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Caffeic acid copigmentation of anthocyanins from Cabernet Sauvignon grape extracts in model systems
Food Chemistry Food Chemistry 100 (2007) 1289–1296 www.elsevier.com/locate/foodchem
Analytical, Nutritional and Clinical Methods
Caffeic acid copigmentation of anthocyanins from Cabernet Sauvignon grape extracts in model systems E.F. Gris, E.A. Ferreira, L.D. Falca˜o, M.T. Bordignon-Luiz
*
Departamento de Cieˆncia e Tecnologia de Alimentos-CAL/CCA/UFSC. Rod. Admar Gonzaga, 1346, CEP: 88034-001 Floriano´polis SC, Brazil Received 24 May 2005; received in revised form 16 September 2005; accepted 23 October 2005
Abstract Caffeic copigmentation of anthocyanins from Cabernet Sauvignon grape extracts in model system and yoghurt model system was investigated, as well as the influence of copigmentation on the stability of these pigments. In the model system, the dependence of anthocyanin stability on added caffeic acid was evaluated at temperatures (4 ± 1 °C and 29 ± 3 °C) and in the presence or absence of light, at two pH values: 3.0 and 4.0; in the yoghurt model system, the stability of the anthocyanins was evaluated at 4 ± 1 °C in the dark. The half-life and percentage of color retention of the anthocyanins in all treatments was calculated. The spectrophotometric results (Dk, DA) revealed that interaction occurred between the crude extract of anthocyanins and caffeic acid suggesting copigmentation. The addition of caffeic acid (1:1 w/v) significantly increased (p < 0.05) the stability of anthocyanins in both model and yoghurt systems. In the model system the temperature and presence of light significantly influenced the stability of anthocyanins (p < 0.05), where the highest values for half-life were obtained for anthocyanins with caffeic acid at pH 3.0, stored in the dark at a temperature of 4 ± 1 °C (6.930 h). In the yoghurt system the caffeic acid increased the half-life time of anthocyanins to 6673 h. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Anthocyanin; Stability; Copigmentation; Caffeic acid
1. Introduction Color is one of the most important attributes of food and beverages. It is appreciated for its intrinsic aesthetic value and as a basis for identification and quality judgment. Synthetic dyes are much used by the food industry because they have higher stability with respect to light, oxygen, temperature, and pH, among other factors; but the presence of these dyes can represent a risk factor to health (Del Giovine & Bocca, 2003; Mazza & Brouillard, 1987). Natural plant colorants have been in high demand by the food industry as substitutes of synthetic dyes for the past decade. The need has stemmed from legal actions against their use and from the consumer concern against synthetic food additives (Francis, 1989). Anthocyanins have a high *
Corresponding author. Tel.: +55 48 331 5376; fax : +55 48 331 9943. E-mail addresses: [email protected] (E.F. Gris), bordign@ cca.ufsc.br (M.T. Bordignon-Luiz). 0308-8146/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2005.10.014
potential for use as natural colorants due to their attractive colors (Mazza & Miniati, 1993); they are not only non-toxic and non-mutagenic (Bridle & Timberlake, 1997), but their pharmacological properties are also well known and account for their therapeutic use (Delgado-Vargas, Jime´nez, & Paredes-Lo´pez, 2000; Lazze´ et al., 2003; Valcheva-Kuzmanova, Borisova, Galunska, Krasnaliev, & Belcheva, 2004). Anthocyanins are vacuolar pigments and their organelles account for the presence of membrane bound bodies called anthocyanoplasts; they are responsible for many of the attractive colors of flowers, fruits and leaves, from scarlet to blue (Brouillard, 1983). Chemically, anthocyanins are flavonoids, and consequently based on a C15 skeleton with a chromane ring bearing a second aromatic ring in position 2 (C6-C3-C6) and with one or more sugar molecules bonded at different hydroxylated positions of the basic structure, the flavylium cation (Mazza & Miniati, 1993). In acidic or neutral media, four anthocyanin structures exist in equilibrium: the flavylium cation, quinoidal base,
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carbinol pseudobase, and chalcone (Hrazdina, 1974). The concentration of each structure has been shown to be dependent upon the pH of the media (Mazza & Miniati, 1993). Besides the changes caused by pH, the color of anthocyanins is determined by the structure and concentration of the pigment, the presence of metallic ions, temperature, enzymes, oxygen, sugars, copigments, and other factors (Dangles & Brouillard, 1992; Hoshino, Matsumoto, & Goto, 1980). All these effects, however, cannot account for the ample variety of colors and hues in the plant world (Brouillard, Mazza, Saad, Gary, & Cheminat, 1989), which is why it was initially assumed, and later confirmed, that different colors are the consequences of complexation, i.e., copigmentation of the flavylium chromophore (Asen, Stewart, & Norris, 1972; Brouillard, 1983). Copigmentation is regarded today as one of the significant factors of structure stabilization, i.e., coloration, of anthocyanins under in vivo conditions (Boulton, 2001; Brouillard et al., 1989); it is a molecular interaction that occurs between anthocyanins and copigments (Gonzalez, Fougerousse, & Brouillard, 2001; Goto & Kondo, 1991). Copigments may be flavonoids, alkaloids, amino acids, organic acids, polysaccharides, metals and anthocyanins themselves, and their basic role is to protect the colored flavylium cation from nucleophilic attack of the water molecule (Asen et al., 1972; Mazza & Brouillard, 1990). Caffeic acid is an organic phenolic molecule. It is part of the plant tissue in which it plays an important role. In addition to having a significant metabolic function, cinnamic acids constitute the main acyl group in the structure of acylated anthocyanins, which are also part of plant tissue (Harborne, 1967). Dimitric-Markovic, Petranovic, and Baranac (2000), evaluated in vitro the intermolecular copigmentation reaction of malvidin 3.5-diglucoside with caffeic acid using UV–Vis spectrophotometry. These authors verified that the organic acid possesses the capacity to participate in copigmentation reactions with anthocyanins and that this process is dependent on the pH of the medium, copigment concentration and temperature. Through the oxidation potential Dimitric-Markovic, Ignjatovic, Markovic, and Baranac (2003a, 2003b), evaluated the antioxidant properties of the copigmentation complexes of malvidin 3.5-diglucoside and caffeic acid. The results confirmed that caffeic acid has the capacity to copigment this anthocyanin and that the antioxidant activity of the complex anthocyanin:caffeic acid was greater than the antioxidant activity of the anthocyanin alone. The scavenging potential and antioxidant properties of caffeic acid in terms of its ability to increase the resistance of low density lipoproteins (LDL) to cholesterol oxidation were reported (Castelluccio et al., 1995; Marinova & Yanishlieva, 2003; Sroka & Cisowski, 2003). Evaluation of the copigmentation reaction and the stability of anthocyanins from an extract of Cabernet Sauvignon grape were realized due to the fact that this grape variety is in ascension in Brazil, especially in Santa Catarina State. The use of the anthocyanins from grape skins of wine indus-
try solid residues is a real possibility under future consideration, as it is currently realized in many countries (Bridle & Timberlake, 1997); the use of a crude extract, which does not depend on high cost processes of purification, could be an alternative use of this industrial residue as a source of anthocyanins for food applications. The objective of the present work was to evaluate the stability of anthocyanins from a crude extract of Cabernet Sauvignon (Vitis vinifera L.) grape skins with caffeic acid under different conditions of temperature (4 ± 1 °C and 29 ± 3 °C), pH (3.0 and 4.0) and in the presence of light in a model system and in a yoghurt model system stored at 4 ± 1 °C in the dark. 2. Materials and methods The study was carried out with red grapes of Cabernet Sauvignon cultivars (V. vinifera L.), harvest 2002, from the Agricultural Research Enterprise (EPAGRI), in the town of Sa˜o Joaquim, 1400 m a.s.l., Santa Catarina, Brazil. For the elaboration of the yoghurt system commercial sugar and milk were used and a thermophilic lactic culture (YC-X11, CHR HANSEN, Sa˜o Paulo, Brazil). The caffeic acid used to evaluate copigmentation was obtained from Fluka (P95.0% HPLC). All other reagents were of analytical grade. 2.1. Anthocyanin crude extract: extraction and quantification One hundred grams of grape skins were macerated overnight in the dark, at 4.0 ± 1 °C, in 400 mL of 0.1% HCl in methanol. The crude extract obtained was filtered through a nylon filter and the skin remains were washed with 100 mL of extracting solvent (Lees & Francis, 1972). Vacuum filtration was carried out on Whatman (no. 2) filter paper using a Bu¨chner funnel and afterwards the extract was concentrated under vacuum (Bu¨chi R-144, Switzerland) at 35 °C to 50% of the methanol initial volume (Wilska-Jeszka & Korzuchowska, 1996). The concentrated extract was filtered through a 0.45-lm Millipore filter to eliminate possible interferences in the spectrophotometric readings (Millipore, Bedford, MA) and kept in an amber flask at 4.0 ± 1 °C. The quantification of total monomeric anthocyanins was determined by the pH-differential methods (Giusti & Worsltad, 2001) using MW = 562.5 and e = 29,500 ((Koeppen & Basson (1966) and Giusti, Saona-Rodrı´guez & Wrolstad, 1999) and expressed in g/L of malvidin 3-glucoside, the main anthocyanins from Cabernet Sauvignon (V. vinifera L.) grapes (Passamonti, Vrhovsek, Vanzo, & Mattivi, 2003; Wulf & Nagel, 1978). 2.2. Stability study 2.2.1. Model system To prepare control samples (model system without caffeic acid) and test samples (model system with caffeic acid), anthocyanic extract dilutions were made in a model system
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of citrate buffer (0.1 M, Merck citric acid – sodium citrate), at both pH 3.0 and 4.0 to obtain absorbance values between 1.000 and 1.200 at maximum absorption wavelength. According to Asen et al. (1972) & Brouillard et al. (1989) copigmentation occurs between the colored forms of anthocyanins (flavylium cation and quinoidal base) and certain copigments. Thus the absorbancy values of the samples were standardized (absorbance at ‘‘zero time’’) at pH 3.0 and pH 4.0, with the aim of standardizing the effect of copigmentation. The concentration of caffeic acid to be added to the anthocyanins (weight (mg)/volume (mL) ratios w/v) was determined from the total solid content of the crude extract (g/L), itself previously determined by drying 3 mL of anthocyanin crude extract at 105 °C until constant weight was reached (AOAC, 1998). Different ratios of caffeic acid were assessed at random up to the maximum concentration of acid diluted in the model system of citrate buffer (0.5:1; 0.8:1 and 1:1 (mg/mL) caffeic acid:anthocyanin crude extract volume). Control and test samples (model system with caffeic acid) were prepared in volumetric vials and added the crude extract with 0.05% potassium sorbate (g/L) to prevent bacterial growth. The volume was completed with the model system of citrate buffer (0.1 M, Merck citric acid–sodium citrate), at pH 3.0 and 4.0; caffeic acid was added to the test samples. The solutions were agitated for 30 min and then transferred to screw-top test tubes and left to rest for 2 h in the dark and at a temperature of 29 ± 3 °C to reach equilibrium (Fuleki & Francis, 1968b). Test and control samples were kept under the following conditions: at 4 ± 1 and 29 ± 3 °C, under a fluorescent lamp (2500 lm) and in the absence of light, in pH 3.0 and 4.0; all samples were maintained in the presence of atmospheric oxygen. The values of sample absorbancy were monitored using three test tubes with screw-tops containing model systems for each of the repetitions throughout the experiment. The absorption spectra of samples were monitored through UV–Vis absorption spectrophotometry in the visible wavelength range from 400 to 700 nm, at regular time intervals of 72 h, until 60% or more of the pigments were degraded. A Hitachi U2010 spectrophotometer (CA, USA) fitted with a 10-mm optical path quartz cell was used in this monitoring. After reaching equilibrium in the dark (2 h), the anthocyanins maximum wavelength was read in the model system, absorbance being characterized as ‘‘zero time’’. Distilled water was used as blank in all of the experiments (Fuleki & Francis, 1968a; Giusti, Saona-Rodriguez, & Wrolstad, 1999). 2.2.2. Yoghurt model system Yoghurt model systems were prepared by adding 5% sugar to milk, which was then heated to 95 °C for 5 min, cooled to 42 °C and inoculated with 0.05% of thermophilic lactic culture (YC-X11, CHR HANSEN, Sa˜o Paulo, Brazil). The samples were incubated at 42 °C until they reached pH 3.8 and transferred to a cold room (4 ± 1 °C)
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for storage. The concentration of total solids of the crude extract (g/L) was used as an initial parameter for the addition of caffeic acid. Control samples (yoghurt model system without caffeic acid) and test samples (yoghurt model system with caffeic acid) were prepared. Control solutions (1:1 v/v crude extract: citrate buffer pH 3.8) and test solutions (1:1:1 w/v/v caffeic acid: crude extract: citrate buffer pH 3.8) were added to yoghurt systems (control and test) in order to obtain a color similar to that of a commercial artificially colored grape flavor yoghurt. The control and test yoghurt model systems were kept at 4 ± 1 °C, in the dark for 45 days (1,080 hours), shelf-life of the yoghurt. The study of pigments stability was carried as follows: 20 g aliquots of each sample were centrifuged (Janetzki K-24, Germany) for 30 min at 8942 g. The supernatant was filtered and used for the reading of sample absorbance at the maximum absorption wavelengths. Distilled water was used as blank in all of the experiments (Fuleki & Francis, 1968a; Giusti et al., 1999). The absorption spectra of the samples were monitored through UV–Vis absorption spectrophotometry in the visible range at regular time intervals of 72 h for 45 days. 2.3. Kinetic study For all samples the half-life time (t1/2) values were calculated according Kirca & Cemeroglu (2003) and the percent color retention percentage (R%) values were calculated according to Katsaboxakis, Papanicolaou, & Melanitou (1998) in accordance with the equations below: lnðAt =A0 Þ ¼ k t t1=2 ¼ ln 0:5 k 1 At % Retention ¼ 100; A0 where t = time (hours); At = final absorbance (‘‘time t’’); A0 = initial absorbance (‘‘time zero’’). 2.4. Statistical analysis In the model system, the experiment was conducted entirely at random, in two repetitions in triplicate, in the Factorial 24 scheme where the factors under analysis were the presence of copigment (caffeic acid), temperature (4 ± 1 and 29 ± 3 °C), presence of light and dark and pH (3.0 and 4.0). In the yoghurt model system, the experiment was conducted with two repetitions in duplicate. The halflife time of all samples was studied by means of analysis of variance (ANOVA) with STATISTICA software, version 6.0 (2001). The Tukey Test was applied whenever a difference was detected among factors at a 5% level of significance. 3. Results and discussion The content of anthocyanins in the anthocyanic crude extract was calculated to be 1.82 g/L, expressed in malvidin
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3-glucoside. The total ratio of solids (g/L) of the crude extract was estimated to be 17.20 g/L. The volume (mL) of anthocyanic crude extract and weight of caffeic acid (mg) added to the buffer solutions are described in Table 1. Fig. 1 illustrates the spectrophotometry of the effect of caffeic acid in absorption spectra of anthocyanins at pH 3.0 and 4.0, where it was verified that caffeic acid promoted an increase in the maximum absorption wavelength (bathochromic effect – Dk) and absorbance (hyperchromic effect – DA). These shifts simultaneous characterize an intermolecular copigmentation reaction (Asen et al., 1972; Davies & Mazza, 1993; Mazza & Brouillard, 1990). An increase of 16.63%, 22.61% and 39.54% was observed in sample absorbance at pH 3.0, respectively, and of 10.72%, 33.65% and 38.01% in sample absorbance at pH 4.0, respectively. The greatest hyperchromic shifts occurred with greatest intensity in the samples with the highest proportion of added caffeic acid, both at pH 3.0 and 4.0, an occurrence repeated with the bathochromic shifts (Fig. 1). According to the results obtained in this experiment, the control samples presented a bathochromic shift of Dk = 2.5 nm with the increase in pH from 3.0 to 4.0. This may have occurred because the existing flavylium cation forms in the crude extract diminished proportionally with the increased pH of the media, causing a slight bathochromic shift in the maximum absorption wavelength (Baranac, Petranovic, & Dimitric-Markovic, 1997). The increase of caffeic acid concentration in anthocyanin solutions (w/v) produced an increase in the hyperchromic and bathochromic effects, a result that agrees with the literature and confirms that copigmentation reaction depends on copigment concentration (Fig. 1) (Mazza & Brouillard, 1990; Wilska-Jeszka & Korzuchowska, 1996). Hyperchromic effects were seen to occur more intensely (p = 0.0325) in solutions at pH 4.0 at concentrations of 0.8:1 and 1:1 (caffeic acid:anthocyanin solutions (mg/mL) (Fig. 1B). According to Mazza & Brouillard (1987), at this pH, anthocyanins are essentially in their hemiacetal (colorless) form, a chemical structure resulting from the hydration of flavylium cations. Wilska-Jeszka & Korzuchowska (1996) affirm that the effect of copigmentation is greater at pH 3.4–3.6 when compared to pH 3.0 because the copigment prevents the hydration reaction and stabilizes the colTable 1 Caffeic acid concentration values and anthocyanin crude extract volumes used in the model system experiment at pH 3.0 and 4.0 pH
Concentration caffeic acid: anthocyanins extract (w/v), in a model system
Anthocyanins extract/in a model system (vial 25 mL)a (mL)
3.0
0.5:1 (8.6 mg/mL) 0.8:1 (13.76 mg/mL) 1:1 (17.20 mg/mL)
0.6 0.6 0.6
4.0
0.5:1 (8.6 mg/mL) 0.8:1 (13.76 mg/mL) 1:1 (17.20 mg/mL)
0.9 0.9 0.9
a
Values used to obtain initial absorbance close to 1.000.
Fig. 1. Effect of caffeic acid addition at maximum absorption wavelength for anthocyanins from grape skins at pH 3.0 (A) and 4.0 (B) at T (°C) = 29 ± 3. 1 = control samples (anthocyanins without caffeic acid) k = 517.5 nm (A) and 520.0 nm (B); 2, 3 and 4 = test samples (anthocyanins:caffeic acid) in concentrations 0.5:1; 0.8:1 and 1:1 (w/v), respectively.
ored form of anthocyanins more intensely in this pH range. Boulton (2001) also affirms that copigmentation occurs with greater intensity at pH 3.6, which justifies the greater intensity of hyperchromic and bathochromic effects at pH 4.0 than at 3.0. The ratio of caffeic acid used in the experiment to check stability was 1:1 caffeic acid:anthocyanin crude extract volume (mg/mL), because greater hyperchromic (DA) and bathochromic (Dk) effects were observed in this ratio in the anthocyanin maximum absorption wavelength at the pH values studied (Fig. 1). The 24 Factorial model was considered well adjusted with a R2 = 0.977 correlation coefficient. The analysis of variance showed that there was a significant difference between different treatments (with and without caffeic acid), temperatures (4 ± 1 and 29 ± 3 °C) and in presence of light and dark, as well as significant interactions between all the factors analyzed. Table 2 shows the t1/2 (h) and percentage retention color (R%) values of control (anthocyanins) and test (anthocyanins and caffeic acid) solutions in different storage conditions. Caffeic acid significantly interfered in the stability of anthocyanins (p = 0.001) promoting a significant increase in the t1/2 values of the samples, except for those
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Table 2 Half-life time and colour retention percentage of model system samples without (control) and with (test) caffeic acid (1:1 w/v) in different storage conditions pH
T (°C)
Light/dark
Half-life (h)
Colour retention (%)
Control sample 3.0
4 ± 1 °C 29 ± 3 °C
4.0
4 ± 1 °C 29 ± 3 °C
** *
Test sample
Control sample
Test sample
*
D L D L
5641 1430 894 232
6930 1413 1711* 306
96 84 76 36
97 83 86 45
D L D L
2309 1060 898 464
6986* 1350* 1896* 693*
90 79 76 58
96 83 88 69
Average of two repetitions in triplicate; L = under light (2500 lm); D = dark. Significantly differ from the control (p < 0.05).
kept at pH 3.0 and in the presence of light (Table 2). As shown in Table 2, there was a greater increase in t1/2 and R% in the samples kept in the dark, at 4 ± 1 °C, at the two pH values studied. These results are in agreement with data previously reported in the literature, where the effect of copigmentation of grape anthocyanins from Cabernet Sauvignon grape skins with tannic acid promoted increased pigment stability (Falca˜o et al., 2004). The different sample storage temperatures significantly influenced the stability of anthocyanins in buffer solution (p = 0.000). The increase in temperature, from 4 ± 1 to 29 ± 3 °C, was seen to quicken the degradation of these pigments in all the samples, as confirmed by the t1/2 and R% values (Table 2). Similar results have been observed by Morais, Ramos, Forga´cs, Cserha´ti, & Oliviera (2002) who assessed the stability of malvidin 3-glucoside from V. vinifera (var. Red globe) grape skins at three different temperatures (24, 32 and 40 °C) where the increase in temperature was seen to quicken the degradation of this pigment. Table 2 also shows that anthocyanin degradation at 29 ± 3 °C, as well as at 4 ± 1 °C, in general, occurred with greater intensity in the control samples, which demonstrates that caffeic acid protected these pigments. These results agree with those found by Dimitric-Markovic et al. (2000) who noticed that the addition of caffeic acid to malvidin 3.5-diglucoside solutions promoted an increase in the color stability of this pigment, even when the solutions were submitted to temperature increases of 22–50 °C. The presence of light significantly interfered in anthocyanin t1/2 values (p = 0.000), thus influencing the stability of these pigments. Anthocyanin degradation was more distinct in the presence of light, an effect heightened when the samples were kept at 29 ± 3 °C (Table 2). Similar results have been found by Falca˜o et al. (2004) who verified that the stability of anthocyanins from Cabernet Sauvignon grape skins was significantly reduced in the presence of light, mainly in samples kept at 29 ± 2 °C. In all the storage conditions evaluated, there was a significant increase in t1/2 and R% values in test samples, except for those kept at pH 3.0 in the presence of light
(Table 2). Caffeic acid was observed conferring more protection to anthocyanins at pH 4.0 than at pH 3.0. A non-significant difference was observed among the t1/2 values of anthocyanins at the pH values studied when these were analyzed in isolation (p = 0.057), but this factor interacted with treatment, temperature and light presence (p = 0.037). Fig. 2A illustrates the interaction between temperature, pH and presence of light. There was no significant difference between t1/2 values of both control and test samples at pH 3.0, in the presence of light at both storage temperatures. However, at pH 4.0, an increase of 290 and 228 hours was observed in the t1/2 values of samples at 4 ± 1 °C (p = 0.021) and at 29 ± 3 °C (p = 0.009), respectively. Fig. 2B illustrates the interaction between temperature, pH and dark. A significant increase of 3332 h was observed in the t1/2 values of control samples at pH 3.0 when compared to samples at pH 4.0 kept at 4 ± 1 °C. This was not observed in test samples. As a whole, the addition of caffeic acid conferred a significant increase in the t1/2 of samples; this effect was more distinct at pH 4.0, at 4 ± 1 °C and in the dark, when an increase of 4,676 hours was observed. For the samples kept at pH 3.0, there was an increase of 1319 h. For samples kept at 29 ± 3 °C, the increase of anthocyanins t1/2 was similar in the two pH levels surveyed, 817 and 961 h at pH 3.0 and 4.0, respectively (2). The control solution used was 0.1 L/0.1 L crude extract: citrate buffer pH 3.8 (1:1) and test solution was 1.720 g/ 0.1 L/0.1 L (1:1:1) caffeic acid: crude extract: citrate buffer pH 3.8) added to 1000 g of the yoghurt model system. In the test samples (anthocyanins and caffeic acid) of the yoghurt model system there was a 7.5 Dk (nm) and a 0.242 DA in relation to control samples (anthocyanins), characterizing anthocyanins copigmentation in this system. Control samples and test samples displayed t1/2 values of 6980 and 13,653 hours and R% of 90% and 95%, respectively. It was observed that the addition of the copigment contributed to an increase in the stability of the pigment
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Fig. 2. Representation of the interaction between temperature (4 ± 1 and 29 ± 3 °C) and pH (3.0 and 4.0) in the model system samples without (control) and with (test) caffeic acid under light presence (A) and in the dark (B).
color (p = 0,000) and to an increase of 6673 h in t1/2 values. These t1/2 results were higher than those obtained by Falca˜o, Gauche, Barros, Drunkler, & Luiz (2003) who noticed that copigmentation of anthocyanins of Cabernet Sauvignon grape skins promoved to tannic acid did not confer a significant increase in the half-life time of anthocyanins in yoghurt model system. Therefore, the results demonstrate that anthocyanins comply with the requirements for dyes in this food model system. They also demonstrate that the addition of caffeic acid fostered a better stabilization of the productÕs color.
4. Conclusion Caffeic acid promoted an increase in the maximum absorption wavelength (bathochromic effect – Dk) and the absorbancy (hyperchromic effect – DA) in the absorption spectra of anthocyanins at pH 3.0 and 4.0, which characterizes an intermolecular copigmentation reaction. Generally, caffeic acid conferred greater stability to anthocyanin solutions under the storage conditions evaluated. The t1/2 maximum values of anthocyanins were verified in the samples with caffeic acid (test samples), kept in
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the dark at 4 ± 1 °C; the minimum values were those of samples kept in the presence of light and at 29 ± 3 °C, which shows that the association of these factors significantly degraded anthocyanins. In the yoghurt model system, anthocyanins presented stability suitable for use as a dye in foods and the addition of caffeic acid conferred an increase of 6673 h to the t1/2 of anthocyanins. References AOAC (1998). Official methods of analysis of the association of official analytical chemists (65th ed., 5th revision). USA: Association of Official Analytical Chemists. Asen, S., Stewart, R. N., & Norris, K. H. (1972). Co-pigmentation of anthocyanins in plant tissues and its effect on color. Phytochemistry, 11(3), 1139–1144. Baranac, J. M., Petranovic, N. A., & Dimitric-Markovic, J. M. (1997). Spectrophometric study of anthocyan copigmentation reactions. 2. Malvin and the nonglycosidized flavone quercetin. Journal of Agricultural and Food Chemistry, 45, 1694–1697. Boulton, R. (2001). The copigmentation of anthocyanins and its role in the color of red wine: A Critical Review. American Journal of Enology and Viticulture, 52(2), 67–80. Bridle, P., & Timberlake, C. F. (1997). Anthocyanins as natural food colours – selected aspects. Food Chemistry, 58(1–2), 103–109. Brouillard, R. (1983). The in vivo expression of anthocyanin colour in plants. Phytochemistry, 22(6), 1311–1323. Brouillard, R., Mazza, G., Saad, A. M., Gary, A., & Cheminat, A. (1989). The copigmentation reaction of anthocyanins: A micropobe for the structural study of aqueous solutions. Journal of American Chemical Society, 111, 2604–2611. Castelluccio, C., Paganga, G., Melikian, N., Bolwell, P. B., Pridham, J., Sampson, J., et al. (1995). Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants. Federation of the European Biochemical Societies Letters, 368, 188–192. Dangles, O., & Brouillard, R. (1992). A spectrophotometric method based on the anthocyanin copigmentation interaction and applied to the quantitative study of molecular complexes. Journal of the Chemical Society Perkin Transactions, 2, 247–257. Davies, A. J., & Mazza, G. (1993). Copigmentation of simple acylated anthocyanins with colourless phenolic compounds. Journal of Agricultural and Food Chemistry, 41(5), 716–720. Del Giovine, L., & Bocca, P. A. (2003). Determination of synthethic dyes in ice-cream by capillary electrophoresis. Food Control, 14, 131–135. Delgado-Vargas, F., Jime´nez, A. R., & Paredes-Lo´pez, O. (2000). Natural pigments: carotenoids, anthocyanins and betalains – characteristics, biosynthesis, processsing and stability. Critical Reviews in Food and Nutrition, 40(3), 173–289. Dimitric-Markovic, J. M., Ignjatovic, L. M. ., Markovic, D. A., & Baranac, J. M. (2003a). Antioxidant capabilities of some organic acids and their co-pigments with malvin – Part I. Journal of Electroanalytical Chemistry, 553, 169–175. Dimitric-Markovic, J. M., Ignjatovic, L. M., Markovic, D. A., & Baranac, J. M. (2003b). Antioxidant capabilities of some organic acids and their co-pigments with malvin – Part II. Journal of Electroanalytical Chemistry, 553, 177–182. Dimitric-Markovic, J. M. D., Petranovic, N. A., & Baranac, J. M. (2000). Spectrophotometric study of the copigmentation of malvin with caffeic and ferulic acids. Journal of Agricultural and Food Chemistry, 48, 5530–5536. Falca˜o, L. D., Gauche, C., Barros, D. M., Drunkler, D. A., & Luiz, M. T. B. (2003). Estabilidade de antocianinas de uvas Cabernet Sauvignon e betalaı´nas de beterraba Asgrow Wonder adicionadas de a´cido taˆnico em iogurte. Revista do Instituto de Laticı´nios Caˆndido Tostes, 38(332), 18–24.
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Falca˜o, L. D., Gauche, C., Barros, D. M., Prudeˆncio, E. S., Gris, E. F., SantÕAnna, E. S., et al. (2004). Stability of anthocyanins from grape (Vitis vinifera L.) skins with tannic acid in a model system. Italian Journal of Food Science, 16(3), 323–332. Francis, F. J. (1989). Food colorants: anthocyanins. CRC Critical Reviews. Food Science and Nutrition, 8, 273–314. Fuleki, T., & Francis, F. J. (1968a). Quantitative methods for anthocyanins. 1. Extraction and determination of total anthocyanins in cranberries. Journal of Food Science, 33(1), 72–77. Fuleki, T., & Francis, F. J. (1968b). Quantitative methods for anthocyanins. 2. Determination of total anthocyanin and degradation index for cranberries juices. Journal of Food Science, 33, 78–83. Giusti, M., Saona-Rodriguez, L. E., & Wrolstad, R. E. (1999). Molar absortivity and color characteristics of acylated and non-acilated pelargonidin-based anthocyanins. Journal of Agricultural and Food Chemistry, 47, 4631–4637. Giusti, M. M., & Worsltad, R. E. (2001). Characterization and measurement of anthocyanins by UV–visible spectroscopy. Current protocols in food analytical chemistry. New York: Wiley. Gonzalez, E., Fougerousse, A., & Brouillard, R. (2001). Two diacylated malvidin glycosides from Petunia hybrida flowers. Phytochemistry, 58, 257–1262. Goto, T., & Kondo, T. (1991). Structure and molecular staking of anthocyanins flower color variation. Angewandte Chemie International Edition, 30, 17–33. Harborne, J. B. (1967). Comparative biochemistry of the flavonoids (pp. 1– 30). New York: Academic Press, In Brouillard, R. (1983). The in vivo expression of anthocyanin colors in plants. Phytochemistry, 22(6), 311– 323.. Hoshino, T., Matsumoto, U., & Goto, T. (1980). The stabilizing effect of the acyl group on the copigmentation of acylated anthocyanins with Cglucosylflavones. Phytochemistry, 19, 663–667. Hrazdina, G. (1974). Reactions of anthocyanins in food products. Lebensmittel Wissenschaft Technologie, 4, 103–108. Katsaboxakis, K., Papanicolaou, D., & Melanitou, M. (1998). Stability of pigmented orange anthocyanins in model and real food system. Italian Journal Food Science, 10, 17–25. Kirca, A., & Cemeroglu, B. (2003). Degradation kinetics of anthocyanins in blood orange juice and concentrate. Food Chemistry, 81, 583–587. Koeppen, B. H., & Basson, D. S. (1966). The anthocyanin pigments of Barlinka grapes. Phytochemistry, 5, 183–187, and Giusti, M., SaonaRodriguez, L. E., & Wrolstad, R. E. (1999). Molar absortivity and color characteristics of acylated and non-acilated pelargonidin-based anthocyanins. Journal of Agricultural and Food Chemistry, 47, 4631– 4637.. Lazze´, M. C., Pizzala, R., Sa´vio, M., Stivala, L. A., Prosperi, E., & Bianchi, L. (2003). Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth muscle and hepatoma cells. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 535, 103–115. Lees, D. H., & Francis, F. G. (1972). Standardization of pigment analysis in cranberries. Hortscience, 7, 83–84. Marinova, E. M., & Yanishlieva, N. V. (2003). Antioxidant activity and mechanism of action of some phenolic acids at ambient and high temperatures. Food Chemistry, 81, 189–197. Mazza, G., & Brouillard, R. (1987). Recent developments in the stabilization of anthocyanins in food products. Food Chemistry, 25, 207–225. Mazza, G., & Brouillard, R. (1990). The mechanism of co-pigmentation of anthocyanins in aqueous solutions. Phytochemistry, 29, 1097–1102. Mazza, G., & Miniati, E. (1993). Anthocyanins in fruits, vegetables and grains. Boca Raton, Florida (USA): CRC Press, 362p. Morais, H., Ramos, C., Forga´cs, E., Cserha´ti, T., & Oliviera, J. (2002). Influence of storage conditions on the stability of monomeric anthocyanins studied by reversed-phase high-performance liquid chromatography. Journal of Chromatography B, 770(1-2), 297–301.
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Passamonti, S., Vrhovsek, U., Vanzo, A., & Mattivi, F. (2003). The stomach as a site for anthocyanins absorption from food. Federation of the European Biochemical Societies Letters, 544, 210–213. Sroka, Z., & Cisowski, W. (2003). Hydrogen peroxide scavenging, antioxidant and anti-radical activity of some phenolic acids. Food and Chemical Toxicology, 41, 753–758. Valcheva-Kuzmanova, S., Borisova, P., Galunska, B., Krasnaliev, I., & Belcheva, A. (2004). Hepatoprotective effect of the natural fruit juice from Aronia melanocarpa on carbon tetrachloride-induced
acute liver damage in rats. Experimental and Toxicologic Pathology, 56, 195–201. Wilska-Jeszka, J., & Korzuchowska, A. (1996). Anthocyanins and chlorogenic acid copigmentation – influence on the colour of strawberry and chokeberry juices. Zeitschrift fu¨r Lebensmitteluntersuchung und – Forschung, 203, 38–42. Wulf, L. W., & Nagel, C. W. (1978). High-pressure liquid chromatographic separation of anthocyanins of Vitis vinifera. American Journal of Enology and Viticulture, 29, 42–49.
Analytical, Nutritional and Clinical Methods
Caffeic acid copigmentation of anthocyanins from Cabernet Sauvignon grape extracts in model systems E.F. Gris, E.A. Ferreira, L.D. Falca˜o, M.T. Bordignon-Luiz
*
Departamento de Cieˆncia e Tecnologia de Alimentos-CAL/CCA/UFSC. Rod. Admar Gonzaga, 1346, CEP: 88034-001 Floriano´polis SC, Brazil Received 24 May 2005; received in revised form 16 September 2005; accepted 23 October 2005
Abstract Caffeic copigmentation of anthocyanins from Cabernet Sauvignon grape extracts in model system and yoghurt model system was investigated, as well as the influence of copigmentation on the stability of these pigments. In the model system, the dependence of anthocyanin stability on added caffeic acid was evaluated at temperatures (4 ± 1 °C and 29 ± 3 °C) and in the presence or absence of light, at two pH values: 3.0 and 4.0; in the yoghurt model system, the stability of the anthocyanins was evaluated at 4 ± 1 °C in the dark. The half-life and percentage of color retention of the anthocyanins in all treatments was calculated. The spectrophotometric results (Dk, DA) revealed that interaction occurred between the crude extract of anthocyanins and caffeic acid suggesting copigmentation. The addition of caffeic acid (1:1 w/v) significantly increased (p < 0.05) the stability of anthocyanins in both model and yoghurt systems. In the model system the temperature and presence of light significantly influenced the stability of anthocyanins (p < 0.05), where the highest values for half-life were obtained for anthocyanins with caffeic acid at pH 3.0, stored in the dark at a temperature of 4 ± 1 °C (6.930 h). In the yoghurt system the caffeic acid increased the half-life time of anthocyanins to 6673 h. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Anthocyanin; Stability; Copigmentation; Caffeic acid
1. Introduction Color is one of the most important attributes of food and beverages. It is appreciated for its intrinsic aesthetic value and as a basis for identification and quality judgment. Synthetic dyes are much used by the food industry because they have higher stability with respect to light, oxygen, temperature, and pH, among other factors; but the presence of these dyes can represent a risk factor to health (Del Giovine & Bocca, 2003; Mazza & Brouillard, 1987). Natural plant colorants have been in high demand by the food industry as substitutes of synthetic dyes for the past decade. The need has stemmed from legal actions against their use and from the consumer concern against synthetic food additives (Francis, 1989). Anthocyanins have a high *
Corresponding author. Tel.: +55 48 331 5376; fax : +55 48 331 9943. E-mail addresses: [email protected] (E.F. Gris), bordign@ cca.ufsc.br (M.T. Bordignon-Luiz). 0308-8146/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2005.10.014
potential for use as natural colorants due to their attractive colors (Mazza & Miniati, 1993); they are not only non-toxic and non-mutagenic (Bridle & Timberlake, 1997), but their pharmacological properties are also well known and account for their therapeutic use (Delgado-Vargas, Jime´nez, & Paredes-Lo´pez, 2000; Lazze´ et al., 2003; Valcheva-Kuzmanova, Borisova, Galunska, Krasnaliev, & Belcheva, 2004). Anthocyanins are vacuolar pigments and their organelles account for the presence of membrane bound bodies called anthocyanoplasts; they are responsible for many of the attractive colors of flowers, fruits and leaves, from scarlet to blue (Brouillard, 1983). Chemically, anthocyanins are flavonoids, and consequently based on a C15 skeleton with a chromane ring bearing a second aromatic ring in position 2 (C6-C3-C6) and with one or more sugar molecules bonded at different hydroxylated positions of the basic structure, the flavylium cation (Mazza & Miniati, 1993). In acidic or neutral media, four anthocyanin structures exist in equilibrium: the flavylium cation, quinoidal base,
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carbinol pseudobase, and chalcone (Hrazdina, 1974). The concentration of each structure has been shown to be dependent upon the pH of the media (Mazza & Miniati, 1993). Besides the changes caused by pH, the color of anthocyanins is determined by the structure and concentration of the pigment, the presence of metallic ions, temperature, enzymes, oxygen, sugars, copigments, and other factors (Dangles & Brouillard, 1992; Hoshino, Matsumoto, & Goto, 1980). All these effects, however, cannot account for the ample variety of colors and hues in the plant world (Brouillard, Mazza, Saad, Gary, & Cheminat, 1989), which is why it was initially assumed, and later confirmed, that different colors are the consequences of complexation, i.e., copigmentation of the flavylium chromophore (Asen, Stewart, & Norris, 1972; Brouillard, 1983). Copigmentation is regarded today as one of the significant factors of structure stabilization, i.e., coloration, of anthocyanins under in vivo conditions (Boulton, 2001; Brouillard et al., 1989); it is a molecular interaction that occurs between anthocyanins and copigments (Gonzalez, Fougerousse, & Brouillard, 2001; Goto & Kondo, 1991). Copigments may be flavonoids, alkaloids, amino acids, organic acids, polysaccharides, metals and anthocyanins themselves, and their basic role is to protect the colored flavylium cation from nucleophilic attack of the water molecule (Asen et al., 1972; Mazza & Brouillard, 1990). Caffeic acid is an organic phenolic molecule. It is part of the plant tissue in which it plays an important role. In addition to having a significant metabolic function, cinnamic acids constitute the main acyl group in the structure of acylated anthocyanins, which are also part of plant tissue (Harborne, 1967). Dimitric-Markovic, Petranovic, and Baranac (2000), evaluated in vitro the intermolecular copigmentation reaction of malvidin 3.5-diglucoside with caffeic acid using UV–Vis spectrophotometry. These authors verified that the organic acid possesses the capacity to participate in copigmentation reactions with anthocyanins and that this process is dependent on the pH of the medium, copigment concentration and temperature. Through the oxidation potential Dimitric-Markovic, Ignjatovic, Markovic, and Baranac (2003a, 2003b), evaluated the antioxidant properties of the copigmentation complexes of malvidin 3.5-diglucoside and caffeic acid. The results confirmed that caffeic acid has the capacity to copigment this anthocyanin and that the antioxidant activity of the complex anthocyanin:caffeic acid was greater than the antioxidant activity of the anthocyanin alone. The scavenging potential and antioxidant properties of caffeic acid in terms of its ability to increase the resistance of low density lipoproteins (LDL) to cholesterol oxidation were reported (Castelluccio et al., 1995; Marinova & Yanishlieva, 2003; Sroka & Cisowski, 2003). Evaluation of the copigmentation reaction and the stability of anthocyanins from an extract of Cabernet Sauvignon grape were realized due to the fact that this grape variety is in ascension in Brazil, especially in Santa Catarina State. The use of the anthocyanins from grape skins of wine indus-
try solid residues is a real possibility under future consideration, as it is currently realized in many countries (Bridle & Timberlake, 1997); the use of a crude extract, which does not depend on high cost processes of purification, could be an alternative use of this industrial residue as a source of anthocyanins for food applications. The objective of the present work was to evaluate the stability of anthocyanins from a crude extract of Cabernet Sauvignon (Vitis vinifera L.) grape skins with caffeic acid under different conditions of temperature (4 ± 1 °C and 29 ± 3 °C), pH (3.0 and 4.0) and in the presence of light in a model system and in a yoghurt model system stored at 4 ± 1 °C in the dark. 2. Materials and methods The study was carried out with red grapes of Cabernet Sauvignon cultivars (V. vinifera L.), harvest 2002, from the Agricultural Research Enterprise (EPAGRI), in the town of Sa˜o Joaquim, 1400 m a.s.l., Santa Catarina, Brazil. For the elaboration of the yoghurt system commercial sugar and milk were used and a thermophilic lactic culture (YC-X11, CHR HANSEN, Sa˜o Paulo, Brazil). The caffeic acid used to evaluate copigmentation was obtained from Fluka (P95.0% HPLC). All other reagents were of analytical grade. 2.1. Anthocyanin crude extract: extraction and quantification One hundred grams of grape skins were macerated overnight in the dark, at 4.0 ± 1 °C, in 400 mL of 0.1% HCl in methanol. The crude extract obtained was filtered through a nylon filter and the skin remains were washed with 100 mL of extracting solvent (Lees & Francis, 1972). Vacuum filtration was carried out on Whatman (no. 2) filter paper using a Bu¨chner funnel and afterwards the extract was concentrated under vacuum (Bu¨chi R-144, Switzerland) at 35 °C to 50% of the methanol initial volume (Wilska-Jeszka & Korzuchowska, 1996). The concentrated extract was filtered through a 0.45-lm Millipore filter to eliminate possible interferences in the spectrophotometric readings (Millipore, Bedford, MA) and kept in an amber flask at 4.0 ± 1 °C. The quantification of total monomeric anthocyanins was determined by the pH-differential methods (Giusti & Worsltad, 2001) using MW = 562.5 and e = 29,500 ((Koeppen & Basson (1966) and Giusti, Saona-Rodrı´guez & Wrolstad, 1999) and expressed in g/L of malvidin 3-glucoside, the main anthocyanins from Cabernet Sauvignon (V. vinifera L.) grapes (Passamonti, Vrhovsek, Vanzo, & Mattivi, 2003; Wulf & Nagel, 1978). 2.2. Stability study 2.2.1. Model system To prepare control samples (model system without caffeic acid) and test samples (model system with caffeic acid), anthocyanic extract dilutions were made in a model system
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of citrate buffer (0.1 M, Merck citric acid – sodium citrate), at both pH 3.0 and 4.0 to obtain absorbance values between 1.000 and 1.200 at maximum absorption wavelength. According to Asen et al. (1972) & Brouillard et al. (1989) copigmentation occurs between the colored forms of anthocyanins (flavylium cation and quinoidal base) and certain copigments. Thus the absorbancy values of the samples were standardized (absorbance at ‘‘zero time’’) at pH 3.0 and pH 4.0, with the aim of standardizing the effect of copigmentation. The concentration of caffeic acid to be added to the anthocyanins (weight (mg)/volume (mL) ratios w/v) was determined from the total solid content of the crude extract (g/L), itself previously determined by drying 3 mL of anthocyanin crude extract at 105 °C until constant weight was reached (AOAC, 1998). Different ratios of caffeic acid were assessed at random up to the maximum concentration of acid diluted in the model system of citrate buffer (0.5:1; 0.8:1 and 1:1 (mg/mL) caffeic acid:anthocyanin crude extract volume). Control and test samples (model system with caffeic acid) were prepared in volumetric vials and added the crude extract with 0.05% potassium sorbate (g/L) to prevent bacterial growth. The volume was completed with the model system of citrate buffer (0.1 M, Merck citric acid–sodium citrate), at pH 3.0 and 4.0; caffeic acid was added to the test samples. The solutions were agitated for 30 min and then transferred to screw-top test tubes and left to rest for 2 h in the dark and at a temperature of 29 ± 3 °C to reach equilibrium (Fuleki & Francis, 1968b). Test and control samples were kept under the following conditions: at 4 ± 1 and 29 ± 3 °C, under a fluorescent lamp (2500 lm) and in the absence of light, in pH 3.0 and 4.0; all samples were maintained in the presence of atmospheric oxygen. The values of sample absorbancy were monitored using three test tubes with screw-tops containing model systems for each of the repetitions throughout the experiment. The absorption spectra of samples were monitored through UV–Vis absorption spectrophotometry in the visible wavelength range from 400 to 700 nm, at regular time intervals of 72 h, until 60% or more of the pigments were degraded. A Hitachi U2010 spectrophotometer (CA, USA) fitted with a 10-mm optical path quartz cell was used in this monitoring. After reaching equilibrium in the dark (2 h), the anthocyanins maximum wavelength was read in the model system, absorbance being characterized as ‘‘zero time’’. Distilled water was used as blank in all of the experiments (Fuleki & Francis, 1968a; Giusti, Saona-Rodriguez, & Wrolstad, 1999). 2.2.2. Yoghurt model system Yoghurt model systems were prepared by adding 5% sugar to milk, which was then heated to 95 °C for 5 min, cooled to 42 °C and inoculated with 0.05% of thermophilic lactic culture (YC-X11, CHR HANSEN, Sa˜o Paulo, Brazil). The samples were incubated at 42 °C until they reached pH 3.8 and transferred to a cold room (4 ± 1 °C)
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for storage. The concentration of total solids of the crude extract (g/L) was used as an initial parameter for the addition of caffeic acid. Control samples (yoghurt model system without caffeic acid) and test samples (yoghurt model system with caffeic acid) were prepared. Control solutions (1:1 v/v crude extract: citrate buffer pH 3.8) and test solutions (1:1:1 w/v/v caffeic acid: crude extract: citrate buffer pH 3.8) were added to yoghurt systems (control and test) in order to obtain a color similar to that of a commercial artificially colored grape flavor yoghurt. The control and test yoghurt model systems were kept at 4 ± 1 °C, in the dark for 45 days (1,080 hours), shelf-life of the yoghurt. The study of pigments stability was carried as follows: 20 g aliquots of each sample were centrifuged (Janetzki K-24, Germany) for 30 min at 8942 g. The supernatant was filtered and used for the reading of sample absorbance at the maximum absorption wavelengths. Distilled water was used as blank in all of the experiments (Fuleki & Francis, 1968a; Giusti et al., 1999). The absorption spectra of the samples were monitored through UV–Vis absorption spectrophotometry in the visible range at regular time intervals of 72 h for 45 days. 2.3. Kinetic study For all samples the half-life time (t1/2) values were calculated according Kirca & Cemeroglu (2003) and the percent color retention percentage (R%) values were calculated according to Katsaboxakis, Papanicolaou, & Melanitou (1998) in accordance with the equations below: lnðAt =A0 Þ ¼ k t t1=2 ¼ ln 0:5 k 1 At % Retention ¼ 100; A0 where t = time (hours); At = final absorbance (‘‘time t’’); A0 = initial absorbance (‘‘time zero’’). 2.4. Statistical analysis In the model system, the experiment was conducted entirely at random, in two repetitions in triplicate, in the Factorial 24 scheme where the factors under analysis were the presence of copigment (caffeic acid), temperature (4 ± 1 and 29 ± 3 °C), presence of light and dark and pH (3.0 and 4.0). In the yoghurt model system, the experiment was conducted with two repetitions in duplicate. The halflife time of all samples was studied by means of analysis of variance (ANOVA) with STATISTICA software, version 6.0 (2001). The Tukey Test was applied whenever a difference was detected among factors at a 5% level of significance. 3. Results and discussion The content of anthocyanins in the anthocyanic crude extract was calculated to be 1.82 g/L, expressed in malvidin
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3-glucoside. The total ratio of solids (g/L) of the crude extract was estimated to be 17.20 g/L. The volume (mL) of anthocyanic crude extract and weight of caffeic acid (mg) added to the buffer solutions are described in Table 1. Fig. 1 illustrates the spectrophotometry of the effect of caffeic acid in absorption spectra of anthocyanins at pH 3.0 and 4.0, where it was verified that caffeic acid promoted an increase in the maximum absorption wavelength (bathochromic effect – Dk) and absorbance (hyperchromic effect – DA). These shifts simultaneous characterize an intermolecular copigmentation reaction (Asen et al., 1972; Davies & Mazza, 1993; Mazza & Brouillard, 1990). An increase of 16.63%, 22.61% and 39.54% was observed in sample absorbance at pH 3.0, respectively, and of 10.72%, 33.65% and 38.01% in sample absorbance at pH 4.0, respectively. The greatest hyperchromic shifts occurred with greatest intensity in the samples with the highest proportion of added caffeic acid, both at pH 3.0 and 4.0, an occurrence repeated with the bathochromic shifts (Fig. 1). According to the results obtained in this experiment, the control samples presented a bathochromic shift of Dk = 2.5 nm with the increase in pH from 3.0 to 4.0. This may have occurred because the existing flavylium cation forms in the crude extract diminished proportionally with the increased pH of the media, causing a slight bathochromic shift in the maximum absorption wavelength (Baranac, Petranovic, & Dimitric-Markovic, 1997). The increase of caffeic acid concentration in anthocyanin solutions (w/v) produced an increase in the hyperchromic and bathochromic effects, a result that agrees with the literature and confirms that copigmentation reaction depends on copigment concentration (Fig. 1) (Mazza & Brouillard, 1990; Wilska-Jeszka & Korzuchowska, 1996). Hyperchromic effects were seen to occur more intensely (p = 0.0325) in solutions at pH 4.0 at concentrations of 0.8:1 and 1:1 (caffeic acid:anthocyanin solutions (mg/mL) (Fig. 1B). According to Mazza & Brouillard (1987), at this pH, anthocyanins are essentially in their hemiacetal (colorless) form, a chemical structure resulting from the hydration of flavylium cations. Wilska-Jeszka & Korzuchowska (1996) affirm that the effect of copigmentation is greater at pH 3.4–3.6 when compared to pH 3.0 because the copigment prevents the hydration reaction and stabilizes the colTable 1 Caffeic acid concentration values and anthocyanin crude extract volumes used in the model system experiment at pH 3.0 and 4.0 pH
Concentration caffeic acid: anthocyanins extract (w/v), in a model system
Anthocyanins extract/in a model system (vial 25 mL)a (mL)
3.0
0.5:1 (8.6 mg/mL) 0.8:1 (13.76 mg/mL) 1:1 (17.20 mg/mL)
0.6 0.6 0.6
4.0
0.5:1 (8.6 mg/mL) 0.8:1 (13.76 mg/mL) 1:1 (17.20 mg/mL)
0.9 0.9 0.9
a
Values used to obtain initial absorbance close to 1.000.
Fig. 1. Effect of caffeic acid addition at maximum absorption wavelength for anthocyanins from grape skins at pH 3.0 (A) and 4.0 (B) at T (°C) = 29 ± 3. 1 = control samples (anthocyanins without caffeic acid) k = 517.5 nm (A) and 520.0 nm (B); 2, 3 and 4 = test samples (anthocyanins:caffeic acid) in concentrations 0.5:1; 0.8:1 and 1:1 (w/v), respectively.
ored form of anthocyanins more intensely in this pH range. Boulton (2001) also affirms that copigmentation occurs with greater intensity at pH 3.6, which justifies the greater intensity of hyperchromic and bathochromic effects at pH 4.0 than at 3.0. The ratio of caffeic acid used in the experiment to check stability was 1:1 caffeic acid:anthocyanin crude extract volume (mg/mL), because greater hyperchromic (DA) and bathochromic (Dk) effects were observed in this ratio in the anthocyanin maximum absorption wavelength at the pH values studied (Fig. 1). The 24 Factorial model was considered well adjusted with a R2 = 0.977 correlation coefficient. The analysis of variance showed that there was a significant difference between different treatments (with and without caffeic acid), temperatures (4 ± 1 and 29 ± 3 °C) and in presence of light and dark, as well as significant interactions between all the factors analyzed. Table 2 shows the t1/2 (h) and percentage retention color (R%) values of control (anthocyanins) and test (anthocyanins and caffeic acid) solutions in different storage conditions. Caffeic acid significantly interfered in the stability of anthocyanins (p = 0.001) promoting a significant increase in the t1/2 values of the samples, except for those
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Table 2 Half-life time and colour retention percentage of model system samples without (control) and with (test) caffeic acid (1:1 w/v) in different storage conditions pH
T (°C)
Light/dark
Half-life (h)
Colour retention (%)
Control sample 3.0
4 ± 1 °C 29 ± 3 °C
4.0
4 ± 1 °C 29 ± 3 °C
** *
Test sample
Control sample
Test sample
*
D L D L
5641 1430 894 232
6930 1413 1711* 306
96 84 76 36
97 83 86 45
D L D L
2309 1060 898 464
6986* 1350* 1896* 693*
90 79 76 58
96 83 88 69
Average of two repetitions in triplicate; L = under light (2500 lm); D = dark. Significantly differ from the control (p < 0.05).
kept at pH 3.0 and in the presence of light (Table 2). As shown in Table 2, there was a greater increase in t1/2 and R% in the samples kept in the dark, at 4 ± 1 °C, at the two pH values studied. These results are in agreement with data previously reported in the literature, where the effect of copigmentation of grape anthocyanins from Cabernet Sauvignon grape skins with tannic acid promoted increased pigment stability (Falca˜o et al., 2004). The different sample storage temperatures significantly influenced the stability of anthocyanins in buffer solution (p = 0.000). The increase in temperature, from 4 ± 1 to 29 ± 3 °C, was seen to quicken the degradation of these pigments in all the samples, as confirmed by the t1/2 and R% values (Table 2). Similar results have been observed by Morais, Ramos, Forga´cs, Cserha´ti, & Oliviera (2002) who assessed the stability of malvidin 3-glucoside from V. vinifera (var. Red globe) grape skins at three different temperatures (24, 32 and 40 °C) where the increase in temperature was seen to quicken the degradation of this pigment. Table 2 also shows that anthocyanin degradation at 29 ± 3 °C, as well as at 4 ± 1 °C, in general, occurred with greater intensity in the control samples, which demonstrates that caffeic acid protected these pigments. These results agree with those found by Dimitric-Markovic et al. (2000) who noticed that the addition of caffeic acid to malvidin 3.5-diglucoside solutions promoted an increase in the color stability of this pigment, even when the solutions were submitted to temperature increases of 22–50 °C. The presence of light significantly interfered in anthocyanin t1/2 values (p = 0.000), thus influencing the stability of these pigments. Anthocyanin degradation was more distinct in the presence of light, an effect heightened when the samples were kept at 29 ± 3 °C (Table 2). Similar results have been found by Falca˜o et al. (2004) who verified that the stability of anthocyanins from Cabernet Sauvignon grape skins was significantly reduced in the presence of light, mainly in samples kept at 29 ± 2 °C. In all the storage conditions evaluated, there was a significant increase in t1/2 and R% values in test samples, except for those kept at pH 3.0 in the presence of light
(Table 2). Caffeic acid was observed conferring more protection to anthocyanins at pH 4.0 than at pH 3.0. A non-significant difference was observed among the t1/2 values of anthocyanins at the pH values studied when these were analyzed in isolation (p = 0.057), but this factor interacted with treatment, temperature and light presence (p = 0.037). Fig. 2A illustrates the interaction between temperature, pH and presence of light. There was no significant difference between t1/2 values of both control and test samples at pH 3.0, in the presence of light at both storage temperatures. However, at pH 4.0, an increase of 290 and 228 hours was observed in the t1/2 values of samples at 4 ± 1 °C (p = 0.021) and at 29 ± 3 °C (p = 0.009), respectively. Fig. 2B illustrates the interaction between temperature, pH and dark. A significant increase of 3332 h was observed in the t1/2 values of control samples at pH 3.0 when compared to samples at pH 4.0 kept at 4 ± 1 °C. This was not observed in test samples. As a whole, the addition of caffeic acid conferred a significant increase in the t1/2 of samples; this effect was more distinct at pH 4.0, at 4 ± 1 °C and in the dark, when an increase of 4,676 hours was observed. For the samples kept at pH 3.0, there was an increase of 1319 h. For samples kept at 29 ± 3 °C, the increase of anthocyanins t1/2 was similar in the two pH levels surveyed, 817 and 961 h at pH 3.0 and 4.0, respectively (2). The control solution used was 0.1 L/0.1 L crude extract: citrate buffer pH 3.8 (1:1) and test solution was 1.720 g/ 0.1 L/0.1 L (1:1:1) caffeic acid: crude extract: citrate buffer pH 3.8) added to 1000 g of the yoghurt model system. In the test samples (anthocyanins and caffeic acid) of the yoghurt model system there was a 7.5 Dk (nm) and a 0.242 DA in relation to control samples (anthocyanins), characterizing anthocyanins copigmentation in this system. Control samples and test samples displayed t1/2 values of 6980 and 13,653 hours and R% of 90% and 95%, respectively. It was observed that the addition of the copigment contributed to an increase in the stability of the pigment
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Fig. 2. Representation of the interaction between temperature (4 ± 1 and 29 ± 3 °C) and pH (3.0 and 4.0) in the model system samples without (control) and with (test) caffeic acid under light presence (A) and in the dark (B).
color (p = 0,000) and to an increase of 6673 h in t1/2 values. These t1/2 results were higher than those obtained by Falca˜o, Gauche, Barros, Drunkler, & Luiz (2003) who noticed that copigmentation of anthocyanins of Cabernet Sauvignon grape skins promoved to tannic acid did not confer a significant increase in the half-life time of anthocyanins in yoghurt model system. Therefore, the results demonstrate that anthocyanins comply with the requirements for dyes in this food model system. They also demonstrate that the addition of caffeic acid fostered a better stabilization of the productÕs color.
4. Conclusion Caffeic acid promoted an increase in the maximum absorption wavelength (bathochromic effect – Dk) and the absorbancy (hyperchromic effect – DA) in the absorption spectra of anthocyanins at pH 3.0 and 4.0, which characterizes an intermolecular copigmentation reaction. Generally, caffeic acid conferred greater stability to anthocyanin solutions under the storage conditions evaluated. The t1/2 maximum values of anthocyanins were verified in the samples with caffeic acid (test samples), kept in
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the dark at 4 ± 1 °C; the minimum values were those of samples kept in the presence of light and at 29 ± 3 °C, which shows that the association of these factors significantly degraded anthocyanins. In the yoghurt model system, anthocyanins presented stability suitable for use as a dye in foods and the addition of caffeic acid conferred an increase of 6673 h to the t1/2 of anthocyanins. References AOAC (1998). Official methods of analysis of the association of official analytical chemists (65th ed., 5th revision). USA: Association of Official Analytical Chemists. Asen, S., Stewart, R. N., & Norris, K. H. (1972). Co-pigmentation of anthocyanins in plant tissues and its effect on color. Phytochemistry, 11(3), 1139–1144. Baranac, J. M., Petranovic, N. A., & Dimitric-Markovic, J. M. (1997). Spectrophometric study of anthocyan copigmentation reactions. 2. Malvin and the nonglycosidized flavone quercetin. Journal of Agricultural and Food Chemistry, 45, 1694–1697. Boulton, R. (2001). The copigmentation of anthocyanins and its role in the color of red wine: A Critical Review. American Journal of Enology and Viticulture, 52(2), 67–80. Bridle, P., & Timberlake, C. F. (1997). Anthocyanins as natural food colours – selected aspects. Food Chemistry, 58(1–2), 103–109. Brouillard, R. (1983). The in vivo expression of anthocyanin colour in plants. Phytochemistry, 22(6), 1311–1323. Brouillard, R., Mazza, G., Saad, A. M., Gary, A., & Cheminat, A. (1989). The copigmentation reaction of anthocyanins: A micropobe for the structural study of aqueous solutions. Journal of American Chemical Society, 111, 2604–2611. Castelluccio, C., Paganga, G., Melikian, N., Bolwell, P. B., Pridham, J., Sampson, J., et al. (1995). Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants. Federation of the European Biochemical Societies Letters, 368, 188–192. Dangles, O., & Brouillard, R. (1992). A spectrophotometric method based on the anthocyanin copigmentation interaction and applied to the quantitative study of molecular complexes. Journal of the Chemical Society Perkin Transactions, 2, 247–257. Davies, A. J., & Mazza, G. (1993). Copigmentation of simple acylated anthocyanins with colourless phenolic compounds. Journal of Agricultural and Food Chemistry, 41(5), 716–720. Del Giovine, L., & Bocca, P. A. (2003). Determination of synthethic dyes in ice-cream by capillary electrophoresis. Food Control, 14, 131–135. Delgado-Vargas, F., Jime´nez, A. R., & Paredes-Lo´pez, O. (2000). Natural pigments: carotenoids, anthocyanins and betalains – characteristics, biosynthesis, processsing and stability. Critical Reviews in Food and Nutrition, 40(3), 173–289. Dimitric-Markovic, J. M., Ignjatovic, L. M. ., Markovic, D. A., & Baranac, J. M. (2003a). Antioxidant capabilities of some organic acids and their co-pigments with malvin – Part I. Journal of Electroanalytical Chemistry, 553, 169–175. Dimitric-Markovic, J. M., Ignjatovic, L. M., Markovic, D. A., & Baranac, J. M. (2003b). Antioxidant capabilities of some organic acids and their co-pigments with malvin – Part II. Journal of Electroanalytical Chemistry, 553, 177–182. Dimitric-Markovic, J. M. D., Petranovic, N. A., & Baranac, J. M. (2000). Spectrophotometric study of the copigmentation of malvin with caffeic and ferulic acids. Journal of Agricultural and Food Chemistry, 48, 5530–5536. Falca˜o, L. D., Gauche, C., Barros, D. M., Drunkler, D. A., & Luiz, M. T. B. (2003). Estabilidade de antocianinas de uvas Cabernet Sauvignon e betalaı´nas de beterraba Asgrow Wonder adicionadas de a´cido taˆnico em iogurte. Revista do Instituto de Laticı´nios Caˆndido Tostes, 38(332), 18–24.
1295
Falca˜o, L. D., Gauche, C., Barros, D. M., Prudeˆncio, E. S., Gris, E. F., SantÕAnna, E. S., et al. (2004). Stability of anthocyanins from grape (Vitis vinifera L.) skins with tannic acid in a model system. Italian Journal of Food Science, 16(3), 323–332. Francis, F. J. (1989). Food colorants: anthocyanins. CRC Critical Reviews. Food Science and Nutrition, 8, 273–314. Fuleki, T., & Francis, F. J. (1968a). Quantitative methods for anthocyanins. 1. Extraction and determination of total anthocyanins in cranberries. Journal of Food Science, 33(1), 72–77. Fuleki, T., & Francis, F. J. (1968b). Quantitative methods for anthocyanins. 2. Determination of total anthocyanin and degradation index for cranberries juices. Journal of Food Science, 33, 78–83. Giusti, M., Saona-Rodriguez, L. E., & Wrolstad, R. E. (1999). Molar absortivity and color characteristics of acylated and non-acilated pelargonidin-based anthocyanins. Journal of Agricultural and Food Chemistry, 47, 4631–4637. Giusti, M. M., & Worsltad, R. E. (2001). Characterization and measurement of anthocyanins by UV–visible spectroscopy. Current protocols in food analytical chemistry. New York: Wiley. Gonzalez, E., Fougerousse, A., & Brouillard, R. (2001). Two diacylated malvidin glycosides from Petunia hybrida flowers. Phytochemistry, 58, 257–1262. Goto, T., & Kondo, T. (1991). Structure and molecular staking of anthocyanins flower color variation. Angewandte Chemie International Edition, 30, 17–33. Harborne, J. B. (1967). Comparative biochemistry of the flavonoids (pp. 1– 30). New York: Academic Press, In Brouillard, R. (1983). The in vivo expression of anthocyanin colors in plants. Phytochemistry, 22(6), 311– 323.. Hoshino, T., Matsumoto, U., & Goto, T. (1980). The stabilizing effect of the acyl group on the copigmentation of acylated anthocyanins with Cglucosylflavones. Phytochemistry, 19, 663–667. Hrazdina, G. (1974). Reactions of anthocyanins in food products. Lebensmittel Wissenschaft Technologie, 4, 103–108. Katsaboxakis, K., Papanicolaou, D., & Melanitou, M. (1998). Stability of pigmented orange anthocyanins in model and real food system. Italian Journal Food Science, 10, 17–25. Kirca, A., & Cemeroglu, B. (2003). Degradation kinetics of anthocyanins in blood orange juice and concentrate. Food Chemistry, 81, 583–587. Koeppen, B. H., & Basson, D. S. (1966). The anthocyanin pigments of Barlinka grapes. Phytochemistry, 5, 183–187, and Giusti, M., SaonaRodriguez, L. E., & Wrolstad, R. E. (1999). Molar absortivity and color characteristics of acylated and non-acilated pelargonidin-based anthocyanins. Journal of Agricultural and Food Chemistry, 47, 4631– 4637.. Lazze´, M. C., Pizzala, R., Sa´vio, M., Stivala, L. A., Prosperi, E., & Bianchi, L. (2003). Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth muscle and hepatoma cells. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, 535, 103–115. Lees, D. H., & Francis, F. G. (1972). Standardization of pigment analysis in cranberries. Hortscience, 7, 83–84. Marinova, E. M., & Yanishlieva, N. V. (2003). Antioxidant activity and mechanism of action of some phenolic acids at ambient and high temperatures. Food Chemistry, 81, 189–197. Mazza, G., & Brouillard, R. (1987). Recent developments in the stabilization of anthocyanins in food products. Food Chemistry, 25, 207–225. Mazza, G., & Brouillard, R. (1990). The mechanism of co-pigmentation of anthocyanins in aqueous solutions. Phytochemistry, 29, 1097–1102. Mazza, G., & Miniati, E. (1993). Anthocyanins in fruits, vegetables and grains. Boca Raton, Florida (USA): CRC Press, 362p. Morais, H., Ramos, C., Forga´cs, E., Cserha´ti, T., & Oliviera, J. (2002). Influence of storage conditions on the stability of monomeric anthocyanins studied by reversed-phase high-performance liquid chromatography. Journal of Chromatography B, 770(1-2), 297–301.
1296
E.F. Gris et al. / Food Chemistry 100 (2007) 1289–1296
Passamonti, S., Vrhovsek, U., Vanzo, A., & Mattivi, F. (2003). The stomach as a site for anthocyanins absorption from food. Federation of the European Biochemical Societies Letters, 544, 210–213. Sroka, Z., & Cisowski, W. (2003). Hydrogen peroxide scavenging, antioxidant and anti-radical activity of some phenolic acids. Food and Chemical Toxicology, 41, 753–758. Valcheva-Kuzmanova, S., Borisova, P., Galunska, B., Krasnaliev, I., & Belcheva, A. (2004). Hepatoprotective effect of the natural fruit juice from Aronia melanocarpa on carbon tetrachloride-induced
acute liver damage in rats. Experimental and Toxicologic Pathology, 56, 195–201. Wilska-Jeszka, J., & Korzuchowska, A. (1996). Anthocyanins and chlorogenic acid copigmentation – influence on the colour of strawberry and chokeberry juices. Zeitschrift fu¨r Lebensmitteluntersuchung und – Forschung, 203, 38–42. Wulf, L. W., & Nagel, C. W. (1978). High-pressure liquid chromatographic separation of anthocyanins of Vitis vinifera. American Journal of Enology and Viticulture, 29, 42–49.