Estudio II: Influencia de la corteza visual so bre las propiedades espaciales de las células del

2.6.6.1.Stability of colorants to cooking conditions

The reactivity of the double bonds in a conjugated system of a colorant molecule will have a bearing on food colour. Food systems are a potential source of reducing agents (donors of electrons which become oxidised as a result) which can cleave double bonds and lead to loss of colour, but the extent to which this occurs can depend on the colorant (Scotter and Castle, 2004). Hydrogen is a reducing agent which can be produced from the reaction of tartaric and citric acids with the metal of cans. High temperatures (110°C to 170°C and above) are reached in confectionery manufacture which can degrade sugars to highly reactive (reducing) agents. In simulated candy manufacture Amaranth was degraded to naphthionic acid and amino R-salt, while Tartrazine and Sunset Yellow were not affected. However low levels of these degradation intermediates (<1%) can already be present in dyes from dye manufacture. Synthetic dyes contained in soft drinks can potentially be reduced by ascorbic acid (AA) which is added as a dietary antioxidant and vitamin supplement, unless AA can be protected from oxidation within the drink by, for example, chelation of metal ion catalysts by sugars. Dissolved oxygen, or tungsten light at pH 5.5 can enhance AA oxidation. The stability of most food dyes to reduction by sulfites which are used as food preservatives, ranges from fair to excellent, with the exception of Indigo carmine, which has poor stability (Scotter and Castle, 2004).

Natural pigments have been the subject of studies investigating the effects of shorter-time cooking processes – microwave cooking and extrusion cooking - on food colorants. The effect of microwave cooking on the level of natural pigments appears to depend on the type of food. Microwave heating reduced total carotenoid content in papaya puree by up to 57%, and in kiwifruit puree loss of chlorophylls a and b was significant (de Ancos et al., 1999). Anthocyanin content of strawberries was unchanged, but this may have been due to more efficient extraction provided by heat-induced cellular disruption. The stability of pigments to microwave cooking relative to other forms of heating could be due to its shorter duration. For

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some green vegetables, levels of pheophytins a and b (the degradation products of chlorophylls a and b) were lower after microwaving and steaming, compared with after boiling (Turkmen et al., 2006). Loss of bixin, the major lipid-soluble carotenoid in annatto, was negligible or nil in biscuits containing added annatto that were cooked by microwave heating for 60 seconds. Losses were higher for conventionally baked cakes and deep fried flour based snacks where heating times were longer (Prabhakara Rao et al., 2005). These latter findings should be viewed with some caution, as each cooking regime used a different recipe; the recipes had different concentrations of added annatto (82 to 340 mg/kg for the biscuits, 113 to 465 mg/kg for cakes and 125 to 513 mg/kg for the deep fried snacks), and for conventional baking and deep frying, bixin losses increased with increasing concentration.

Some natural pigments show good stability to the thermal and physical stresses of extrusion cooking, where the maximum temperatures reached can be higher than those in microwave cooking. 94% of the norbixin (the water-soluble pigment from annatto) added to rice flour and water was retained after extrusion at 155°C, as determined by thin layer chromatography. Retention levels of bixin decreased from 74% in annatto before extrusion to 72% and 69% after extrusion at 125°C and 155°C respectively. Degradation products from oil-soluble turmeric accounted for 27% and 38% of the colorant at the two temperatures. Under the same conditions beet was the least stable, with only 29% of the original colorant remaining at 155°C (Maga and Kim, 1990).

2.6.6.2.Colorant content and other contributors to colour appearance

In some cases the level of colorant present in a food is seen in the final appearance of the food. Rice flour and water mixtures containing beet that were extruded at 125°C retained 63% of the original colorant and visually had a characteristic red colour. On increasing the extrusion temperature to 155°C measured lightness (L*) increased from 64.6 to 76.2 units, yellowness (b*) from 3.8 to 8.6 units, and redness (a*) decreased from 19.0 to 10.3 units, in line with a visually observed change in colour to a very faded pink (Maga and Kim, 1990). The significant decreases found in the levels of chlorophylls a and b in kiwifruit puree after microwave cooking

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were accompanied by a significant change in instrumentally measured colour (as indicated by by ΔE*) relative to the untreated puree. High correlations (r2 0.89 to 0.96) were found between chlorophyll levels and measured chroma (de Ancos et al., 1999).

At other times colorant level is not necessarily a good indicator of final colour, due to the colour contributed by other processes. Significant changes in the lightness of strawberry puree were found for some microwave cooking conditions, but these were not strongly related to anthocyanin content. It is likely that the colour change was the result of browning, given that polyphenol oxidase and peroxidase (responsible for enzymic browning in fruit) in strawberry showed resistance to inactivation under the same conditions (de Ancos et al., 1999). The change in the greenness of vegetables after cooking could be due to changes in light scattering as water replaces intracellular air (Hutchings, 1999), rather than to a change in the level of chlorophylls. Despite a difference in the amount of pigment degradation products in rice flour and water extruded with oil-soluble turmeric at 125°C and 155°C (27% and 38% respectively), yellow values were unchanged between the two temperatures (at 30.3 and 30.5 units respectively) suggesting that degradation products from oil-soluble turmeric were themselves coloured (Maga and Kim, 1990).

2.6.6.3.Colorant-ingredient interactions

The distribution of colour within a food is an indication of binding or interaction with food components, such as polysaccharides, starches and proteins. These interactions depend on the structure and physical characteristics of the components, which determine the potential for electrostatic interactions, or for hydrogen and van der Waals bonding. For proteins, these interactions can be enhanced by heating. Heating at 60oC helped to preserve bands in SDS- PAGE electrophoresis when proteins were stained with Sunset Yellow and Allura Red, and made otherwise very light stains produced at low and high pH conditions more visible (Badaruddin et al., 2007; Umer Abdullah et al., 2008). For Sunset Yellow, heating may have enhanced electrostatic binding of the dye with protein via SO3

2-

groups on the dye, with heat- induced denaturation and unfolding of protein molecules exposing more binding sites. The

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coloration of reticulated waxy corn starch by the natural red colorants Cochineal Carmine and Beet Red could be explained by electrostatic interactions between the positively-charged pigment compounds and negatively-charged phosphoric groups in the starch (Berset et al., 1995). Anionic (negatively-charged) polysaccharides (carrageenans, pectins, alginic acid) were found to be ineffective removers of dyes from solutions mimicking dyehouse effluent, owing to electrostatic repulsion between the polysaccharides and the anionic dye molecules. Although non-ionic, the galactomannans locust bean gum, guar gum and cassia gum performed very well as effluent dye removers. Strong chain interactions between galactomannan molecules are prevented by their branched galactose residues, meaning galactomannans are available for hydrogen bonding with dyes (Blackburn, 2004). The non-ionic starch, by comparison, was an ineffective effluent dye remover, as temperatures much higher than the one used (20°C) would have been needed to break the inter- and intra- molecular hydrogen bonding between amylose and amylopectin chains in starch for these to have become available for bonding with dye molecules.

The binding of dyes to components can be enhanced by the addition of other agents. In textile dyeing, mordants are used to increase the affinity of dyes for the substrate. Metal salt-based mordants form complexes with dyes, and tannins increase adsorptivity via hydrogen bonding and van der Waals forces (Bechtold et al., 2007). The addition of positively-charged electrolytes may overcome the electrostatic repulsion between negatively-charged dye and polysaccharide molecules in dye effluent, but this will depend on the strength of these charges (Blackburn, 2004).

For starches, another property that could influence dye binding is the size of the starch grains. Native corn, native wheat and reticulated waxy corn starches retained higher levels of Cochineal Carmine and Beet Red (as determined from the absorbance of the supernatant), than did potato starch, yet potato starch was as strongly coloured as the other starches. The was possibly due to the saturation of colorant binding sites on the potato starch grains, given the larger size of these

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grains and their lower specific area (total surface area per unit mass, or per unit volume (Berset

et al., 1995).