phosphates, phytates, and oxalates in spinach can complex iron and thus compromise its bioavailability. Carbonyls present in avour adjuncts can react with pyridoxamine to form inactive Schi bases, and pyridoxal could react with amines similarly. Schroeder (1971) reports losses of natural pyridoxal and pyridoxamine, as well as of pantothenic acid, biotin, folacin, choline, and inositol in food processing, where some of the vitamin B6 group losses were thought possibly to be due to interactions.
Copper and iron

Other than from the use of copper complexes of chlorophylls and chlorophyllins, the use of copper with respect to food additives is very small. Conversely, iron is added to many foods. Because their reactions are reported to be somewhat similar and more detrimental to other nutrients than is generally recognized, they are here discussed together. Ascorbic acid is known to be destroyed by copper. AA is rst converted to DHAA which itself is unstable, degrading further to diketogulonic acid, which has no bioactivity. Conversely, Cu2 is reduced by AA to lower oxidation states, as is Fe3 to Fe2 (Laurence and Ellis 1972). According to Jukes (1974), Cu can reduce peroxides (and hydroperoxides) to form a copper Cu2 compound and an oxygen-containing free radical, which can abstract hydrogen from a variety of compounds to form carbon free radicals. The carbon free radicals can then be oxidized by Cu2 compounds to liberate Cu. In addition, alkyl radicals can be generated directly from the reaction between Cu and diacylperoxides. The interactions of Fe3 and Cu2 with phenolic antioxidants have also been examined (Cort 1974, Cort et al. 1974). Cu2 reacts with tocopherol to form the p-tocopherolquinone, which has no vitamin E or antioxidant activity. Reduction of Fe3 to Fe2, with the
Biological eects
The interactions of food additives and nutrients have been reviewed by Vanderveen (1988), who focussed largely on food additives as dened by the US FDA, i.e. as functional additives, and did not, for instance, include colouring materials. The review is a general
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one of the types of interactions between food additives and nutrients that have been identied. The author addresses the criteria by which food additives should be reviewed for approval. Additive nutrient interactions were categorized in terms of the following biological eects, which in some cases may occur simultaneously: . Decreased nutrient absorption, e.g. EDTA chelation of mineral elements, and the interaction of caeine with riboavin (Jusko and Levy 1975). . Enhanced nutrient absorption, e.g. the EDTAmediated mobilization of Zn, Mn, Fe, the AAenhanced bioavailability of Fe, and the enhanced (albeit conjectural) bioavailability of Ca by citric and malic acids. . Altered nutrient metabolism, e.g. the metabolism of BHT by cytochrome P450 enzymes and implication of its role in the inhibition of vitamin K absorption; the interaction of vitamin B6 with monosodium glutamate, and the basis of Chinese restaurant syndrome (Folkers et al. 1984). . Altered nutrient excretion, e.g. the enhanced excretion of vitamin C following consumption of hemicellulose (Keltz 1978). . Nutrient destruction, e.g. the addition of pHlowering substances which may cause the destruction of the polyglutamate forms of folacin, whereas the addition of pH-raising substances can increase the destruction of thiamine (Sauberlich 1985). Basu et al. (1984) reported on the in vitro eect of AA on the spontaneous reduction of sodium nitrite concentration in reaction mixtures.
Main outcomes and recommendations

It is well established that food additives (and to a lesser extent their degradation products) can interact with one another and with food constituents to form an array of new products. In some instances, such products have been identied, whereas, in others they are poorly characterized or remain of unknown structure. The consequences of such interactions are the main consideration here, with food safety implications being of particular concern. From the information and data in the key references and other important articles relating to additiveadditive and additivefood component interactions, it is clear that the bulk of the research eort in this area has targeted ve main classes of food additive: the sulfur (IV) species of preservatives, synthetic food colouring materials, nitrate and nitrite, ascorbic acid, and sorbic acid, though the consequences of these interactions are by no means fully understood as yet. The availability of this information in the literature has been summarized in table 5. The eects of pH, temperature, light, reactive species, oxygen, and other parameters on the reactivity of these food additives have been discussed. Other important aspects that have been addressed include the formation of toxic reaction products and mutagenic compounds, protective eects and inhibition, kinetics and mechanisms, and where appropriate the likelihood and signicance of additive reactions in food and model systems. The amount of available information on the interactions of certain additives, which may have the potential for interaction is sparse, e.g. intense sweeteners, preservatives, gallate esters,
Table 5. Summary of published literature on additiveadditive interactions.

Ascorbic Nitrates/ Sorbic acid/ Sulfur (IV) Synthetic Natural food acid nitrites sorbates oxo-anions dyes colours Ascorbic acid Nitrates/nitrites Sorbic acid/sorbates Sulfur (IV) oxo-anions Synthetic dyes Natural food colours Other additives intense sweeteners, benzoate, metal cations vitamins, phenols, antioxidants, amines, thiols vitamins, olens, amino acids acids, alkalis, preservatives, sugars metal cations, preservatives, antioxidants, sugars, acids, alkalis
None found; sparse; signicant.
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sorbitol, mannitol, polyoxyethylene stabilizers, and monosodium glutamate. (Note: because of the considerable amount of complex information available on the chemistry of caramels, this group of food additives was not included, except where appropriate to discussions on other additives.) The main conclusions to be drawn from this review in terms of future requirements are the following: . Research eort in this area has been largely targeted towards ve main classes of food additive: the sulfur (IV) species of preservatives, synthetic food colouring materials, nitrate and nitrite, ascorbic acid, and sorbic acid. Even so, the consequences of their interactions are not fully understood. . Information on certain additives, which have the potential for interaction is sparse and these may merit further studies, e.g. . intense sweeteners (saccharin, acesulfam K, aspartame, cyclamate, NHDC); . preservatives ( p-hydroxybenzoates, biphenyl, thiabendazole, o-phenylphenol); . gallate esters; . sorbitol, mannitol; . polyoxyethylene stabilizers; . monosodium glutamate; and . others, e.g. bulk additives, modied atmosphere gases. . There is a clear requirement for a coordinated approach to research on food additiveadditive interactions, especially for those additives that may be of concern. . There is a need to develop our understanding of the important parameters that dictate the outcome of additive interactions. This might be achieved by using the literature data within the framework of the review essentially as a training set to evolve the framework into a diagnostic tool. . There is a need to formalize our understanding of the type of information required to indicate where further studies (if any) are required to allow a strategic assessment of additive interactions. Other workers have sought to formalize schemes for assessing priorities for safety (toxicity) testing of food additives, based upon a quantitative structure activity relationship (QSAR) approach. Phillips et al. (1987), for instance, used a modied decision tree scheme, based on that of Cramer, Ford and Hall, to allow more chemical structures to be considered and to take into account advances in toxicology.
The majority of food additives (then) permitted in either the UK, USA or Canada were processed through a modied decision tree questionnaire and their classication compared with available chronic toxicity data. Additives were then assigned to three toxicity classes, low, moderate, or high. Although less discriminating than originally suggested, it was concluded that the decision tree approach remains a useful method for classifying compounds in terms of their probable toxicity and that further modications to the tree could be made in the light of the occurrence of incorrect assignments. This type of approach is therefore worthy of consideration, especially for assessing the potential reactivity of groups of additives that have similar chemical functionality. As mentioned above, it is feasible to regard the information summarized here as a training set to develop our understanding of the important parameters that dictate the outcome of additive interactions. This could be achieved by assessing the data within a framework, which could develop into a diagnostic tool. The framework (and the relational database in which it would operate) would formalize our understanding of what information is needed and so would automatically indicate where further studies (if any) are required to allow a strategic assessment of additive interactions. In order to predict and assess the outcome of additiveadditive or additivefood encounters, a provisional set of reaction parameters must be assembled. With a basic framework in place, the data obtained from the literature could be used as a Teaching Set to identify any factors overlooked or, conversely, factors proposed that have in fact negligible consequence. Thus, a multilevel frame work operating systematically may be developed along the following lines: Level 1. Description of food additives: . Concentration (e.g. bulk additives, gums and bulk sweeteners versus trace additives, colours and preservatives). . Reactivity (e.g. electrophilic, nucleophilic, redox-active, free-radical precursor, chelator, light or heat-sensitive). Level 2. Description of food components: . Concentration (e.g. macro-constituents, lipids, proteins and carbohydrates versus micro constituents, vitamins and minerals). . Reactivity (e.g. electrophilic, nucleophilic, redox-active, free-radical precursor).
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Level 3. Likelihood of encounter: . Permitted additivefood combinations (according to EU Directives): . Occurrence. . Concentration range. . Actual food-additive combinations (according to Food Standards Agency [FSA] database on additive usage): . Occurrence. . Concentration range. Level 4. Likelihood of interaction during processing and storage: . Chemical compatibility. . Reactivity of the additive with the additive or food component that it encounters, e.g. nucleophile-electrophile, oxidant-oxidizable substrate, free-radical precursor-autoxidizable substrate, nitrosating agent-nitrosatable substrate. . Time, temperature, pH, water activity. The types of food in which the additive is permitted give the reaction conditions that should be considered with respect to pH and water activity. The time and temperature parameters can be taken from the foreseeable conditions of use (e.g. a chilled dairy product or an extrusion-formed and heat-dried snack product). It is likely that knowledge will be incomplete in this area. The use of chemical modelling to predict the products that may be formed is a foreseeable requirement, as is the use of, for instance, NMR and radio-tracer experiments, to determine the fate of reactive additives. Level 5. Consequence of the interaction: . Known or predictable products that can be assessed toxicologically (e.g. by QSAR). . Unknown and unpredictable products that require identication. . Known or unknown products that nevertheless are bound to macromolecules and not absorbed.
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Acknowledgement
Financial support for this work was provided by the UK Food Standards Agency.
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Food Additives and Contaminants, Vol. 21, No. 2 (February 2004), pp.

Chemical interactions between additives in foodstus: a review

(Received 3 March 2003; revised 15 September 2003; accepted 30 September 2003)

M. J. Scotter and L. Castle

Chemical interactions between additives in foodstus

Reactions of nitrate and nitrite with specic additives

Nitrates and nitrites

M. J. Scotter and L. Castle

DHNO CH3 COO H2 O:

Chemical interactions between additives in foodstus

OH NO2 BHA-NO2 OCH 3

Synthetic food dyes

M. J. Scotter and L. Castle

Table 1. EU-permitted synthetic food colouring materials with numerical ADIs.

(I) O 1[O ] 2 N H (II) N . O (III) O

Chemical interactions between additives in foodstus

Interaction of synthetic food dyes with other food additives

M. J. Scotter and L. Castle

Chemical interactions between additives in foodstus

M. J. Scotter and L. Castle

Other food colours

Chemical interactions between additives in foodstus

M. J. Scotter and L. Castle

Chemical interactions between additives in foodstus

Sulfur (IV) species SO2 and sultes

M. J. Scotter and L. Castle

Chemical reactivity of sulfur (IV)

Oxidation of sulfur (IV)

Free and bound sulfur (IV)

Chemical interactions between additives in foodstus

Inhibition of non-enzymic browning (NEB)

Inhibition of enzymic browning (EB)

Other sulfur (IV)food component interactions of interest

! RCSO 3 CH2 COH O:

M. J. Scotter and L. Castle

O Vitamin K 1 free radical mechanism

Figure 8. Reactions of S(IV) with K vitamins.

Eects of sulfur (IV) on avourings

Chemical interactions between additives in foodstus

Group C C C C C C CHO C C CHO CHO CHO C C CHO

Carrot Potato Cabbage

Sorbates and other carboxylic acid preservatives

Reaction with ascorbic acid (AA) (see also below)

M. J. Scotter and L. Castle

Reactions with thiols

Reaction with amines

Chemical interactions between additives in foodstus

Transition metal ions

Amino acids and proteins

Reaction of sorbic acid and analogous food components with nitrite

Sugars and glycerol

M. J. Scotter and L. Castle

OH O O NO/NO 2 (b) O + (c) HN

Chemical interactions between additives in foodstus

O H3C NO2 H3C OH N HO

Reaction with nitrite during curing

Reaction with benzoate

Reaction with intense sweeteners

M. J. Scotter and L. Castle

Experimental conditions given in the text.

Other additives Intense sweeteners (see also above)

Chemical interactions between additives in foodstus