Anthocyanins

Anthocyanins D.R. Kammerer WALA Heilmittel GmbH, Bad Boll/Eckwälden, Germany

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Abstract Among water-soluble natural pigments, anthocyanins play by far the most important role due to their widespread occurrence, imparting attractive hues and bright colors to many flowers, vegetables, fruits, and grains. However, their limited stability upon processing and storage is still a major challenge for industrial manufacturers. Consequently, synthetic pigments have frequently been preferred to improve visual appearance and restore initial color shades of processed foods. Due to health concerns associated with the application of synthetic dyes, modern food processing aims at stabilizing genuine pigments (eg, by adding anthocyanins and anthocyanin-rich juice concentrates as natural colorants and coloring foodstuffs, respectively), which exhibit superior stability and high consumer acceptance. This chapter provides a general overview of anthocyanins and their occurrence in the plant kingdom, with particular focus on food plants, structural features, color properties, and intrinsic and extrinsic factors that have an impact on the stability of these putative health-beneficial components. Such parameters having a significant impact on anthocyanin stability are of particular relevance for systematic process optimization that aims at color stabilization in the modern food industry. Keywords: Browning, Color stability, Coloring foodstuff, Intramolecular and intermolecular copigmentation, Pigment degradation, Processing, Storage.

1.  Structural Diversity of Anthocyanins and Their Occurrence in Food Plants Anthocyanins form a subgroup of flavonoids, which are characterized by their typical C6dC3dC6 structural backbone. They are almost ubiquitously found in higher plants, with the exception of 10 plant families of the Caryophyllales, where betalains may be found. The high structural diversity is based on more than 30 anthocyanidins, which have been unambiguously identified so far (Bueno et al., 2012). Among these aglycones, six compounds—cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin—are the most common representatives, the basic structures of which are depicted in Fig. 3.1 (Mazza and Miniati, 1993). These aglycones differ in their hydroxylation and methylation patterns, which also goes along with differing color shades, ranging from an orange-red of pelargonidin to blue-violet of delphinidin. Generally, hydroxylation of the aglycone causes a bathochromic shift, whereas methylation is associated with a hypsochromic shift (Stintzing and Carle, 2004). With only few exceptions, such as 3-deoxyanthocyanidins Handbook on Natural Pigments in Food and Beverages. http://dx.doi.org/10.1016/B978-0-08-100371-8.00003-8 Copyright © 2016 Elsevier Ltd. All rights reserved.

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Handbook on Natural Pigments in Food and Beverages R1 R2 O+

HO

R3 OH

OH

Pelargonidin Cyanidin Delphinidin Peonidin Petunidin Malvidin

R1

R2

R3

H OH OH OCH3 OCH3 OCH3

OH OH OH OH OH OH

H H OH H OH OCH3

Figure 3.1  Structures of the six most frequently occurring anthocyanidins.

occurring in red-skinned bananas, sorghum, and black tea, the aglycones do not occur in free form but are generally accumulated in plant vacuoles as glycosides. Anthocyanidins may be substituted at different positions with one or more saccharide moieties, thus giving rise mainly to 3-glycosides and 3,5-glycosides. Among sugar substituents, glucose, rhamnose, xylose, galactose and arabinose are predominant. Further, disaccharides, such as rutinose, sambubiose, lathyrose, sophorose, and more complex saccharide moieties are also found in anthocyanins. In addition, the saccharide substituents may be esterified with aliphatic and aromatic organic acids. Among the former, acetic, malonic, succinic, oxalic, tartaric, and malic acids have been identified, whereas coumaric, caffeic, ferulic, and p-hydroxybenzoic acids have been found among the latter (Delgado-Vargas et al., 2000). With certain structural features, acylated anthocyanins may show enhanced stability with regard to decolorization of the molecule upon pH modification, which is referred to as intramolecular copigmentation (see Section 2.3). More than 60% of anthocyanins reported in the literature are acylated compounds. Due to the variability of saccharide moieties that may be linked to anthocyanidins, varying positions of the ether linkage and the possibility of acylating the sugar moieties with aliphatic and aromatic acids, a high number of different structures results, which is usually 15–20 times greater than the number of anthocyanidins (Mazza and Brouillard, 1987). In addition, further nonanthocyanin flavonoid glycosides may be covalently bound to anthocyanins, thus forming even more complex pigments. Fresh plant material usually contains a limited number of anthocyanins. However, there are also examples being characterized by more complex pigment profiles consisting of a wide range of individual compounds, such as in grapes (Kammerer et al., 2004). Different plants and plant parts usually exhibit unique anthocyanin profiles. Therefore, pigment patterns may be used as chemotaxonomic markers (Bueno et al., 2012). Anthocyanin concentrations in most fruits and vegetables have been reported to range from 0.1% up to 1% of dry weight. However, marked differences in contents

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may be observed between different plant species and even between different cultivars of the same species, which is due to genetic factors, light, and temperature conditions during growth, agronomic factors, and maturity (Bueno et al., 2012). As an example, plums are known to contain comparatively low levels of anthocyanins. Pigment levels in plums may vary in a wide range of 20–250 mg/kg. In contrast, various berries are richest in anthocyanins, with chokeberries and elderberries showing contents of 5000– 10,000 and 2000–10,000 mg/kg, respectively (Clifford, 2000). Because anthocyanins are mostly accumulated in outer plant parts such as epidermal cell layers, small-sized fruits characterized by a high surface-to-volume ratio are often particularly rich in anthocyanins. This high pigment concentration usually brings about enhanced pigment stability, which is due to interaction of anthocyanins with themselves, thus protecting them from hydration or degradation (see Sections 2.2 and 2.3). For this reason, the aforementioned examples exhibit superior color stability as compared to plums or strawberries, for example, which have significantly lower pigment contents.

2.  Intrinsic and Extrinsic Factors Having an Impact on Color Evolution and Anthocyanin Stability 2.1   Compound Structure Generally, the color of nonacylated and monoacylated anthocyanins is largely determined by the substitution pattern of the aglycone B-ring. An increasing number of hydroxyl groups (pelargonidin → cyanidin → delphinidin) causes a bathochromic shift, whereas increasing methylation (cyanidin  malvidin) brings about a → peonidin →  hypsochromic shift. Acylation with cinnamic acids causes a bathochromic shift of the pigment, which can be observed as bluish colors; however, the type of acylating moiety and the position where the acyl group is attached to the saccharide is also of particular relevance for color evolution and spectral characteristics. Furthermore, acylation with cinnamic acids usually goes along with lower visual detection thresholds, thus indicating higher tinctorial strengths. This is known as a hyperchromic effect and could be demonstrated both for isolated anthocyanins and pigments in complex plant crude extracts (Stintzing et al., 2002). Anthocyanin color is also known to strongly depend on the solvent system. As an example, methanolic solutions of pelargonidin glycosides reveal a hyperchromic effect with higher chroma values, at the same time producing lower hue values as compared to aqueous solutions. However, these effects appear to depend on the degree of acylation and on anthocyanin concentration (Giusti et al., 1999). However, such phenomena may be neglected when anthocyanin color in food is evaluated, except for commodities with significant proportions of solvents other than water, such as in red wine. Color intensity of individual compounds may be deduced from visual detection thresholds, which is defined as the minimal pigment concentration at which a difference between purified water and an anthocyanin solution can still be visually observed. Furthermore, color activity values may be determined, which are defined as the ratio of pigment concentrations to the detection thresholds of the individual

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pigments. Finally, the color contribution of individual compounds to the overall color can be given as the percentage of the color activity value in relation to the dilution factor of an anthocyanin extract, thus allowing the identification of key colorants (Degenhardt et al., 2000; Hofmann, 1998; Stintzing et al., 2002). As an example of the application of this concept to the color of foods, red wine anthocyanins were evaluated. Monomeric anthocyanins were found to make up 57–69% of the color of young red wines, whereas the contribution of the latter to aged wines is negligible due to the formation of oligomeric and polymeric derivatives upon storage. The monoglucosides were found to exhibit the highest color intensities, which could be deduced from lowest visual detection thresholds, whereas a second saccharide moiety on the aglycone or acylation of the saccharide moiety increased detection thresholds at the natural pH of red wine (ie, at a pH value of around 3.6; Degenhardt et al., 2000). These findings are partially in contrast to previous investigations of the color and spectral characteristics of pelargonidin-based anthocyanins performed at pH 1, where a drop in the molar absorptivity of pelargonidin monoglycosides as compared to the pelargonidin aglycone was observed. In contrast, diglycosides seemed to evoke a hyperchromic effect (Giusti et al., 1999). Such contrasting reports underline the necessity to clearly define conditions as close as possible to those of the final product when color evaluation is performed; the color and spectral characteristics of anthocyanins are a complex interaction of compound structure, concentration, pH value, temperature, metal ions, solvent type, and the occurrence of nonanthocyanin phenolics and further copigments.

2.2  pH Value The structure of anthocyanins and, consequently, their color quality, intensity, and stability markedly depend on pH value. Only under highly acidic conditions (pH ∼1), isolated anthocyanins exist in their flavylium cation (phenyl-2-benzopyrylium) form exhibiting their bright red color. Upon pH increase, anthocyanins are converted into colorless hemiacetal structures as a result of a nucleophilic attack of water in position C-2. In a subsequent step, ring fission may occur, bringing about the formation of yellowish E- and Z-chalcones (Fig. 3.2). From a practical point of view, hydration constants (pKH values) of anthocyanins are of particular relevance for food applications, describing the equilibrium reaction between flavylium cations and the corresponding hemiacetal structures. As an example, anthocyanins substituted at the 5-position reveal markedly lowered hydration constants, whereas a higher number of sugar moieties at the 3-position increases hydration constants to a minor degree. Most efficiently with regard to anthocyanin stabilization, acylation with phenolic acids was shown to substantially raise hydration constants, which translates into enhanced stability of anthocyanins at the pH value of most food commodities as far as the nucleophilic attack of water is concerned, forming colorless hemiacetal structures. The type of acylating moiety markedly affects the degree of this stabilizing effect (Stintzing et al., 2002). Furthermore, flavylium cations may be deprotonated upon pH increase, yielding neutral quinoidal bases exhibiting purplish shades and anionic quinoidal bases with bluish colors (Fig. 3.2) (Stintzing and Carle, 2004).

Anthocyanins

65 R3ʹ

R3ʹ OH

OH O

HO

HO

R5ʹ

OH

HO O

HO

R5ʹ

OGlc OH E-chalcone (yellowish)

OGlc OH Hemiacetal (colorless)

OH OGlc

R3ʹ

OH

R5ʹ O OH Z-chalcone (yellowish)

+ (–H /+H2O) KH

R3ʹ OH +

HO

O

R5ʹ OGlc

OH Flavylium cation (red) KA

+

+

(–H )

KA

KA (–H+)

R3ʹ

R3ʹ OH HO

O

R5ʹ OGlc

O

O

O

R5ʹ

HO

OGlc OH Neutral quinoidal bases (purplish)

+

O

R3ʹ

–

O

+

R5ʹ OGlc

(–H ) R3ʹ –

O O

O

R5ʹ OGlc

R5ʹ

OH

+

O

O

OGlc

(–H ) R3ʹ

O

R3ʹ

OH

(–H )

HO

(–H )

OH Anionic quinoidal bases (bluish)

–

O

O

O R5ʹ

OGlc OH

Figure 3.2  Structural transformation of anthocyanins depending on pH value. After Stintzing, F.C., Carle, R., 2004. Functional properties of anthocyanins and betalains in plants, food, and in human nutrition. Trends in Food Science & Technology 15, 19–38; reprinted with permission.

2.3  Intramolecular and Intermolecular Copigmentation, Self-Association Association mechanisms, comprising both intramolecular copigmentation, intermolecular copigmentation, and self-association, significantly improve compound stability and, accordingly, color retention. These stabilization mechanisms depend on the type and concentration of the participating compounds as well as pH value, temperature, and solvent type. Copigmentation phenomena are unique to anthocyanins and have not been observed so far for any other polyphenol subclass or class of nonphenolic compounds (Brouillard et al., 2010). Generally, copigmentation phenomena are associated with a bathochromic shift in the absorption spectrum of the corresponding anthocyanin or plant extract and a hyperchromic effect. Further, copigmentation becomes obvious using 1H nuclear magnetic resonance techniques, providing evidence of vertical stacking of anthocyanidins and their copigments (Brouillard et al., 2010). Intramolecular copigmentation involves the interaction of the anthocyanidin backbone, with a copigment forming part

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of the anthocyanin itself. This is achieved by the alignment of aromatic acyl moieties of acylated anthocyanins with the anthocyanidin core structure, thus sterically hindering the nucleophilic attack of water at positions 2 or 4 of the anthocyanidin, leading to the colorless hemiacetal forms (Brouillard, 1983). Structural features determine the effectiveness of this intramolecular interaction, which is brought about by ππ-interaction of the acyl moiety with the aromatic backbone of the anthocyanidin. The saccharides act as spacers between the acyl moiety and the anthocyanidin backbone, allowing the aforementioned alignment of both molecule parts. A spacer consisting of two or more monosaccharide units is regarded as a prerequisite for efficient stabilization. Anthocyanins with two or more aromatic acyl moieties exhibit excellent pigment stability, which is due to the formation of so-called sandwich-type structures—that is, the possibility of embedding the anthocyanidin core between two acyl moieties (Dangles et al., 1993). Consequently, anthocyanins acylated with aromatic acids exhibit superior stability as compared to their nonacylated counterparts, which can, among others, be deduced from increased hydration constants as compared to their nonacylated counterparts (see Section 2.2). The overall process is spontaneous and exothermic (Escribano-Bailon and Santos-Buelga, 2012; Kammerer et al., 2007). A comparable effect can be brought about by further colorless compounds, which are not bound to the anthocyanin molecule—so-called copigments. Mainly van der Waals forces, hydrophobic effects and ionic interactions have been suggested as driving forces for this type of interaction with either the flavylium cation or the quinoidal base form of anthocyanins (Cavalcanti et al., 2011). The protective effect is also based on steric hindrance of the nucleophilic attack of water. However, in contrast to intramolecular copigmentation, the present phenomenon is based on the interaction of two different molecules. Consequently, the latter stabilization mechanism is less effective than intramolecular copigmentation due to the entropic advantage of the copigment being attached to the anthocyanidin nucleus and the nonrequirement of bringing together chromophores and copigments in the case of intramolecular effects (Brouillard et al., 2010). Copigments may belong to a wide range of different compound classes, such as colorless phenolic compounds (ie, both flavonoids and phenolic acids), alkaloids, amino acids, purines, and organic acids. Furthermore, a high copigment-to-pigment ratio is favorable for enhancing pigment stability (Mazza and Brouillard, 1987). Copigmentation effects are more pronounced at lower temperatures and are reduced upon heating, becoming negligible at temperatures close to the boiling point of water (Dangles and Brouillard, 1992), which is of particular interest for food preparation involving thermal processing steps. Generally, formation constants were determined for this type of molecular association, being not larger than 100–300 M−1, and thus characterizing copigmentation as weak interaction allowing the existence of a chemical equilibrium between the complexed and noncomplexed forms (Brouillard et al., 2010). Copigmentation effects are also responsible for the nonlinear deviation from Beer’s law, which must be taken into consideration whenever anthocyanins contents are to be measured spectrophotometrically (Boulton, 2001). Moreover, anthocyanin stabilization may be brought about by a mechanism referred to as self-association. That is, the aforementioned steric hindrance of the hydration of the anthocyanidin aglycone in position 2 is accomplished by the association of

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several anthocyanin molecules via hydrophobic interactions of their aromatic nuclei. By implication, this effect is concentration-dependent; upon increasing anthocyanin concentrations, a bathochromic shift can be observed (Cavalcanti et al., 2011).

2.4   Metal Complexation Anthocyanins exhibiting two or more vicinal hydroxyl functions (ie, mainly cyanidin, delphinidin, and petunidin glycosides) may form complexes with di- or trivalent cations, such as Fe3+, Al3+, Mg2+, Sn2+ or Cu2+. This complex formation goes along with the stabilization of the anthocyanins, but also with a significant bathochromic shift of the absorption spectrum. Naturally occurring complex pigment structures (eg, in plant flowers) have been characterized. These metalloanthocyanins, such as commelinin in Commelina communis L., may be composed of several anthocyanin molecules, metal ions, and nonanthocyanin copigments, thus forming supramolecular complexes that are characterized by high stability in their intact plant cell environment and in concentrated solutions; however, the pigments easily dissociate and become colorless upon dilution with water. Another familiar example of blue colors formed by metal complexation of anthocyanins may be observed in hydrangea (Hydrangea macrophylla (Thunb.) Ser.), where the blue pigment of the sepals is formed by delphinidin 3-glucoside through complexation with Al3+ (Yoshida et al., 2009).

2.5  Interaction of Anthocyanins With Food Hydrocolloids The interaction of anthocyanins with polymeric food components has long been postulated; however, analytical difficulties in analyzing such phenomena and in a more detailed characterization of the polymeric compounds involved in such interactions have hampered thorough and unambiguous conclusions. In a very early approach, Bayer and coworkers characterized a pigment from blue cornflower, being composed of a metalloanthocyanin based on cyanidin-glucoside, which interacts with a pectin-like substance. The latter was assumed to be mediated via carboxylic functions of the hydrocolloid acting as a ligand of the anthocyanin-metal ion chelate (Bayer et al., 1966). In further studies, assumptions were made that anthocyanins may only be adsorbed onto polymeric plant constituents, thus excluding chemical binding (Asen et al., 1970). More recent investigations into the interaction of polysaccharides with anthocyanins revealed a stabilizing effect of sodium alginate, pectin, and corn starch when added to certain anthocyanin solutions (Hubbermann et al., 2006). Meanwhile, much more detailed studies have been performed, revealing the stabilizing potential of pectins when added to black currant anthocyanins in model solutions, with amidated pectins revealing most pronounced effects and citrus pectins performing better than apple pectins with regard to anthocyanin retention upon storage (Buchweitz et al., 2013). Interestingly, certain pectins, such as from sugar beet, may also be applied to generate and stabilize anthocyanin-based blue colors upon storage. A particular pectic fraction isolated from sugar beet pectin brought about a bathochromic shift of up to 50 nm, thus yielding appealing intense gentian-blue colors due to the formation of

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metalloanthocyanins, which were stabilized by interaction with the pectic compounds and avoided precipitation of anthocyanin complexes. The highest bathochromic shifts and most intense blue colors were obtained in model solutions with delphinidin 3-glucoside, exhibiting a pyrogallol moiety, as compared to cyanidin 3-glucoside and petunidin 3-glucoside, which are characterized by two vicinal hydroxyl functions, also allowing complex formation with metal ions (Buchweitz et al., 2012a,b). Consequently, this type of interaction and stabilization may be exploited for the application of anthocyanin-based blue food colorants.

2.6  Deposition of Anthocyanins in Plant Cells Anthocyanins are water-soluble pigments accumulated in plant vacuoles. However, the pigments are not necessarily distributed homogeneously in the vacuoles. With the detection of so-called anthocyanic vacuolar inclusions (AVIs), it has become obvious that anthocyanins may also be highly accumulated in certain parts within the vacuole, whereas others may be almost devoid of anthocyanins. AVIs are intravacuolar structures, which allow the spatial accumulation of anthocyanins. They have been described in more than 50 plant species, occurring in flowers, fruits, and vegetables. The occurrence of AVIs has a significant effect on the color of the corresponding plant part. A markedly changed color hue and bathochromic shifts have been found to be due to the occurrence of such structures. This has been studied in detail for the color of rose flowers, exhibiting a color shift to more intense blue shades upon progressive plant development (Gonnet, 2003). Investigations into the structure revealed these bodies to contain lipid components (ie, anthocyanins being associated with membrane structures). In addition, long-chain tannins and low amounts of protein and further unidentified organic compounds were detected in these AVIs (Conn et al., 2010). Thorough analysis of the vacuoles formed in sweet potato suspension cultures revealed the accumulation of significant amounts of a vacuolar protein, which seemed to participate in the formation of AVIs in potato cells (Nozue et al., 1995). The density of AVIs in fruit skins (eg, grapes) markedly affects skin coloration and contributes to the diversity of colors in the plant kingdom. Furthermore, accumulation of anthocyanins in AVIs appears to be structure-dependent. Acylated anthocyanins are preferably incorporated into AVIs. Thus, high proportions of acylated compounds may result in the formation of higher amounts of AVI (Mizuno et al., 2006). AVIs of Eustoma grandiflorum petals were analyzed and three different forms were detected—vesicle-like, rod-like, and irregular shaped forms; all of them consisted of membranous and thread structures throughout, while membranes encompassing the AVIs were not observed. Such differences in the AVI composition of different plants indicate that, although anthocyanin biosynthetic pathways together with their regulation have been elucidated in great detail, the mechanisms of anthocyanin accumulation within vacuoles are still poorly understood (Zhang et al., 2006). Such supramolecular structures exhibit interesting features with regard to the evolution of a wide range of color shades based on a limited array of compound structures, and also with regard to the resistance of anthocyanins toward degradation reactions. Although AVIs are expected to be at least partially degraded upon extraction from

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their natural matrix in the course of pigment extraction for the recovery of natural colorants, the elucidation of their supramolecular structure and stability behavior may help in the biomimetic formulation of pigments for industrial food applications, exerting improved stability and desired color attributes.

2.7  Color Range in Intact Plant Matrices and in Processed Foods Anthocyanins are responsible for the color of innumerable fruits, vegetables, flowers, and grains. Their color palette ranges from orange to bright red and deep blue to violet. The high diversity of colors found in nature based on a limited number of base structures may not solely be explained by pH variation, as previously assumed by Willstätter (1914), but by their interaction with further plant constituents (eg, nonanthocyanin phenolic compounds, metal ions, polymeric hydrocolloids), thus forming supramolecular complexes, and by differences in their spatial distribution, and finally, pH value differences. Further hues observed in fruits and vegetables are achieved by the co-occurrence of anthocyanins and nonanthocyanin pigments, such as chlorophylls and carotenoids in the same tissue, which has been observed in apples and eggplants (Stintzing and Carle, 2004). The aforementioned complex structures composed of anthocyanins and nonanthocyanin components are hardly retained upon extraction. Therefore, anthocyanin color in processed foods and of coloring extracts from anthocyanic matrices is mostly limited to red tints. Only more recent studies of anthocyanin interactions in processed foods with polymeric hydrocolloids and metal ions have pointed out ways to produce stable anthocyanin-based blue food colorants (Buchweitz et al., 2012a,b, 2013).

3.  Factors Affecting Anthocyanin Stability Upon Processing and Storage Anthocyanins are characterized by their high reactivity and concomitant susceptibility toward hydration and oxidation. Additionally, they show interactions and undergo reactions with further food components, such as ascorbic acid, sulfur dioxide, saccharides, and their degradation products. In addition, their susceptibility toward enzymatic degradation makes the stabilization of anthocyanins in plant-based foods or their application as food colorants as an alternative to synthetic dyes a real challenge (Jackman et al., 1987). Among the factors and measures during postharvest treatment and upon food processing markedly contributing to changes of the genuine anthocyanin profile, the formation of novel compounds, and the loss of pigments, the following may be itemized: winemaking, wine aging and storage, maturity stage and postharvest ripening, freezing and cold storage, thermal treatment and storage at elevated temperatures, comminution and pressing techniques, clarification, filtration and concentration, juice production, enzymatic treatment, peeling, extraction, drying, fermentation, preand postharvest dip treatment or spraying, 1-methylcyclopropene treatment, germination, postharvest ultraviolet irradiation, and controlled and modified atmosphere storage (Amarowicz et al., 2009).

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3.1  Genuine Plant Enzyme Activities and Technical Enzyme Preparations Technical enzyme preparations are commonly applied in modern food processing, aiming at the maximization of juice yields in the course of fruit and vegetable juice preparation and vinification, and for easier pressing and improved clarification. Moreover, enhanced release of secondary metabolites, such as phenolic compounds for their enrichment in the juice fraction, is considered a major advantage of such treatment. When applied to red grape mashes, pectinolytic enzymes bring about improved red wine color and enhanced visual color intensity (Guadalupe et al., 2007; Pardo et al., 1999). Cell wall degrading enzymes are further applied for improving the recovery of phenolic compounds, such as anthocyanins from the by-products of plant food processing, as a contribution to sustainable agricultural production. Such by-products are particularly rich in plant phenolics, which are only poorly extracted upon juice recovery. For this purpose, pectinolytic enzyme preparations are commercially available, which may be combined with cellulolytic and hemicellulolytic enzymes. Because these are technical preparations, the presence of glycosidase side activities must be carefully excluded to minimize pigment losses as a result of anthocyanin hydrolysis releasing the corresponding aglycones, which are rapidly degraded (Kammerer and Carle, 2009; Kammerer et al., 2005; Sacchi et al., 2005). Moreover, blanching has been shown in fruit juice technology of anthocyanic fruits to enhance anthocyanin yields, which is not only due to an increase of fruit skin permeability, but also to the initial thermal inactivation of genuine plant enzymes, such as polyphenoloxidases, peroxidases, or hydrolases, which cause significant pigment losses if not completely inactivated (Holzwarth et al., 2012a; Rossi et al., 2003). Lowered enzymatic activities may also be achieved by drying, thus reducing aw values, which also minimizes unwanted chemical reactions and microbial spoilage. However, drying temperature must be carefully monitored because pigment retention may significantly differ depending on this latter parameter (Del Caro et al., 2004).

3.2  General Effects of aw Value and Interaction With Saccharides High sugar concentrations translating into low water activity values of processed foods have been reported to be favorable with regard to anthocyanin stability (Castañedo-Ovando et al., 2009). Thus, attempts to reduce saccharide contents of low caloric products, such as of fruit spreads as an alternative to their corresponding jams, may bring about lower pigment retention. As an example, strawberry fruit spreads exhibiting water activity values of around 0.95 generally showed lower pigment stability upon storage than strawberry jams (aw ∼0.86), which was attributed to their differing water activities, whereas both gelled products exhibited enhanced pigment retention as compared to strawberry pureés with significantly higher aw values (Holzwarth et al., 2012a, 2013). With regard to the effects of individual saccharides on anthocyanin stability, contradictory results have been reported in literature. On the one hand, increased pigment losses in the presence of saccharides have been reported, and the extent

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was found to depend on the sugar type. On the other hand, sugar concentrations in processed foods above 20% were shown to enhance anthocyanin stability due to the lowering effect on water activity (Sadilova et al., 2009; Tsai et al., 2004). Moreover, saccharides may exert an indirect effect on anthocyanin stability via their degradation products formed in the course of Maillard reactions upon thermal treatment, such as 5-hydroxymethylfurfural and further furfural derivatives (Debicky-Pospisil et al., 1983; Jiménez et al., 2010; Sloan et al., 1969). Systematic investigations revealed the stability of individual compounds in the presence of saccharides to depend on their chemical structures and the food matrix. Presumably, polymeric matrix compounds may exert protective effects (Sadilova et al., 2009).

3.3  Effects of Light on Anthocyanin Stability Light has two significantly different effects on anthocyanins and anthocyanin stability. In living tissues, light is an essential factor for anthocyanin biosynthesis, thus stimulating pigment accumulation. This has also been observed when evaluating anthocyanin production by plant tissue cultures. However, light has also a marked pigment-degrading effect during storage of processed foods (Delgado-Vargas et al., 2000; Markakis, 1982). As an example, anthocyanin contents of pectin and gelatin gels colored with anthocyanin extracts recovered from red grape pomace decreased more rapidly and color retention was worse when the aforementioned products were stored under illumination, whereas samples stored in the dark revealed remarkably higher pigment stability (Maier et al., 2009). Similar findings were made for strawberry jams and spreads, which showed markedly higher losses of monomeric anthocyanins upon storage under illumination with a concomitant increase of the proportion of polymeric pigments (Holzwarth et al., 2013). Apart from these general observations, some more specific effects have been found for acylated anthocyanins: in pigments acylated with a coumaric acid moiety, light-induced trans-cis-isomerization of the exocyclic double bond caused a color change and stabilization (George et al., 2001). Such findings may be exploited in future approaches aiming at pigment stabilization upon processing and storage, which must be accompanied by the search of novel pigment sources with promising anthocyanin profiles and contents.

3.4  Susceptibility of Anthocyanins Toward Thermal Treatment Anthocyanins are known to be susceptible to thermal treatment (ie, pronounced pigment losses and color fading or browning), especially in the presence of oxygen, as may be observed upon the application of more rigorous time-temperature regimes in the course of food processing. Consequently, systematic evaluation of compound stability under various processing and storage conditions has been performed, aiming at maximal pigment retention as well as minimizing anthocyanin degradation and polymerization. Just to mention a few examples of such studies, the anthocyanin half-life values of processed foods were found to significantly decrease at elevated storage temperatures. The latter was demonstrated among others for fermented black carrot juices

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(Turker et al., 2004). Expectedly, frozen storage of anthocyanic foods maximizes pigment retention, also for a prolonged period of time. However, the freezing and thawing methods appear to be of particular relevance with regard to subsequent pigment loss upon frozen storage and thawing (Holzwarth et al., 2012b; Sahari et al., 2004). Thermal treatment of foods, such as blanching, pasteurization, and sterilization, are the most frequently applied methods for food preservation. However, these methods are mostly associated with a significant loss of anthocyanins, which is due to their thermolability. As an exception, blanching as an initial step during juice processing may contribute to enhanced anthocyanin yields, which is attributed to the thermal inactivation of pigment-degrading enzymes and to enhanced fruit skin permeability, thus increasing extraction yields (Rossi et al., 2003). In contrast, more rigorous thermal treatment inevitably goes along with anthocyanin losses. The evaluation of the extent of pigment degradation must also carefully consider the matrix of the heated foods, since high saccharide contents may result in the formation of hydroxymethylfurfural and further furfural derivatives, as well as end products of the caramelization process and Maillard reaction, which may interact with anthocyanins, resulting in higher pigment losses (Tsai et al., 2005). Treatment of preparations containing anthocyanins at elevated temperatures has frequently been found to reveal pigment degradation, which follows a first-order reaction kinetics (Amaraowicz et al., 2009). Drying is another effective tool for stabilizing foods toward chemical and enzymatic reactions and microbial spoilage. In this context, the drying temperature was demonstrated to have a marked effect on anthocyanin stability. Higher temperatures applied for drying were found to result in higher pigment losses (Del Caro et al., 2004). This was substantiated when comparing different drying methods, such as microwave-vacuum drying, freeze drying, and convective drying. Methods with lower thermal impact on fruits yielded dried products with higher anthocyanin contents (Böhm et al., 2006). Consequently, the optimization of time-temperature regimes applied during food processing is of utmost importance for maximizing pigment and color retention.

3.5  Ascorbic Acid Effects on Anthocyanin Stability Contradictory results have been reported for the effects of ascorbic acid on pigment stability. Numerous studies revealed enhanced pigment degradation in the presence of ascorbic acid, with the underlying mechanisms still being speculative. Cleavage of the pyrylium ring as a result of H2O2 formation through oxidation of ascorbic acid as well as the formation of anthocyanin-ascorbic acid condensation products and concomitant color fading have been postulated (Sadilova et al., 2009). In contrast, protective effects on anthocyanins have been observed as well. Compound structure of individual anthocyanins was found to have a marked effect on their reactivity toward ascorbic acid and, consequently, on their resistance toward color fading. In the presence of metal ions, complexes between anthocyanins and ascorbic acid may be formed, presumably protecting ascorbic acid against oxidation (Sadilova et al., 2009). Protective effects caused by ascorbic acid may also be due to its redox potential, thus reducing anthocyanin o-quinones and protecting them from polymerization reactions (Cavalcanti et al., 2011).

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3.6  Sulfite Application in Food Processing and Its Effects on Anthocyanin Color Sulfites are applied in food processing as antioxidants and preserving agents, among others, for the production of dried fruits or in vinification. However, their application goes along with discoloration of anthocyanins, which is due to a reversible nucleophilic addition of sulfite to the anthocyanidin backbone, mainly in position C4 of the C-ring, resulting in colorless sulfonic acid derivatives. Consequently, anthocyanins “blocked” in C4 position, such as in vitisins, exhibit greater resistance to sulfite-induced color loss (Delgado-Vargas et al., 2000; Stintzing and Carle, 2004). Due to the reversible nature of sulfonic acid formation and because the reaction products reveal higher hydrophilicity than their corresponding anthocyanins, sulfite addition has often been applied for enhancing pigment yields upon extraction, such as for the recovery of oenocyanin (E 163) from grape pomace. Subsequently, sulfite is thermally removed from the extracts to recover anthocyanin color. However, quantitative removal of sulfite is impossible in practice, leaving trace amounts in the resulting pigment extracts. This is of particular concern because pseudo-allergenic reactions caused by foods with added sulfites have been described, thus requiring labeling. Hence, novel strategies for cost-efficient recovery of anthocyanins without the application of sulfites have been developed, such as enzyme-assisted cell wall degradation using pectinolytic and cellulolytic enzyme preparations (Kammerer et al., 2014).

3.7  Anthocyanin Stabilization by Technological Means: Microencapsulation Pigment stability in processed foods may also be enhanced by technological processing. Among the most promising techniques, microencapsulation has been thoroughly studied. A wide range of different encapsulation techniques have been described; however, only a few have been evaluated with regard to anthocyanin stabilization. Spray drying has most frequently been applied for anthocyanin encapsulation. Obviously, anthocyanin stability is generally increased upon encapsulation, irrespective of the technique applied. However, processing conditions may have a significant impact on pigment retention. As an example, higher air inlet temperatures during spray drying of anthocyanin-containing extracts caused higher pigment losses (Ersus and Yurdagel, 2007). Maltodextrins are commonly applied as coating materials for anthocyanin stabilization; however, further polymers, such as pectin, starch, glucan, arabic gum, sodium alginate, curdlan, and whey protein isolate have also been studied. Besides imparting enhanced stability to anthocyanins, encapsulation technologies may also be applied to allow controlled release of anthocyanins in the gastrointestinal tract, which is of particular interest given the putative health-beneficial properties of anthocyanins. Consequently, encapsulated anthocyanins have been studied in simulated gastrointestinal models for assessing their bioavailability (Cavalcanti et al., 2011; Kandansamy and Somasundaram, 2012; Robert and Fredes, 2015).

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3.8  Reactions of Anthocyanins With Other Food Components, Oxygen and Metal Ions Anthocyanins are reactive components, which may undergo a wide range of reactions with different food components naturally occurring in plants or which are formed as a result of fermentation processes. Such modifications of genuine plant components have been thoroughly studied for the formation of derivatives from anthocyanins in the course of vinification and wine aging. The pigment profile of red grapes and of the corresponding red wines, especially after extended storage, significantly differs, which is due to oxidation and polymerization reactions as well as reactions of genuine anthocyanins with further grape constituents and fermentation products. During aging, the content of monomeric anthocyanins decreases significantly (Brenna et al., 2005). This decrease usually follows a first-order kinetics being associated with the formation of oxidation and condensation products, which also have a marked impact on the color attributes of the wines. Investigations of the behavior of individual anthocyanins during aging demonstrated individual compounds to reveal differing stabilities and reaction rates. Due to the complex phenolic profile of red grapes and young red wines and the multitude of potential reactions, aged red wines exhibit an even more complex composition (Monagas et al., 2005). Thus, far more than 100 anthocyanin-derived components may be detected in aged red wines. These belong to four different pigment families— anthocyanins, pyranoanthocyanins, direct flavanol-anthocyanin condensation products, and acetaldehyde-mediated flavanol-anthocyanin condensation products, such as vitisins and portisins (Alcade-Eon et al., 2006). The formation of the latter, involving further phenolic constituents of grapes and the corresponding wines as well as fermentation products such as acetaldehyde, go along with a change of color hues from a bright red of grape juices and young wines to a more red-brown tint of aged wines. The underlying mechanisms have been studied in great detail (Es-Safi and Cheynier, 2004; Fulcrand et al., 2006). The findings of such anthocyanin derivatives in plant extracts other than from grape, such as anthocyanin-flavanol condensation products in black currant extracts (McDougall et al., 2005), underlines the fact that anthocyanins in general may be regarded as reactive compounds. Hence, a fairly complex pigment profile is to be expected upon extended storage of anthocyanin containing products. Vinification and wine aging have been thoroughly assessed with regard to the effects of individual parameters on color evolution and anthocyanin stability. Among these parameters, the ripening stage of the grapes, the maceration technique, pressing parameters, alcoholic and malolactic fermentation, the fining of wines, the duration of storage, the type of container (ie, storage in oak barrels or stainless steel tanks), the addition of oak chips for enhanced aging, micro-oxygenation, and further factors may play significant roles (Amarowicz et al., 2009; Kammerer and Carle, 2009). In general, the storage of foods containing anthocyanins under aerobic conditions reveals greater pigment and color losses as compared to almost anaerobic conditions, as oxidative processes are prevented in the latter case (Andrés-Bello et al., 2013). Furthermore, the presence of oxygen must be considered together with the application of various time-temperature regimes when evaluating potential pigment losses because

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high temperatures have been shown to be particularly detrimental to anthocyanins under aerobic conditions (Cavalcanti et al., 2011). One further parameter that must be considered in this context is the presence of metal ions, which may catalyze anthocyanin oxidation and degradation (Stintzing and Carle, 2004).

3.9  Typical Anthocyanin Degradation Pathways Under aerobic conditions, anthocyanins tend to oxidize upon storage. Such oxidation processes are accelerated when anthocyanins are heated in the presence of oxygen (see Section 3.8), which usually has marked effects on color quality. Alternatively, anthocyanins may be degraded under anaerobic conditions, yielding colorless low-molecular reaction products. To systematically monitor the latter pathway, acylated and nonacylated anthocyanins were isolated and heated under strongly acidic conditions (pH 1.0) at 95°C. Such treatment causes anthocyanin hydrolysis, showing successive loss of sugar moieties, with pentoses being more readily split off than hexoses. Acylated anthocyanins are also hydrolyzed under such conditions, releasing partially glycosylated anthocyanidins and hydroxycinnamic acid glycosides. Anthocyanidins are highly unstable and further degraded to colorless breakdown products, such as phloroglucinaldehyde, 4-hydroxybenzoic acid, and protocatechuic acid. At pH 3.5, differing degradation pathways may be observed. Among others, chalcone glycosides were observed in heat-treated anthocyanin isolates and fission of the pyrylium ring initiated anthocyanin degradation. Final reaction products observed under these conditions were phloroglu­ cinaldehyde and phenolic acids as remainders of the A- and B-ring. Consequently, these reactions go along with significant color fading (Sadilova et al., 2006, 2007).

4.  Future Perspectives With regard to the aforementioned adverse effects of synthetic pigment ingestion on human health, it becomes quite obvious that the application of natural pigments in processed foods and their stabilization toward degradation and oxidation reactions are of increasing importance in modern food production. This trend is enhanced by intense research efforts systematically studying anthocyanin stability under well-defined processing conditions. Furthermore, the color palette of red and orange shades is extended, thus also producing stable violet and blue colors in processed foods as derived from the appealing colors found in flowers, fruits, and vegetables. This enhanced application goes along with research findings of recent decades, indicating that a range of secondary metabolites, among them anthocyanins, may exert health-beneficial properties upon ingestion, even though unambiguous evidence for most effects still needs to be adduced (Bueno et al., 2012). Nevertheless, anthocyanins have been demonstrated in numerous studies to be absorbed upon ingestion. However, their bioavailability appears to be poor as compared to other phenolic compounds. Usually, absorption rates of anthocyanins, expressed as relative urinary excretion rates, are below 1%. In general, absorption and biotransformation, the type of metabolites formed, their activity, tissue distribution, and accumulation are still largely unknown (Netzel et al., 2008; Santos-Buelga et al., 2014).

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Processing of foods rich in anthocyanins may affect their absorption, for example, due to partial hydrolysis of anthocyanidin-glycosides. As an example, the latter parameter as deduced from the urinary excretion of individual compounds was markedly affected by cooking of food and significantly depended on the compound structure (Kurilich et al., 2005). Food components added during processing or produced (eg, upon fermentation), such as ethanol, as well as constituents naturally present, such as saccharides, may also influence bioavailability (Frank et al., 2003; Mülleder et al., 2002). Moreover, high interindividual metabolic variability aggravates the prediction of anthocyanin bioavailability in each individual case (Cermak et al., 2009). Despite these difficulties, the putative health-beneficial properties of anthocyanins are expected to further boost the interest in anthocyanins aiming at their stabilization in their natural matrix as well as their relevance as natural pigments applied in food production. For this purpose, novel processing strategies are continuously developed considering all aspects reviewed in this introductory chapter, which may have detrimental effects on anthocyanins. Such considerations will also have to cover intermolecular and especially intramolecular copigmentation effects, which can be exploited to significantly stabilize anthocyanins in processed foods. Such strategies go hand in hand with the search for novel pigment sources possessing auspicious anthocyanin profiles and contents, which might thus be exploited for the recovery of pigment extracts exhibiting superior color stability and pigment retention during food processing and storage.

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Kammerer, D.R., Kammerer, J., Valet, R., Carle, R., 2014. Recovery of polyphenols from the by-products of plant food processing and application as valuable food ingredients. Food Research International 65, 2–12. Kandansamy, K., Somasundaram, P.D., 2012. Microencapsulation of colors by spray drying – a review. International Journal of Food Engineering 8, 17 pp. Kurilich, A.C., Clevidence, B.A., Britz, S.J., Simon, P.W., Novotny, J.A., 2005. Plasma and urinary responses are lower for acylated vs nonacylated anthocyanins from raw and cooked purple carrots. Journal of Agricultural and Food Chemistry 53, 6537–6542. Maier, T., Fromm, M., Schieber, A., Kammerer, D.R., Carle, R., 2009. Process and storage stability of anthocyanins and non-anthocyanin phenolics in pectin and gelatin gels enriched with grape pomace extracts. European Food Research and Technology 229, 949–960. Markakis, P., 1982. Stability of anthocyanins in foods. In: Markakis, P. (Ed.), Anthocyanins as Food Colors. Academic Press Inc., New York, pp. 163–178. Mazza, G., Brouillard, R., 1987. Recent developments in the stabilization of anthocyanins in food products. Food Chemistry 25, 207–225. Mazza, G., Miniati, E., 1993. Anthocyanins in Fruits, Vegetables, and Grains. CRC Press, Boca Raton/Ann Arbor/London/Tokyo. McDougall, G.J., Gordon, S., Brennan, R., Stewart, D., 2005. Anthocyanin-flavanol condensation products from black currant (Ribes nigrum L.). Journal of Agricultural and Food Chemistry 53, 7878–7885. Mizuno, H., Hirano, K., Okamoto, G., 2006. Effect of anthocyanin composition in grape skin on anthocyanic vacuolar inclusion development and skin coloration. Vitis 45, 173–177. Monagas, M., Bartolomé, B., Gómez-Cordovéz, C., 2005. Updated knowledge about the presence of phenolic compounds in wine. Critical Reviews in Food Science and Nutrition 45, 85–118. Mülleder, U., Murkovic, M., Pfannhauser, W., 2002. Urinary excretion of cyanidin glycosides. Journal of Biochemical and Biophysical Methods 53, 61–66. Netzel, M., Netzel, G., Maier, T., Kammerer, D.R., Carle, R., Schieber, A., Bitsch, I., Bitsch, R., 2008. Polyphenole aus Trauben – Erste Ergebnisse aus Metabolisierungsstudien mit Traubentresterextrakten und Probanden. Flüssiges Obst 75, 240–246. Nozue, M., Kubo, H., Nishimura, M., Yasuda, H., 1995. Detection and characterization of a vacuolar protein (VP24) in anthocyanin-producing cells of sweet potato in suspension culture. Plant and Cell Physiology 36, 883–889. Pardo, F., Salinas, M.R., Alonso, G.L., Navarro, G., Huerta, M.D., 1999. Effect of diverse enzyme preparations on the extraction and evolution of phenolic compounds in red wines. Food Chemistry 67, 135–142. Robert, P., Fredes, C., 2015. The encapsulation of anthocyanins from berry-type fruits. Trends in foods. Molecules 20, 5875–5888. Rossi, M., Giussani, E., Morelli, R., Lo Scalzo, R., Nani, R.C., Torreggiani, D., 2003. Effect of fruit blanching on phenolics and radical scavenging activity of highbush blueberry juice. Food Research International 36, 999–1005. Sacchi, K.L., Bisson, L.F., Adams, D.O., 2005. A review of the effect of wine-making techniques on phenolic extraction in red wines. American Journal of Enology and Viticulture 56, 197–206. Sadilova, E., Stintzing, F.C., Carle, R., 2006. Thermal degradation of acylated and non-acylated anthocyanins. Journal of Food Science 71, 504–512. Sadilova, E., Carle, R., Stintzing, F.C., 2007. Thermal degradation of anthocyanins and its impact on color and in vitro antioxidant capacity. Molecular Nutrition and Food Research 51, 1461–1471. Sadilova, E., Stintzing, F.C., Kammerer, D.R., Carle, R., 2009. Matrix dependent impact of sugar and ascorbic acid addition on color and anthocyanin stability of black carrot, elderberry

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