Bárbara Nieva-Echevarría

2,6-Di-Tert-Butyl-Hydroxytoluene and Its Metabolites in Foods

2,6-Di-tert-butyl-hydroxytoluene (BHT, E-321) is a synthetic phenolic antioxidant which has been widely used as an additive in the food, cosmetic, and plastic industries for the last 70 y. Although it is considered safe for human health at authorized levels, its ubiquitous presence and the controversial toxicological data reported are of great concern for consumers. In recent years, special attention has been paid to these 14 metabolites or degradation products: BHT-CH2 OH, BHT-CHO, BHT-COOH, BHT-Q, BHT-QM, DBP, BHT-OH, BHT-OOH, TBP, BHQ, BHT-OH(t), BHT-OH(t)QM, 2-BHT, and 2-BHT-QM. These derived compounds could pose a human health risk from a food safety point of view, but they have been little studied. In this context, this review deals with the occurrence, origin, and fate of BHT in foodstuffs, its biotransformation into metabolites, their toxicological implications, their antioxidant and prooxidant properties, the analytical determination of metabolites in foods, and human dietary exposure. Moreover, noncontrolled additional sources of exposure to BHT and its metabolites are highlighted. These include their carryover from feed to fish, poultry and eggs, their presence in smoke flavorings, their migration from plastic pipelines and packaging to water and food, and their presence in natural environments, from which they can reach the food chain.

Usefulness of 1H NMR in assessing the extent of lipid digestion

Proton Nuclear Magnetic Resonance ((1)H NMR) is proved to be, for the first time, a very useful technique in monitoring the extent of lipid hydrolysis in digestion processes. Sunflower oil and minced fish flesh, as model foods, were subjected to different in vitro digestion experiments and the lipolysis levels reached were evaluated using (1)H NMR spectral data. Simple observation of the spectra gives very valuable information about the extent of the lipolysis and enables a rapid discrimination among samples having different hydrolysis degree. Equations were developed to quantify all the lipolytic products, and either referred to acyl groups plus fatty acids, or to glyceryl structures. The main hydrolysis products were 1,2-diglycerides, 2-monoglycerides, glycerol and fatty acids, although small proportions of 1,3-diglycerides and of 1-monoglycerides were also found. With this methodology, determination of the extent of lipid digestion in its different definitions can be made. It has been shown that these definitions are not equivalent, which is evidence for the need for a consensus in this regard.

A study by 1H NMR on the influence of some factors affecting lipid in vitro digestion

This article focuses on the impact of several experimental factors, including gastric acidification, intestinal transit time, presence of gastric lipase, sample/digestive fluids ratio, concentration and nature of the enzymes in intestinal juice, and bile concentration, on the extent of in vitro lipolysis when using a static model that simulates human digestion processes in mouth, stomach and small intestine. The study was carried out by Proton Nuclear Magnetic Resonance ((1)H NMR). This technique provides a complete molecular picture of lipolysis, evidencing for the first time, whether preferential hydrolysis of certain glycerides over others occurs. A lipolysis degree similar to that reported in vivo was reached by varying certain variables within a physiological range; among them, bile concentration was found to be crucial. The holistic view of this (1)H NMR study provides information of paramount importance to design sound in vitro digestion models to determine the bioaccessibility and bioavailability of lipophilic compounds.

Changes provoked by boiling, steaming and sous-vide cooking in the lipid and volatile profile of European sea bass

This study aims to shed light on the changes provoked by boiling, steaming and sous-vide cooking on the lipids and volatile profile of farmed and wild European sea bass meat. None of the cooking techniques provoked changes due to hydrolytic or oxidation processes detectable by 1H NMR on sea bass lipids. The lipid profile of main and minor lipidic components was maintained after cooking. However, study by SPME-GC/MS evidenced that steaming and sous-vide cooking modified the volatile profile of sea bass meat, especially in farmed specimens. The compounds generated came from the occurrence, to a very small extent, of lipid and protein degradation. By contrast, boiling scarcely modified the initial characteristics of raw sea bass. Thus, from a sensory point of view and considering the odour-active compounds generated, steaming and sous-vide cooking provoked more noticeable changes than boiling, especially in farmed sea bass meat.

1H NMR and SPME-GC/MS study of hydrolysis, oxidation and other reactions occurring during in vitro digestion of non-oxidized and oxidized sunflower oil. Formation of hydroxy-octadecadienoates

Both fresh and slightly oxidized sunflower oils, as models of omega-6 rich lipids, were submitted to in vitro gastrointestinal digestion and studied by 1H NMR and SPME-GC/MS. Changes in lipolysis degree, lipid composition and oxidative level were studied by 1H NMR. Three quantitative approaches were used and several equations were newly developed. In oxidized oil digestates slightly lower hydrolysis and a higher advance of oxidation took place during digestion. This latter was evidenced by a greater decrease of lipid unsaturation degree and enhanced generation of oxidation products (cis,trans-hydroperoxy-octadecadienoates, cis,trans- and trans,trans-hydroxy-octadecadienoates). For the first time, the generation of hydroxy-octadecadienoates during in vitro digestion is reported. Furthermore, SPME-GC/MS study of non-digested and digested samples headspaces confirmed that lipid oxidation occurred: abundances of volatile markers increased (including potentially toxic alpha,beta-unsaturated aldehydes), especially in oxidized oils digestates. Markers of Maillard-type and esterification reactions were also detected in the digestates.

Fish in Vitro Digestion: Influence of Fish Salting on the Extent of Lipolysis, Oxidation, and Other Reactions

A study of the various chemical reactions which take place during fish in vitro digestion and the potential effect of fish salting on their extent is addressed for the first time. Farmed European sea bass fillets, raw, brine-salted or dry-salted, were digested using a gastrointestinal in vitro model. Fish lipid extracts before and after digestion were analyzed by 1H NMR, and the headspace composition of the digestates was investigated by SPME-GC/MS. During digestion, not only lipolysis, but also fish lipid oxidation took place. This latter was evidenced by the generation of conjugated dienes supported on chains having also hydroperoxy- and hydroxy-groups (primary oxidation compounds), by the increase of volatile secondary oxidation products, and by the decrease of the antioxidant 2,6-di-tert-butyl-hydroxytoluene (BHT). Likewise, esterification and Maillard-type reactions also occurred. Salting, and especially dry-salting, enhanced all these reactions, except for lipolysis, during digestion.

Effect of liquid smoking on lipid hydrolysis and oxidation reactions during in vitro gastrointestinal digestion of European sea bass

The effect of smoking using liquid smoke flavourings on the hydrolysis and oxidation of European sea bass lipids during in vitro digestion was investigated. The techniques used were 1H NMR and SPME-GC/MS. The former proved that liquid smoking does not influence the extent of lipolysis, but prevents those lipid oxidation reactions that occur during in vitro digestion of unsmoked samples, giving rise to cis,trans-conjugated dienes associated with hydroperoxy/hydroxy groups. SPME-GC/MS corroborated the results obtained by 1H NMR in relation to the antioxidant effect of smoking under gastrointestinal conditions. Smoked sea bass digests showed lower abundances of volatile oxidation markers derived from omega-3 and omega-6 lipids than unsmoked ones. Moreover, the lowest values were found in the digests of sea bass samples smoked with the flavouring showing the highest phenolic content. For the first time, the bioaccessibility of smoke flavouring components was evidenced, some of them well-known for displaying antioxidant activity.

Behaviour of non-oxidized and oxidized flaxseed oils, as models of omega-3 rich lipids, during in vitro digestion. Occurrence of epoxidation reactions

Fresh and partially oxidized flaxseed oil, as models of omega-3 rich lipids, were submitted to in vitro gastrointestinal digestion. Hydrolysis level, lipid composition and oxidative status of the samples before and after digestion were studied by Proton Nuclear Magnetic Resonance (1H NMR) and Solid Phase Microextraction-Gas Chromatography/Mass Spectrometry (SPME-GC/MS). Although a great degree of lipolysis was reached in both kinds of samples after digestion, it was somewhat lower in the digests of oxidized flaxseed oil. The occurrence of lipid oxidation during digestion was evidenced by decreased unsaturated lipids and increased primary and secondary oxidation products, especially in oxidized samples. In these latter, linolenic-derived monoepoxy-octadecadienoates were the main oxidation products generated. SPME-GC/MS study showed the highest abundances of highly reactive alkadienals (C5-C10), alkatrienals (C9-C10) and linolenic-derived 4,5-epoxy-2-heptenals in the headspace of oxidized flaxseed oil digests. Volatile markers of Maillard-type reactions were also detected.

Polyunsaturated lipids and vitamin A oxidation during cod liver oil in vitro gastrointestinal digestion. Antioxidant effect of added BHT

The extent of cod liver oil hydrolysis and oxidation during in vitro gastrointestinal digestion was investigated by Proton Nuclear Magnetic Resonance (1H NMR) and Solid Phase Microextraction-Gas Chromatography/Mass Spectrometry (SPME-GC/MS). These techniques evidenced the degradation of polyunsaturated ω-3 and ω-6 lipids and, for the first time, that of vitamin A, naturally present in cod liver oil. Cis,trans-conjugated dienes associated with hydroperoxides, as well as monoepoxides, cis,trans-2,4-alkadienals, 4-hydroperoxy- and 4-hydroxy-2-alkenals, and several vitamin A derived metabolites were generated. Moreover, the effect of the addition of the synthetic antioxidant 2,6-di-tert-butyl-hydroxytoluene (BHT) at 20 and 800ppm was tackled. Both techniques showed BHT to be efficient in limiting oxidation reactions during digestion, almost inhibiting them at 800ppm. Therefore, the simultaneous intake of antioxidants with cod liver oil should be considered, in order to increase polyunsaturated lipid and vitamin A bioaccessibility and avoid formation of toxic oxidation compounds like oxygenated alpha,beta-unsaturated aldehydes.

Effects of different cooking methods on the lipids and volatile components of farmed and wild European sea bass ( Dicentrarchus labrax )

European sea bass is very popular in the Mediterranean area, although very little is known about the possible different behaviours of farmed and wild samples during cooking. This study addresses the effect of microwave cooking, salt-crusted and conventional oven baking on the lipids and volatile profile of farmed and wild sea bass. Proton Nuclear Magnetic Resonance did not detect that hydrolysis or oxidation of lipidic components had taken place. However, Solid Phase Microextraction-Gas Chromatography/Mass Spectrometry evidenced that polyunsaturated acyl group oxidation and Maillard-type reactions occurred to a very slight extent, yielding a wide variety of volatile odour-active compounds. Conventional baking enriched fish volatile profile to a higher extent than the other two techniques assayed. In fact, 15 Maillard reaction-derived compounds (pyrroles, alkylpyrazines, alkylthiophenes and 2-ethylpyridine) were only detected in oven-baked samples. Regardless of the cooking method applied, farmed sea bass showed a much richer aromatic profile than did wild samples, having 6-fold higher lipid content than the latter.