14C-sorbic acid distribution in the aqueous and non-aqueous extracts of cooked millet dough (fura).

Millet flour was used to prepare the West African food, fura, preserved with sorbic acid. The lipid and protein fractions which appeared to bind sorbic acid or its degradation products were identified with the help of 14C-sorbic acid. Fractionation of the lipids and proteins from fura showed the 14C to be associated with the petroleum spirit crude fat extract (28 +/- 5%), the water-soluble (6.2 +/- 0.7%), the ethanol-soluble (4.5 +/- 0.7%), the salt-soluble (3.5 +/- 0.6%) and the acetic acid-soluble components (0.9 +/- 0.3%). Dialysis of the ethanol-, salt- and acetic acid-soluble fractions suggested that 14C activity tended to be associated with the low molecular weight components of these fractions.

Reaction of sorbic acid in millet and sorghum doughs: reaction with thiols.

The fate of sorbic acid in pearl millet and sorghum doughs on cooking and storage is reported. A solution of sorbic acid added to the cereal flours gave a dough, which was cooked to give the product 'fura'. A significant amount (40%) of the sorbic acid was not recovered from the dough and fura on extraction with methanol. The use of acidified methanol leads to a quantitative recovery of that proportion of the preservative which otherwise becomes unavailable during the cooking of the dough and evidence is presented to suggest that sorbic acid-thiol adducts are formed at that stage.

Chemistry of sulphiting agents in food.

The main reason for the reactivity of sulphites in food is the nucleophilicity of the sulphite ion. The factors which determine the activity of this nucleophile are summarized and critically evaluated for concentrated systems, e.g. dehydrated foods. The distinction between free and bound sulphite is explained, and reversible binding of the additive in beverages and dehydrated foods is discussed with reference to simple theory of chemical equilibrium. The inhibition of non-enzymic browning reactions accounts for a large proportion of sulphite which undergoes irreversible reaction in concentrated foods. The mechanisms of reactions between sulphite species and intermediates in the model Maillard reaction, glucose+glycine, are considered in depth together with supporting kinetic data. An interesting feature is the fact that sulphites seem to catalyse the reactions they are added to control. Implications of this to the level of use of sulphite are discussed. Reaction products from the inhibition of Maillard browning include 3,4-dideoxy-4-sulphohexosulose which is formed initially and polymeric substances which arise from the reaction of sulphites with melanoidins. A proportion of sulphite added to food becomes converted to sulphate. Mechanisms of autoxidation are critically appraised in view of the presence of considerable concentrations of antioxidants in foods. The autoxidation of sulphite involves reactive free radical intermediates which include effective oxidizing agents. Thus, a pro-oxidant effect by the additive is possible and demonstrable in model system experiments.

Kinetics of the sulphite-inhibited browning of fructose.

Sulphite species, S(IV), inhibit the non-enzymic browning of fructose-amino acid mixtures. Inhibition of browning is accompanied by a loss of S(IV). The kinetics of the reaction of S(IV) in the system: fructose-glycine-S(IV) are described in detail; two distinct mechanisms have been identified. One involves only fructose in the rate determining step. The other requires fructose and both the glycine and S(IV). Some amines (e.g. taurine and ethanolamine) can markedly increase the rate of the S(IV)-independent step. Arginine and lysine are particularly effective for increasing the rate of reaction of S(IV) in the S(IV)-dependent reaction.

Reaction of sorbic acid in wheat flour doughs: reaction with thiols.

The general characteristics of the reaction between sorbic acid and thiols are reviewed. Cysteine adds to the conjugated diene in position 5 to form the substituted 3-hexenoic acid. This is labile in acid solution, yielding a quantitative amount of sorbic acid. When wheat flour doughs are treated with sorbic acid and heated, a significant amount of the sorbic acid is not recovered on extraction with methanol. The use of acidified methanol leads to a quantitative recovery of the preservation and evidence is presented to suggest that sorbic acid-thiol adducts are formed. This is the first report of 'reversibly bound' sorbic acid in a food.

Kinetics of the inhibition of ascorbic acid browning by sulphite.

Despite differences in the structures of aldoses and ascorbic acid, ASA, the non-enzymic browning of the latter involves intermediates similar to those found in Maillard browning. The kinetics of the sulphite-inhibited browning of ASA suggest that, under anaerobic conditions, the rate of reaction of sulphite species, S(IV), is of first order with respect to S(IV). The possibility that S(IV) could catalyse the hydrolysis of the lactone ring of ASA is considered by reference to D-glucono-delta-lactone. Evidence is presented to suggest that, under aerobic conditions, autoxidation of ASA leads to the oxidation of S(IV). The composition of melanoidins from ASA-glycine mixtures is compared with that from arabinose-glycine; the former contain 2 x 5 times more ASA-derived residues per glycine molecule than arabinose-derived residues per glycine molecule in the latter. The implications of these findings to the mechanism of ASA browning are discussed.

Kinetics of the reaction of sorbic acid with sulphite species.

Under aerobic conditions, the reaction of sorbic acid with sulphite species, S(IV), involves a pH-dependent oxidative mechanism with the loss of S(IV). Sorbic acid is essential to the process. In the absence of air, a much slower nucleophilic addition of sulphite ion to undissociated sorbic acid takes place.

Kinetics and mechanism of the reaction between 3-deoxyhexosulose and thiols.

The kinetics of the reaction of 3-deoxyhexosulose, DH, with mercaptoethanol, ME, and glutathione, GSH, resemble those of the DH-sulphite reaction, but the stoichiometry of the DH-thiol reaction is 1:2 unlike that of the DH-sulphite reaction which is 1:1. However, the rate determining step in all these reactions is the spontaneous conversion of DH to a reactive intermediate, followed by a rapid reaction of this intermediate with the nucleophile. This is also true of the reaction between DH and N-acetylcysteine, NAC, but this thiol is less reactive than ME or GSH and less than one mole of NAC reacts with each mole of DH. Evidence for instability of NAC at pH 5.5 is presented. Aminothiols (cysteine, homocysteine, cysteamine) undergo a fast reaction with DH followed by a slow release of thiol. The initial reaction is probably formation of a thiazolidine. In the case of cysteine and homocysteine it is suggested that the subsequent slow step is a Strecker degradation reaction. The kinetic behaviour of thiols in cabbage homogenates is reported.

Formation of sulphate ion during the dehydration of sulphited vegetables.

A reverse isotope dilution procedure was used to measure the formation of sulphate ion during the air dehydration of sulphited cabbage, carrot and potato at 80 degrees C. The conversion of sulphite to sulphate was found to be of first order with respect to sulphite. The formation of sulphate in the vegetables during dehydration accounts for only part of the sulphate in the dehydrated products; the remainder comes from the scald liquor. The technique for sulphate determination is critically evaluated and the observed yields discussed.

Inhibition of browning by sulfites.

The present state of understanding of the mechanisms by which sulfites inhibit browning reactions in food is reviewed. The difficulties of specifying the composition of sulfur(IV) oxospecies in sulfited foods arise from the existence of labile equilibria between SO2, HSO3-, SO3(2-) and S2O5(2-), whose position depends on concentration, ionic strength and the presence of non-electrolytes. A proportion of the additive is also found in a reversibly bound form. The main reason why sulfites are able to inhibit a wide range of browning reactions is the nucleophilic reactivity of sulfite ion. The mechanism of reactions between sulfite species and intermediates in the model Maillard browning reaction, glucose + glycine, are considered in depth and are supported by kinetic data. A most interesting feature is the fact that sulfites seem to catalyse the reactions they are added to control. Reaction products include 3,4-dideoxy-4-sulfohexosulose which is formed initially and polymeric substances arise from the reaction of sulfite species with melanoidins. It is found that melanoidins from glucose + glycine react with sulfite to such an extent that one sulfur atom is incorporated for every two glucose molecules used to make up the polymer. The mechanisms of inhibition of ascorbic acid browning, enzymic browning and lipid browning are reviewed briefly. The known toxicological consequences of the formation of reaction products when sulfites are used for the control of Maillard browning give little cause for concern. Little is known of the implications of the formation of reaction products during the inhibition of other forms of browning. Consideration of the requirements for alternatives to sulfites is given.

Nucleophilic reactions of sorbic acid.

The conjugated dienoic acid structure of sorbic acid renders it susceptible to nucleophilic attack. Nucleophiles known to react with sorbic acid include sulphite ion and amines. These attack the molecule in position 5 and, in the cse of amines, cyclization to form substituted dihydropyridones may follow. Recent investigations show that thiols in general can also add to sorbic acid. Cysteine, for example, reacts slowly with sorbic acid at 80 degrees C and pH 5.5, leading to 5-substituted 3-hexenoic acid. In general, reaction products are difficult to isolate from aqueous reaction mixtures as they are susceptible to acid- and base-catalysed hydrolysis. A synthesis of model compounds may be carried out by reaction of sorbate esters with the appropriate thiol (or its ester if it is an acid) in the presence of the corresponding sodium alkoxide. It is interesting that alkyl thiols give di-adducts with sorbate ester whilst low molecular weight thiols containing an oxygen atom give a monoadduct. The mechanism of this reaction and its implications to the preparation of samples for toxicological evaluation are discussed. The reaction of sorbic acid with nitrite ion is unusual and its mechanism is considered.

Effect of surfactants and dispersed components on the activity and reactivity of sorbic acid.

In common with many other carboxylic acids, sorbic acid shows significant solubility in aqueous and non-aqueous solvents. The presence of a non-aqueous phase (e.g. fat) can markedly affect the concentration of the preservative in the aqueous phase. Solute distribution between the two phases is pH- and concentration-dependent. The presence of dissolved surfactants in the aqueous phase will also affect the activity of sorbic acid. This effect is due to the partitioning of the solute into surfactant micelles. The presence of dispersed components and surfactant micelles also has a marked effect on the reactivity of sorbic acid. Whereas thiols react slowly with sorbic acid, the rate of reaction is increased many-fold by the addition of low molecular weight surfactants. The mechanism of this catalysis will be explained. It has been suggested that sorbic acid inhibits enzymes by reacting with sulphydryl groups of the proteins. Kinetic data from model system studies suggest that the sorbic acid-thiol reaction may be too slow for it to be an obvious means of enzyme inhibition. However, this does not take account of possible catalysis of the reaction in the microenvironment of the protein, perhaps in a manner similar to that identified with low molecular weight surfactants.

Counter-current chromatography of black tea infusions.

Counter-current chromatography using a multilayer coil planet centrifuge, with solvent system ethyl acetate-butanol-water, permits the separation of black tea infusions into fractions which include pure SII and a mixture of SI and SIa thearubigins. Good resolution of several components of the infusion may be achieved in elution times of 1 to 2 h. The appearance of chromatograms is altered on decaffeinating the infusion. The effect of stationary phase composition is considered. Resolution of the peaks improves with butanol content.