David S. Robinson

Lipoxygenases and the quality of foods

The significance of plant lipoxygenases for food quality is reviewed, with particular reference to the enzymes from pea and soybean. Various aspects of the sources of the enzymes, their activities, substrate and product specificities, and co-oxidation potential are discussed in the context of food quality and shelf life. The sequences of lipoxygenases, predicted from DNA sequences, from different plants, are compared and the significance of sequence differences assessed in relation to enzyme specificity and the three-dimensional structure of soybean lipoxygenase-1. A novel scheme is proposed for the mechanism of the lipoxygenase-catalysed dioxygenation of polyunsaturated fatty acids in which two different pathways are suggested for the anaerobic and aerobic oxidations.

FREE RADICALS IN FOODS

During the last decade increasing attention has been given to the role of free radicals in biological oxidations. The subject has been of increasing interest to both the food scientist and the physiologist. Free radical scavengers in the form of both indigenous and added antioxidants are necessary for the successful preservation of food; free radicals are increasingly being implicated in the onset of, among others, ischaemic heart disease and for protection against these diseases it is suggested that the dietary intake of the antioxidant vitamins should be increased especially for diets high in polyunsaturated fats. Convenience and snack foods which absorb substantial amounts of frying oils are being increasingly consumed. Since poly-unsaturated fatty acids are particularly susceptible to oxidation by free radicals during the storage, cooking and frying of foods, the potential risk of exposure to lipid degradation products is likely to have increased. In foods the non-enzymic and lipoxy-genase catalysed oxidation of polyunsaturated fatty acids, beta-carotene and vitamin A can result in the loss of essential nutrients and the development of off-flavours.

2,5-Dimethyl-4-hydroxy-2H-furan-3-one and its derivatives: analysis, synthesis and biosynthesis—a review

Dimethyl-4-hydroxy-2H-furan-3-one is believed to be a key flavour constituent in many fruits and baked foods. The analy- tical and organic methods applied to the analysis of DMHF and its derivatives, synthesis and biosynthesis are reviewed. Possibilities for further elucidation of biosynthetic pathways and the biotechnological production of DMHF are considered. # 1999 Elsevier Science Ltd. All rights reserved. 1. The analysis of 2,5-dimethyl-4-hydroxy-2H-furan-3- one and its derivatives 2,5-Dimethyl-4-hydroxy-2H-furan-3-one (DMHF) is widely distributed in nature and has a very low flavour threshold value in water (410 ˇ5 mg/kg); hence, its eAect on food aroma is considerable (Latrasse, 1991). 2,5-Dimethyl-4-hydroxy-2H-furan-3-one occurs in nature in four forms (Fig. 1): 2,5-dimethyl-4-hydroxy-2H-furan- 3-one glucoside (DMHF glucoside), 2,5-dimethyl-4- hydroxy-2H-furan-3-one 6 0 -O-malonyl-b-D-glucopyrano- side (DMHF malonyl-glucoside), 2,5-dimethyl-4-meth- oxy-2H-furan-3-one (mesifuran) as well as the free aglycone (Latrasse, 1991; Mayerl, Naf, & Thomas, 1989; Roscher, Herderich, SteAen, Schreier, & Schwab, 1996a).

Superoxide dismutases in foods. A review

Abstract The importance of superoxide dismutase (SOD) in providing a defence against the superoxide radical in living systems has been increasingly established during the last twenty years. More recently, the occurrence and role of SOD in post-harvest and post-mortem foods and its significance as a natural antioxidant have been studied. This review highlights the importance of superoxide dismutase in foods. Current knowledge in aspects such as assay methods and the potential role of superoxide dismutase in the preservation of quality of harvested fruits and vegetables is reviewed.

Kinetics of thermal inactivation of pea seed lipoxygenases and the effect of additives on their thermostability

Abstract Mature pea seeds contain two major lipoxygenases (LOX) isoenzymes designated LOX-2 and LOX-3. The thermal inactivation of crude pea LOX and the recombinant LOX (rLOX) were studied. Heat-inactivation plots for crude extracts of pea LOX were linear from which thermodynamic activation parameters, ΔH # , ΔS # sand ΔG # have been estimated. The enzymatic activity was relatively stable with a respective half-life ( t 1/2 ) at 60 °C of 54.2 min for LOX from pea ( Pisum sativum L. cv. Birte) or 18.4 min for a mutant line lacking LOX-2. At 50°C the thermostability of LOX-3 present in crude extracts of the mutant strain ( t 1/2 =66.8 min) was 90% greater than purified recombinant LOX-3 (rLOX-3; t 1/2 =34.6 min). However, rLOX-3 was more heat-stable than rLOX-2. Both rLOX-3 and pea mutant line lacking LOX-2 possessed considerable thermostability at 60°C ( t 1/2 =16.5 min and 18.4 min, respectively). Even at the higher temperatures of 70°C the t 1/2 values were 84 and 51, respectively. It is suggested that LOX in crude enzyme extracts was stabilised at 50°C due to protection by other constituents, possibly including starch and proteins. Separate tests at 70°C in the presence of additives (polyols, detergents and small ions) showed that sucrose was the most effective stabiliser and increased the stability of pea LOX by 400–600%.

The effect of heat on cabbage and Brussels sprout peroxidase enzymes

Abstract Preparations of soluble, ionically bound and covalently bound peroxidases extracted from cabbage and Brussels sprout have been obtained. The effect of heating each fraction at 60°C, 65°C, 70°C and 75°C on peroxidase activity has been studied. Biphasic inactivation kinetics were observed in all cases, except for Spring cabbage ionically bound peroxidases, which were also found to be less heat stable. Heat inactivation energies for the inactivation of the peroxidases in each fraction have been calculated. Following inactivation, incubation at 30°C allowed regeneration of some of the inactivated peroxidase enzymes in the case of each of the three soluble fractions, but only Brussels sprout ionically bound peroxidases showed detectable regeneration.

Hydrogen donor specificity of mango isoperoxidases

Abstract Purified mango cationic and anionic isoperoxidases catalyse the oxidation of guaiacol, o-dianisidine and 2,2′-azino-di-3-ethyl-benzothiazoline sulphonate (ABTS) at different rates. For an anionic isoperoxidase (A1) o-dianisidine is the most susceptible substrate, whereas for a cationic isoperoxidase (C1) ABTS is oxidised more rapidly. For indoleacetic acid (IAA), mixtures of anionic isoperoxidases catalysed the oxidation at higher rates than cationic isoperoxidases relative to their peroxidase activity towards o-dianisidine. For four purified isoenzymes the greatest rate of oxidation of IAA was found for A1. For dihydroxyfumarate (DHFA), mixtures of cationic isoperoxidases catalysed the oxidation at approximately twice that observed for anionic isoperoxidases per unit of peroxidase activity. Likewise, purified mango cationic isoperoxidases (C1 and C2) oxidised DHFA at a rate three times greater than that for the anionic isoperoxidases of identical peroxidase activity towards o-dianisidine. The mango anionic peroxidases have a greater molecular mass than the cationic isoenzymes. Molecular weights determined by gel filtration of 40 000, 44 000, 22 000 and 27 000 were found for mango isoperoxidases A1, A2, C1 and C2.

The thermostability of purified mango isoperoxidases

Abstract Small amounts of purified mango (var. Chaunsa ) anionic and cationic isoperoxidases have been obtained by ion-exchange chromatography. It has been shown that peroxidase activity present in crude extracts of mango pulp is less stable to heat than the enzymic activity of highly purified individual mango isoperoxidases. For the purified isoperoxidases heat-inactivation is still nonlinear. It is suggested that this may be due to microheterogeneity in covalently bound oligosaccharide residues at the molecular level. Isoperoxidase activity did not regenerate after heat treatment of crude mango extracts or purified isoenzymes.

Purification and heat stability of Brussels sprout peroxidase isoenzymes

Abstract Using gel filtration and ion-exchange chromatography, a total of four peroxidase isoenzymes were isolated from extracts of Brussels sprouts. The isoenzymes were found to vary in their substrate specificities and heat stability properties. Three of the isoenzymes showed biphasic inactivation in response to heating, while the fourth isoenzyme was relatively heat labile and inactivated in a more linear manner with time. Regeneration following heat inactivation was not observed for the isolated isoenzymes.

The thermostability of purified isoperoxidases from Brassica oleracea VAR. gemmifera

Abstract The thermostabilities of four previously purified isoperoxidases from Brussels sprouts ( Brassica oleracea VAR. gemmifera .)have been determined. The heating time periods selected (10 s – 0.5 min intervals) are comparable to those used during commercial blanching. Non-linear regression (NLR) equation fitting, using common goodness of fit criteria (low chi-squared value, high regression coefficient and low residuals) points to a mechanism of peroxidase heat inactivation involving two consecutive reactions during the initial periods of heating. In the consecutive model, native peroxidase ( E 0 ) is converted into a partially active from ( E 1 ) and then into the inactivated enzyme ( E 2 ) during short periods of heating. The order of calculated decimal reduction times for the two anionic (A1 and A2) and two cationic (C1 and C2) Brussels sprouts isoperoxidases was A 1⩾ C 1> A 2> C 2. Calculated concentration changes for E 0 , E 1 and E 2 during heat inactivation were quite different for the four isoperoxidase preparations and indicated the generation of more stable forms of partially denatured peroxidases. The anionic isoenzymes showed greater regeneration of enzymatic activity after heat treatment and this could have been due to their greater ability to regain previously liberated haem. ©