Table 812

Stability of Anthocyanin Pigments in Model Systems under Different Conditions

Variable Associated Characteristics pH Acidic pH favors the appearance of the colored forms

Most anthocyanins are fully colored at pH < 4

Temperature The formation of chalcone is favored when temperatures are increased

Oxygen and hydrogen These compounds easily oxidize anthocyanins; the effect is strengthened peroxide when the oxidant agents are accompanied by ascorbic acid

Light Anthocyanins are generally unstable under light conditions

C-5 substituted anthocyanins are more susceptible to photochemical decomposition

Acylation The hydrolysis of acylated pigments is hindered and the formation of quinonoidal colored bases is favored; these anthocyanins show better stability at higher pH values than those without acylation At the C-4 position, such as vitisins, anthocyanins show high stability and resistance to color loss induced by the effect of SO2 and high pH values (>4)

Source: Adapted from Delgado-Vargas et al. (2000).17

maximum absorption is maintained up to 12. On the other hand, pigments of group II have similar behavior to those of the group I in the pH range 1 to 8.1, but a dramatic hypsochromic shift is observed between pH 8.1 and 8.6. It is well known that hydroxyl substitution in ring B produces bathochromic shifts at relative acid pH values. The same tendency is observed with the increased number of methoxyl groups on the aglycone B ring, and with the second group, but only in the range of pH 1.0 to 8.1, with increasing number of hydroxyl and/or methoxyl groups in the B ring. The absorptivities are highest at pH 1 for all pigments and decrease toward pH 5. Thus, the colorful flavylium form dominates at pH 1, and the occurrence of colorless carbinol pseudobase forms increases toward pH 5.0. At pH 6, a hyperchro-mic effect is observed until local maxima are achieved at pH 8.1 to 9.8, although at this range of values it could be expected that anthocyanins were in their quinon-oidal and quinonoidal anion forms. The analysis of the ratio between the maximum in the acid and alkaline region shows that peonidin- and malvidin-3-glucosides have a favorable color intensity at alkaline pH, i.e., anthocyanins with no hydroxyl groups in ortho-positions to each other and one or more methoxyl groups. However, this rule is not obeyed with more complex anthocyanins. It has been established that maximum stability of anthocyanins is at pH 1 to 3 and 10°C (70% after 60 days) and lower at higher pH. However, stability of some anthocyanins is improved at higher pH (8 to 9); pelargonidin-, peonidin-, and malvidin-3-glucosides display 30 to 60% stability after 8 days at these alkaline pH values. From a structural point of view, it seems that the presence of only one free hydroxyl group in the B-ring of the group I anthocyanins seems to favor the stability of the bluish equilibrium forms occurring at alkaline pH values, which is further enhanced by the presence of additional methoxyl groups. On the other hand, group II shows some stability in the pH 5 to 6 range.51

Other factors are also important in the stability of anthocyanins and in general they must be processed and stored at low temperatures, with low availability of O2, and out of light (Table 8.12).40 It has been observed that loss of anthocyanin color is the result of water-addition to C2 of the flavylium cation, converting it into a colorless hemiacetal. Interestingly, anthocyanin acylation stabilizes the pigment against hydrolytic processes, allowing preferential formation of the blue quinonoidal bases, and consequently improving stability at high pH values. The most stable anthocyanins are acylated with several cinnamic acid residues. It has been established that anthocyanins with a single cinnamoyl residue attached do not undergo the rapid pH-dependent hydration reaction and loss of color that is characteristic of nonacy-lated anthocyanins. In particular, it has been shown that acylated anthocyanins with benzoic acid display a greater tendency to form hemiacetals than do the anthocyanins acylated with the corresponding cinnamic acids, which identifies the importance of the exocyclic double bond in color retention. Cinnamic acid acylation has a considerable impact on spectral and color characteristics, causing a bathochromic shift of ^max. Anthocyanin-based colorants approved for use in the United States, such as grape skin, red cabbage, and black carrot extracts, exhibit a red-purple hue at pH values above 3. The presence of additional acylation with cinnamic acids produces a bathochromic shift in the ^max of the pigment, with a slight bluing effect. In addition, sugar substitution also plays an important role, with a hypsochromic shift caused by the presence of glycosylation. It has been clearly established that all acylated pigments show increased absorptivity in all major absorption bands (280, 320, and 500 nm regions) when they are dissolved in acidified methanol. However, a decrease in absorption is observed in the 400- to 440-nm region, decreasing the A400-440/Amax ratio.

Anthocyanins with glycosidic substitutions at position 3 exhibit a ratio between the maximum absorbance in the UV region and the maximum in the visible region that is almost twice as great as that of anthocyanins with glycosidic substitution at position 5 or both positions 3 and 5. It has been mentioned that disaccharides as a substituent group in position 3 of the chromophore exhibit a large drop in their absorptivity. It is clear that the acyl group is not attached to the flavylium nucleus but both are the opposite ends of a disaccharide chain and consequently the number of double bonds in resonance of the whole molecule is not increased. Thus, the copigmentation effect is not directly explainable. Moreover, this phenomenon has been interpreted in terms of intermolecular stacking with the acyl group near the flavylium nucleus in the stacked form, the acyl group away from the flavylium nucleus in the unstacked form, and these two forms in equilibrium with each other. As can be clearly established, small differences in anthocyanin chemical structure can have a critical impact on color and tinctorial strength of anthocyanin extracts.52,53 Acylated anthocyanins induce resistance to other factors such as heat, light, and SO2. Vitisins isolated from Vitis vinifera are acylated at C4, improving characteristics of color and stability; their resistance to sulfur dioxide and high pH values have been observed, and consequently they have been proposed as food colorants (Table 8.12).54 The improved characteristics of acylated anthocyanins and particularly their stability at pH > 4 have spurred the development of studies to find or to synthesize new acylated pigments (Figure 8.6).40,55 Malonylated anthocyanins have been identified in purple sunflower seeds, which could be an excellent source of red cyanidin derivatives to be applied in food and pharmaceutical uses.56 By comparing the anthocyanins of Sambucus nigra and S. canadiensis, it has been established that acylation at 5-position gives high stability to heat treatment, and light stability follows the order of acylated diglucosides > nonacylated diglucosides > monoglu-cosides.57 The same behavior is observed for the C5 acylated anthocyanins from red radish.58 Studies with anthocyanin pigments produced by tissue culture of cells of Ajuga pyramidalis show improved stability of its main pigment 3-O-(6-O-(E)-feru-lyl)-2-O-((6-O-(£)-ferulyl)-P-D-glucopyranosyl-P-D-glucopyranosylj-5-O-(6-O-malonyl)-P-D-glucopyranosylcyanidin, in relation with that produced in vivo. A better stability of this pigment may be associated with the in vitro production of copigmenting agents such as flavonols, phenolic acids, and tannins.59

Generation of synthetic anthocyanins has been attempted by reacting anthocyanins and flavonols with acetaldehyde. With this approach it is observed that color increases up to seven times. It has been suggested that acetaldehyde forms a bridge between the flavonoids. Thus, the improved color could be associated with polymeric color.1,3,60 The reaction of malvidin 3-monoglucoside and procyanidin B2 in the presence of acetaldehyde (15°C/4 months) produces three new pigments with visible spectra showing a bathochromic displacement, in relation to the original anthocya-nin. Interestingly, these compounds show improved stability and have similar characteristics with those isolated from wines.61

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