Anthocyanins as Natural Pigments in Beverages

ANTHOCYANINS AS NATURAL PIGMENTS IN BEVERAGES

12

A. Morata, C. López, W. Tesfaye, C. González, C. Escott Technical University of Madrid, Madrid, Spain

12.1  Relevance of Anthocyanins Anthocyanins are a group of water-soluble polyphenolic pigments that confer red-orange to blue-purple color to plant organs such as fruits, flowers, and leaves. They constitute one of the most widespread families of natural pigments in the Plant Kingdom, with carotenoids and tetrapyrrole derivatives (Delgado-Vargas et al., 2000). Chemically, anthocyanins belong to a class of polyphenols called flavonoids, which constitute the largest group, representing 60% of total dietary polyphenols (Nichenametla et al., 2006). Flavonoids have a common basic structure consisting of two aromatic rings linked by three carbon atoms that form an oxygenated heterocycle (Wolfe and Liu, 2008), leading to a tricyclic (C6C3C6) skeleton. The fused aromatic ring is called the A ring, the phenyl constituent is the B ring, and the heterocycle is known as the C ring. According to structural differences, which include variations in the number/substitution pattern of the hydroxyl and methoxy groups and the presence of a C2C3 double bond in the C ring, several subclasses of flavonoids can be considered, including the most important flavonols, flavones, isoflavones, flavonones, flavanols, and anthocyanins (Bueno et al., 2012). Anthocyanins are polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium or flavylium salts (Kong et al., 2003). Unlike other subgroups of flavonoids, anthocyanins have a positive charge in their structure at acidic pH. The anthocyanin molecule naturally occurs in plants as a glycoside, where the aglycon (called anthocyanidin) is bound to a sugar group, glucose and rhamnose being the most frequent ones (Mazza and Miniati, 1993). Glycosylation renders the molecule less reactive and more water soluble, increasing the polarity and stability and decreasing its capacity to interact with other macromolecules (Corradini et  al., 2011). Frequently, the sugar residues are acylated with aromatic (p-coumaric, caffeic, ferulic, sinapic, gallic, or p-hydroxybenzoic acids) or aliphatic acids (malonic, acetic, Value-Added Ingredients and Enrichments of Beverages. https://doi.org/10.1016/B978-0-12-816687-1.00012-6 © 2019 Elsevier Inc. All rights reserved.

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384  Chapter 12  Anthocyanins as Natural Pigments in Beverages

malic, succinic, tartaric, and oxalic acids), further improving anthocyanin stability, especially in extreme conditions of pH or temperature (Mazza et al., 2004; Zhang et al., 2014). Approximately 50% of anthocyanins in nature are acylated (Wallace and Giusti, 2015). The difference between anthocyanins is due to the number of hydroxyl and methoxy groups, the number and nature of sugars attached to the molecule, the position of the sugar bond, and the nature and number of aromatic acids attached to sugars. To date, 27 anthocyanidins were observed in nature (Wallace and Giusti, 2015), 6 of which are widely distributed, representing 90% of all anthocyanidins identified: pelargonidin (Pl), cyanidin (Cy), peonidin (Pe), delphinidin (Dp), petunidin (Pt), and malvidin (Mv) (Table 12.1). Their glycosylation and acylation renders more than 700 structurally different anthocyanin derivatives (Wallace and Giusti, 2015), which differ in color, stability, interactions with other compounds, bioavailability, and health-­ promoting effects (Zhang et al., 2014). The intensity and color provided by anthocyanins depend on the number of hydroxyl and methoxyl groups: methoxyl groups increase the bluish shade tone, whereas hydroxyl groups increase the redness (Heredia et al., 1998).

12.1.1 Synthesis of Anthocyanins Anthocyanins are considered an important group of phytochemicals, that is, chemical compounds produced by plants, which may have an influence on health, but are not essential nutrients (El Gharras, 2009; Bueno et al., 2012). Anthocyanins exist in almost every plant, in different organs as fruits, flowers, stems, leaves, and roots (Brouillard, 1982). They are especially present in flowers and fruits, with the highest concentrations found in red grapes and berries. According to

Table 12.1  Structural Differences of Six Most Common Anthocyanidins (Wallace and Giusti, 2015) Anthocyanidin

Abbreviation

R1

R2

λmax (nm)

Color

[M]+/aglycon (m/z)

Pelargonidin Cyanidin Peonidin Delphinidin Petunidin Malvidin

Pl Cy Pe Dp Pt Mv

H OH OCH3 OH OCH3 OCH3

H H H OH OH OCH3

494 506 506 508 508 510

Orange Orange-red Orange-red Red Red Bluish-red

– 449/287 463/301 465/303 479/317 493/331

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Wu et al. (2006), out of several food products screened including fruits, vegetables, nuts and dried fruits, and spices, among others, those foods where anthocyanins were found are pistachios, black and small red beans, cabbages, eggplant, lettuces, onions, red radishes, and several fruits such as apples, blackberries, blueberries, cherries, chokeberries, cranberries, black and red currant, elderberries, gooseberries, grapes, nectarines, peaches, plums, raspberries, and strawberries. They are usually found in the vacuolar solution of epidermal cells, although they can be located in discrete regions of the cellular vacuoles, called anthocyanoplasts (Pecket and Small, 1980). Cyanidin-3-glucoside is the most predominant anthocyanin found in nature (Wallace and Giusti, 2015). Pelargonidin, cyanidin, and delphinidin are more common in fruits, whereas peonidin, petunidin, and malvidin are more frequent in flowers (Table 12.2).

Table 12.2  Natural Sources of Anthocyanins Source

Main Anthocyanin

Anthocyanin Content

References

Pl-3-glu (77%–90%) Pl-3-rut (6%–11%) Cy-3-glu (3%–10%) Cy-3-rut (24%–52%) Cy 3-xy-rut (37%–58%)

200–600 mg/kg 2.3–4.5 g/kg (DW)

Lopes da Silva et al. (2007) Wang and Lin (2000)

9.5 g/kg (DW)

Cy-3-soph (60%) Cy-3-gluru (19%) Cy-3-glu (13%) Cy-3-rut (7%) Cy-3-glu Cy-3-rut Cy-3-glu Cy-3-rut Cy-3-glu Cy-3-rut Cy-3-rut (53%–73%) Pe-3-rut (7%–38%) Cy-3-glu (2%–18%) Cy-3-xyl (5%–8%) Pe-3-glu (0%–1%) Cy-3-rut (88%–93%) Cy-3-glu (6%–9%)

2.6–5.7 g/kg (DW)

Tian et al. (2006) Tulio et al. (2008) Wang and Lin (2000) Wang and Lin (2000) Ludwig et al. (2015)

FRUITS

Strawberry

Black raspberry

Red raspberry

Blackberry Mulberry Acai berry Plum

Sweet cherry

8–10 g/kg (DW) 1.2–2.1 g/kg (DW) 1.4 g/kg (DW)

Wang and Lin (2000) Pojer et al. (2013) Bae and Suh (2007)

366 mg/kg (FW)

Yamaguchi et al. (2015) Rogez et al. (2011) Usenik et al. (2009)

1.15 g/kg (FW)

Mozetic et al. (2004)

Continued

386  Chapter 12  Anthocyanins as Natural Pigments in Beverages

Table 12.2  Natural Sources of Anthocyanins—cont’d Source

Main Anthocyanin

Anthocyanin Content

References

Pomegranate

Cy-3-diglu Cy-3,5-diglu Pl-3-diglu Dp-3-diglu Dp-3,5-diglu Cy-3-pen Pe-3-glu

1.38 g/kg

Kulkarni and Aradhya (2005) Legua et al. (2016)

1.63 g/kg

Cy-3-gal Cy-3-ara Cy-3-glu Mv-3-glu Mv-3-cou-glu Pt-3-glu Pe-3-glu Cy-3-glu Cy-3-ma-glu

8 g/kg (FW)

Pojer et al. (2013) Cassidy (2018) Pojer et al. (2013) Pérez-Jiménez et al. (2010) Pérez-Jiménez et al. (2010) Ferrandino et al. (2017) Rosas et al. (2017)

3.2–4 g/kg (DW)

Fabroni et al. (2016)

Feruloyl-cy-xyl-glu-galac (43%–84%) Cy-xyl-glu-gal Dp-3,5-diglu Cy-3,5-diglu Cy-3-glu Pt-3-glu (56%) Dp-3-glu (34%) Mv-3-glu (10%) Cy-3-diglu-5-glu Pt-cou-rut-glu Pe-cou-rut-glu

0.045–17.4 g/kg (DW)

Kammerer et al. (2004) Gras et al. (2015)

300 mg/kg (FW)

Zhang et al. (2016)

32 g/kg (DW)

Mojica et al. (2017)

1.1–6.3 g/kg (DM) 286 mg/kg (FW)

Wiczkowski et al. (2014) Heinonen et al. (2016)

Cy-3-glu Pl-3-glu Pe-3-glu Cy-3-glu Pl-3-glu Pe-3-glu Cy-3-glu Pe-3-glu

4.9 g/kg (DW)

Li et al. (2017)

0.49 g/kg (DW)

Li et al. (2017)

Blueberry Chokeberry Black elderberry Red grape

Blood orange

7.9–13 g/kg 0.8–2 g/kg

VEGETABLES

Black carrot

Red onion

Black bean

Red cabbage Purple potato CEREALS

Purple corn

Blue corn

Purple rice

Das et al. (2016)

Chapter 12  Anthocyanins as Natural Pigments in Beverages   387

Table 12.2  Natural Sources of Anthocyanins—cont’d Source

Main Anthocyanin

Anthocyanin Content

References

Black rice

Cy-3-glu

3.3 g/kg

Das et al. (2016) Abdel-Aal et al. (2006)

Cy-3-gal Cy-3-glu

22–430 mg/kg (DW)

Bellomo and Fallico (2007)

144 mg/kg (DW)

Benvenuti et al. (2016)

NUTS

Pistachio EDIBLE FLOWERS

Red petunia

Anthocyanidins: Pl: pelargonidin; Cy: cyanidin; Pe: peonidin; Dp: delphinidin; Pt: petunidin; Mv: malvidin; (Acyl)glycosides: ara: arabinoside; gal: galactoside; glu: glucoside; diglu: diglucoside; ma-glu: malonyl-glucoside; gluru: glucorutinoside; pen: pentoside; rut: rutinoside; courut-glu: coumaryl-rutinoside-glucoside; soph: sophoroside; xyl: xyloside; xy-rut: xylosyl-rutinoside; cou-glu: coumaryl-glucoside; xyl-galac: xylosylgalactoside; xyl-glu-gal: xylosyl-glucosyl-galactoside; DW: dry weight; FW: fresh weight.

It is known that anthocyanins play relevant roles in plants: (i) they assist in plant propagation by attracting pollinators and seed dispersers, as they provide color to many fruits and vegetables (Santos-Buelga et  al., 2014). Anthocyanins accumulate in anthers (masculine organs) and pistils (feminine organs) of flowers, providing them attractive colors. (ii) Also, anthocyanins collaborate in plant defense mechanisms against biotic and environmental stress factors. Anthocyanins also contribute to the resistance of plants to insect attack. For example, cyanidin 3-glucoside, quercetin, and quercetin 3-glucoside, which are present in cotton leaves, were toxic to the larvae Heliothis virescens, causing a marked inhibition to their growth (Hedin et al., 1983). Plants synthesize anthocyanins under stress conditions, such as infections, wounding, ultraviolet irradiation, ozone, pollutants, etc. The antioxidant capacity of these compounds protects plants against oxidative damage that can be caused by, for example, high irradiation; moreover, anthocyanins are synthesized by the plant when it is infected by pathogens, due to their antibacterial capabilities (Gould et al., 2002; Olsen et al., 2009). Anthocyanin content in plants is highly influenced by environmental factors (rainfall, sun exposure, altitude, diurnal temperature difference, wind), edaphic factors (soil type, mineral content), and growth conditions (cultivation techniques, fertilizer, irrigation). Moreover, anthocyanin concentration increases during ripening. Storage and transport conditions as well as processing technology can modify anthocyanin levels in manufactured foods (Mushtaq and Wani, 2013).

388  Chapter 12  Anthocyanins as Natural Pigments in Beverages

Anthocyanins are produced by a secondary metabolic pathway in plants, which is a branch of the flavonoid pathway (i.e., shikimate/ phenylpropanoid pathway). They are synthesized as a result of the enzymatic reaction of three molecules of malonyl CoA obtained in the fatty acid metabolism and one of p-coumaroyl CoA derived from phenylalanine by chalcone synthase. After a cascade of enzymatic reactions catalyzed by four further enzymes, pelargonidin is synthesized (Zhang et al., 2014). However, most plants hydroxylate this molecule to cyanidin by flavonoid 3′ hydroxylase, or to delphinidin using flavonoid 3′4′ hydroxylase (Zhang et al., 2014). The acknowledgment of the enzymes involved in anthocyanidin synthesis and developments in metabolic engineering of genes coding those enzymes constitute the bases for the regulation of anthocyanin synthesis with the purpose of obtaining anthocyanin- rich varieties (Maligeppagol et  al., 2013; Santos-Buelga et al., 2014; Yan et al., 2005). Butelli et al. (2008) expressed two transcription factors from the snapdragon Antirrhinum majus in tomato, obtaining an accumulation of anthocyanins at concentrations comparable to the levels found in blackberries and blueberries. Expression of the two transgenes enhanced the antioxidant capacity of tomato fruit threefold and peel and flesh increased their coloration to an intense purple tone.

12.1.2  Biological Functions Nowadays, anthocyanins have a high nutritional interest due to their contribution to human health. There are lots of evidences in the literature of the health benefits provided by an anthocyanin-rich diet (Fang, 2015; Fernandes et al., 2017; Jamar et al., 2017; Kong et al., 2003; Lila et al., 2016; Pojer et al., 2013; Santos-Buelga et al., 2014). Early reports suggested that the health-promoting effects of anthocyanins were exclusively the result of their antioxidant properties. However, recent studies have assessed that anthocyanins also play roles on antiinflammatory, cell signaling, and gene expression pathways (Lila et al., 2016). Anthocyanins have strong antioxidant capacity, so that they could potentially prevent free-radical-related injury. Their chemical structure is adequate to donate hydrogens or electrons to free radicals, as well as to scavenge and move them through their aromatic structure. They are able to scavenge a wide range of reactive oxygen (ROO•, O2, O2•−, OH•), nitrogen (NO•) and chlorine species, and alkyl and peroxyl free radicals generating a stable phenoxyl radical. The antioxidant capacity derives from the presence of hydroxyl groups in positions 3′ and 4′ of the B ring, which provides stability to the formed radical. Moreover, free hydroxyl groups in positions 3 (ring C) and 5 (ring A) act as electron donors (Heim et  al., 2002). In  vitro studies have demonstrated that the antioxidant effect of anthocyanins depends on their chemical structure, and efficiency increases when increasing

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the number of hydroxyl groups in ring B. However, the antioxidant activity of cyanidin decreases with the presence of glycoside groups in position 3 of ring C (Seeram and Nair, 2002). Besides, the catechol moiety in the B ring, the oxonium ion in the C ring, as well as the hydroxylation and methylation pattern, acylation, and glycosylation, influence the antioxidant property (Pojer et al., 2013). Diverse studies have assessed that the oxidative activity of anthocyanins and aglycones is equivalent to that of classic antioxidants such as vitamins C and E; also, they appear to be better antioxidants than α-tocopherol (Fukumoto and Mazza, 2000). Moreover, Heinonen et al. (1998) demonstrated a linear correlation between the anthocyanin content and antioxidant effect in some fruits such as blackberries, red raspberries, black raspberries, and strawberries. In  vivo tests done with healthy volunteers have shown that the daily intake of anthocyanins from acai pulp and juice produces antioxidant activity in plasma and urine (Mertens-Talcott et  al., 2008). The volunteers had taken 7 mL/kg of the acai berry products in the morning after night fasting and plasma and urine samples were analyzed during 12 and 24 hours after consumption, revealing the antioxidant activity increase. Inflammation is the protective response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. The stimulation of inflammation is due to cyclooxygenase (COX) enzymes that convert arachidonic acid to prostaglandins. The expression of the COX-1 isozyme is common in most tissues; however, COX-2 is upregulated in inflamed cells, and this regulation is mediated by cytokines (BowenForbes et  al., 2010). Cyanidin-3-glucoside-rich extracts have been found to have an effect on the expression of genes that regulate immune, inflammatory, and apoptotic processes; this effect has been observed when cyanidin affects NF-κB, a protein complex that has an important role in the transcriptional activity occurring in the nucleus (PascualTeresa, 2014). This effect has also been related to a downregulation of COX-2 and nitric oxide synthase (iNOS) responsible for antiinflammatory and immune response to different inducers. Some studies have demonstrated the inhibitory effect of anthocyanins: Bowen-Forbes et al. (2010) observed that extracts of the Jamaican Rubus spp. resulted in moderate COX inhibitory activity (27.5%–33.1%) at concentrations of 100 μg/mL in hexane in in vitro assays. Intuyod et al. (2014) provided an anthocyanin-rich extract (cyanidin and delphinidin) to hamsters in an in vivo experiment and found that anthocyanins decreased the accumulation of fibrous tissue and reduced inflammatory levels with no effect on motor function. Delphinidin and cyanidin have been shown to inhibit COX-2 expression, while pelargonidin, peonidin, and malvidin did not (Pojer et al., 2013). The antioxidant capacity of anthocyanins is related to their preventive function in different diseases, which are produced by the presence

390  Chapter 12  Anthocyanins as Natural Pigments in Beverages

of reactive oxidative species causing cell damage at various sites such as membranes, cytoplasm, and nucleus. Considering their antiinflammatory activity, anthocyanins could participate in the treatment of illnesses involving tissue inflammation. Benefits were observed in the prevention or treatment of noncommunicable chronic diseases such as cardiovascular, neurological, and cognitive alterations, cancer, obesity, or diabetes, as well as other pathologic processes such as aging or vision alterations. A number of studies have demonstrated that consumption of polyphenols reduces the incidence of coronary heart diseases, which are due to platelet aggregation, hypertension, high-plasma low-density lipoprotein (LDL) cholesterol, and vascular endothelium dysfunction. The cardioprotective effect of anthocyanins may be associated with the increase of serum antioxidant capacity, which protects against LDL oxidation, and antiinflammatory and antiplatelet activities (Erlund et al., 2008; Thompson et al., 2017). Some authors evaluated the effect of the antioxidant activity of berries on the oxidation of LDL, obtaining that chokeberry and grape extract decreased total cholesterol, LDL and triglyceride levels, whereas it increased high-density lipoprotein (HDL) cholesterol (Kong et  al., 2003; Valcheva-Kuzmanova et  al., 2007), thus preventing atherosclerosis. Hassellund et al. (2013) observed an increase in the HDL cholesterol and sugar in plasma of prehypertensive men after anthocyanins intake as well as an increase in polyphenols in plasma, although no other beneficial effect in the short term was observed. Thompson et al. (2017) evaluated the effect of anthocyanin supplementation (320 mg/ day) in 26 pro-thrombotic overweight and obese individuals, observing the reduction of platelet aggregate formation by 29%. The antimutagenic and anticarcinogenic properties of anthocyanins were revealed in a high number of in vitro and in vivo assays. Prevention of cancer development is essential, and identifying the compounds that can inhibit tumor cell propagation can be a crucial process. With that purpose, the production of hydroperoxide or increment in the DNA synthesis should be controlled. The antioxidant capacity of anthocyanins was demonstrated to diminish hydroperoxide levels in in  vivo assays when rats were maintained on a vitamin E-deficient diet for 12 weeks in order to enhance susceptibility to oxidative damage, and then repleted with anthocyanidin extracts. Consumption of anthocyanins improved plasma antioxidant capacity and decreased hydroperoxide and 8-oxo-deoxyguanosine concentrations in liver, which are indicators of lipid peroxidation and DNA damage, respectively (Ramírez-Tortosa et al., 2001). Different studies have assessed that some berry extract and isolated anthocyanins help to prevent neurological and cognitive alterations. It has been hypothesized that anthocyanins may exert protective effects on cognition, including memory and executive processing, either

Chapter 12  Anthocyanins as Natural Pigments in Beverages   391

through a direct effect on brain function or indirectly by reducing blood pressure (Kent et al., 2017). Anthocyanins cross the hematoencephalic barrier and join the DNA molecule in the hippocampus and cerebral cortex, thus stabilizing it against oxidative damage (Passamonti et al., 2005). Shukitt-Hale et al. (2005) showed that cognitive behavior and neuronal functions can be improved when supplementing rats diet with strawberries and blueberries. Kent et  al. (2017) demonstrated that daily consumption of an anthocyanin-rich cherry juice improved verbal fluency, as well as short-term and long-term memory of 49 older adults (+70 year) with mild-to-moderate dementia. It has been suggested that anthocyanins have the capacity to improve vision, following several ways: (i) improving night vision by increasing retinal pigments; (ii) improving circulation within retina capillaries; (iii) decreasing degeneration and diabetic retinopathy; (iv) preventing glaucoma and other vision diseases (Pojer et al., 2013). Ohgami et  al. (2005) supplied anthocyanin-rich extracts to rats with ocular deficiency, observing a reduction of inflammation and visual acuity increase. Recently, Nakamura et  al. (2014) evaluated the effect of delphinidin 3,5-O-diglucoside, contained in the maqui berry, for the prevention of dry eye disease. The anthocyanin was found to suppress reactive oxygen species formation from lacrimal gland tissue and preserved tear secretion. Obesity is an inflammatory disease associated with the imbalance of energy inlet/outlet and is characterized by the excessive accumulation of adipose tissue; hypertrophy of the adipose tissue leads to metabolic dysfunctions through the production of adipocytokines. Type-2 diabetes is associated with insulin deficiency, which causes high glucose levels in blood. Anthocyanins interact with adiponectin, which is one of the most important adipocytokines, attenuating adipocyte dysfunction (Jamar et al., 2017; Gowd et al., 2017). Tsuda and coworkers (2004, 2006) experimented with adipocytes isolated from adipose tissue of rats and humans, observing that anthocyanins enhance adipocytokine (adiponectin and leptin) secretion, although the mechanism should be elucidated. On the other hand, a high number of polyphenols, including cyanidin, were demonstrated to inhibit the synthesis of α-glucosidase, which is one of the key enzymes responsible for the digestion of dietary carbohydrates into glucose. Inhibition decreases glucose concentration in blood and, therefore, the risk of diabetes (Tadera et al., 2006). The antioxidant capacity of anthocyanins is responsible for their antiaging properties, as the most accepted theory for aging is the damage of DNA, proteins, lipids, and other cellular constituents by free radical/oxidative stress (Soto et al., 2015). Several studies have demonstrated the effect of anthocyanin consumption in preventing skin and brain aging (Rojo et al., 2013; Soto et al., 2015; Wei et al., 2017).

392  Chapter 12  Anthocyanins as Natural Pigments in Beverages

12.1.3 Bioavailability Studies concerning anthocyanin human benefits and anthocyanin bioavailability constituted a paradox. It is generally known that compounds must be bioavailable in order to interact with the human metabolism and reach tissues and organs. However, lots of works pointed out a very poor anthocyanin bioavailability, reporting urinary excretions below 1% of the ingested amounts (Frank et al., 2003). Recent studies have demonstrated that bioavailability was greatly underestimated due to different reasons: (i) anthocyanins are supposed to suffer some modifications in the gastrointestinal tract and also in some tissues, and derived metabolites could maintain the same health benefits better than the native compounds, most of them being unknown; (ii) very low concentrations of anthocyanins and their metabolites are obtained in blood and urine, below the standard analytical techniques sensitivity (Lila et al., 2016). In recent years, a lot of studies were done in order to elucidate the real bioavailability of anthocyanins and their derivatives, which implies developing metabolic mechanisms and improving analytical technologies. After ingestion, compounds pass through the oral cavity and gastrointestinal tract, where they can be directly absorbed or metabolized. In this pathway, anthocyanins interact with other compounds, proteins, and enzymes, as β-glucosidase which catalyzed deglycosylation yielding anthocyanin aglycone. Also, anthocyanins can suffer glucuronidation, methylation, or sulfation, especially in the small intestine, thus decreasing the amount of native form that can be directly absorbed (Lila et al., 2016). Anthocyanins have been found in low concentration in plasma serum by Murkovic et al. (2000) after ingestion, suggesting that anthocyanin glycosides may diffuse into the bloodstream for potential bioactivity. On the other hand, Czank et al. (2013) have recently studied the bioavailability of anthocyanins in humans using 13C-labeled compounds, obtaining a bioavailability of 12.38%, where 0.18% was recovered from blood, 5.37% from urine, and 6.91% from breath. Another 32.13% was found in feces. Bioavailability depends on the anthocyanin chemical structure, as it determines chemical stability against different pH values, absorption rate, or reactivity against intestinal microorganisms (Lila et al., 2016). The effect of food matrix on anthocyanin bioavailability has not yet been elucidated. Serafini et al. (2009) analyzed the antioxidant properties of blueberry consumption combined with milk and suggested that the combined ingestion caused a decrease of bioavailability and, thus, a reduction of beneficial antioxidant effects in plasma. Other authors observed a relative bioavailability of red wine anthocyanins within 57.1%–76.3% compared to red grape juice anthocyanins, suggesting that the presence of ethanol could decrease the availability of these compounds in healthy volunteers (Frank et  al., 2003). Several

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technologies are being developed in order to improve anthocyanin stability and/or bioavailability. One of them is the encapsulation of compounds, which involves mechanisms for developing compound microparticles, and also in vitro and in vivo stability studies in order to determine the release of anthocyanins from capsules in the human body (Robert and Fredes, 2015).

12.1.4 Relevant Applications of Anthocyanins Anthocyanins are natural compounds that have been extensively studied due to their demonstrated health-beneficial effects as well as variable color. They are increasing in popularity not only for the food industry but also for other sectors such as the cosmetic industry. Anthocyanins are considered in many studies as food pigments because of their variable color while they also have positive effects on human health (Cassidy, 2018). Anthocyanins demonstrate a high potential to be used as natural colorants due to their attractive orange, red, and purple colors and water solubility that allows their incorporation into aqueous food systems (Brouillard, 1982; Mazza and Miniati, 1993). They may serve as an alternative to the use of synthetic colorants, increasing beneficial polyphenol consumption (Wallace and Giusti, 2015). Rapid food technology advances permitted their extraction, identification and, addition to food and beverages as natural colorants, functional ingredients, or food supplements. Anthocyanins are part of the flavonoids, and these account for 39% of the antioxidant compounds consumed in a diet (Yang et al., 2011). Total daily-consumed antioxidants in the United States are estimated at 503 mg, 75% of which comes from fresh food and 25% from supplements. The consumption of anthocyanins in diet is quite variable, and estimations vary within a wide range, from 3 to 150 mg/day depending on the country and nutrition habits. In the United States, the average daily intake has been estimated at 12.5 mg/day (Wu et  al., 2006); in Europe, the estimation was also in the scale of 10 mg/day (Andersen and Jordheim, 2013), although Finnish adults consume 47 mg/day (due to high berry intake). Consumption in the Mediterranean countries was also observed to be higher due to higher intake of red wine, corresponding to 46% of the total anthocyanidin intake (Drossard et  al., 2011). Daily consumption of one to two portions of strawberries, blueberries, or raspberries could have significant effects on CVD (Cassidy et al., 2013). Important sources in the diet of US adults aged more than 20 include berries (20%), wine (16%), grapes (11%), red/ purple vegetables (8%), 100% noncitrus juice (6%), yogurt (6%), and other food sources (33%) (Wallace and Giusti, 2015). Anthocyanins are not essential nutrients, and no deficiency disorder has been associated with the lack of anthocyanin consumption.

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Toxicological effects were also not observed (Wallace and Giusti, 2015). However, dietary reference intakes do not currently exist for anthocyanins in the United States, Canada, or the European Union. China defined a proposed anthocyanin level of 50 mg/day (Chinese Nutrition Society, 2013). Other countries just recommend the consumption of colorful fruits and vegetables. Cosmetic consumers are demanding the introduction of natural ingredients in skin care products. Polyphenolic compounds are being considered due to their antioxidant, antimicrobial, and antiinflammatory actions that provide antiaging effect and ultraviolet radiation protection. Grape and wine making by-products, with up to 70% of polyphenols of the grape, are gaining interest as a source of compounds for new cosmetic formulations (Barbulova et  al., 2015; Soto et al., 2015).

12.2  Anthocyanin Properties Anthocyanins are molecules that can evolve depending on chemical conditions to other derivatives with different chemical structure (Fig. 12.1) and properties in terms of color. Anthocyanins behave as pH colorimetric indicators because both their color intensity and the maximum wavelength of adsorption depend on proton concentration. At low pH, the main specie is the flavylium cation with a positive charge and heteroaromaticity in the central ring (pyrano ring). Under those conditions, the maximum wavelength of absorption is 518–528 nm (green light); depending on the substitution pattern in the B ring, R1 and R2 can be H, OH, or OCH3 groups. Pyrylium cation solutions show an intense red color. When pH increases at neutral or basic values, the pyrylium form evolves to a quinoidal base absorbing at higher wavelengths and expressing a blue-violet color. Flavylium cation is sensitive to nucleophilic addition. Hydration reactions occur mainly in carbon 2 (Fig.  12.1). This reaction breaks the aromaticity of the pyrylium ring and it also produces the loss of adsorption properties, transforming the structure in an uncolored carbinol-pseudobase (Fig. 12.2). Spontaneously, this structure evolves to an open chalcone by tautomeric ring-chain equilibrium (Fig. 12.1). The chalcone shows a light yellow color. Flavylium cation is also sensitive to the nucleophilic addition of bisulfite anion. The reaction is produced in C4 position of the pyrylium ring that is the most electrophilic position. Sulfur dioxide is commonly used in the food industry as an antioxidant and is bacteriostatic. When SO2 gas reacts with water, it produces sulfurous acid, which once dissociated releases bisulfite anion (Fig.  12.3). Bisulfite behaves as a strong nucleophilic reagent able to bond in position 4, so it breaks the

Chapter 12  Anthocyanins as Natural Pigments in Beverages   395

R1 OH O

HO

OH

H

R2 O-Gl

Polymeric pigments SO2 bleaching resistant

SO3H

[CH3CHO] / pH<1

R1

[SO2] 2'

HO

8

7

O

5

4

OH 4'

B

+

5'

2

A

6

Hydratation –H+

1

3'

3

R2

HO O

OH

Flavylium cation pH<1

O

OH OH

pH<1

OH

R1 OH O

HO

R1

–H +

OH

OH

R2

O

O

O-Gl

Uncolored Carbinol-pseudo-base

OH

OH

Tautomeric ring-chain equilibrium

R2

O-Gl Blue-violet Quinoidal-base

R1 OH

OH O

HO

R2 O-Gl OH

Light yellow

Chalcone

Fig. 12.1  Chemical processes affecting anthocyanins in solution or food products depending on pH, hydration, sulfites, or polymerization reactions with other flavonoids.

aromaticity and the pyrylium ring loses the possibility to absorb light and is transformed into a noncolored molecule. This reaction is reversible and common when we add SO2 to foods with anthocyanins; at the beginning, there is a loss of color but it is slowly recovered to later reach the initial color. When the media is strongly acidic (pH 1–2), all reactions described in Fig. 12.1 are displaced to the flavylium cation so the color intensity increases strongly (Fig.  12.4). The hyperchromic effect in a solution of 100 mg/L of malvidin-3-O-glucoside in slightly acidic water (test tube number 1) makes that, after the addition of concentrate HCl in test tube number 2, the absorbance increases more than five times

396  Chapter 12  Anthocyanins as Natural Pigments in Beverages

H

H

O

H2O OCH3

OCH3

OH

OH O

HO

O

HO

+

C

HO O OH

OCH3 H OH

H O

H H

OH HO O

OH CH 2OH

OH

OCH3 H OH

H O

H H

OH CH 2OH

Uncolored molecule

OCH3 OH +

O

HO

HO O OH

OCH3 H OH

H O

H

OH CH 2OH

H Malvidin Malvidin-3-O-glucoside

Fig. 12.2  Discoloration by water nucleophilic attack at position C2.

OCH 3

OCH 3

OH

OH +

O

HO

HO O

H

OH

O H H

O

HO

OCH 3 H OH

HO O

+

C

OH CH 2OH

OH

H

OCH 3 H OH

OH CH 2OH

O H H

Malvidin Malvidin-3-O-glucoside

OCH 3 OH O

HO

OH

H O H2SO3

S O

SO2

+

O

H O S

+

+

H

O

OH2

Fig. 12.3  Scheme of the malvidin-3-glucoside bleaching mechanism by SO2.

HO O OH

H

H

OCH 3 H OH

O

OSO2H

Uncolored molecule

H H

OH CH 2OH

Chapter 12  Anthocyanins as Natural Pigments in Beverages   397

(Fig. 12.4B). Also observable is the bleaching effect of SO2 in test tube number 3 and how the spectra do not show absorbance at 520 nm because of the formation of the uncolored adduct between bisulfite and the anthocyanin (Fig. 12.4B). When CaCO3 is added in test tube 4, the increase in pH produces a light violet-blue color (test tube 4) because of the evolution from the flavylium cation structure to the quinoidal base (Fig. 12.4A). Color intensity is again pH dependent. Lastly, in aqueous solutions, the observed red color typically corresponds to the flavylium cation, which is just around 10% of the total anthocyanin content. The other structures support the pool of reactions that anthocyanins have in aqueous media (Fig. 12.1).

12.3  Polymerization of Anthocyanins Timberlake and Bridle (1976) have described the rapid condensation of anthocyanins with flavanols by adding acetaldehyde to the fermenting model media, while Singleton and Trouslade (1992)

Fig. 12.4  Effect of pH and sulfur dioxide in color (A) and visible absorption spectra (B) of anthocyanins.

398  Chapter 12  Anthocyanins as Natural Pigments in Beverages

observed that the polymeric pigments present in young wine differ from those present in old aged red wines. The difference in polyphenolic composition in wines may not just be due to the formation of polymeric pigments over time, but may also be due to the oxidation and hydrolysis processes that happen in wines during aging (Arnous et al., 2001). Nonetheless, regarding the polymerization occurring during fermentation and wine aging, there might be different paths in which anthocyanins can form oligomeric structures with more stable properties than its monomeric precursor anthocyanins. The formation of oligomers and polymers, as well as pyranoanthocyanins, in red wine takes place when yeast metabolites such as acetaldehyde (Escott et al., 2016; Morata et al., 2007), pyruvic acid (Morata et  al., 2007), and vinylphenols from hydroxycinnamate decarboxylase (HCDC) activity (Morata et  al., 2012) interact with anthocyanins and proanthocyanidins extracted from skins and seeds during maceration of red grapes. The polymerization process could also take place when other aldehydes besides acetaldehyde are in solution; in this way, it has been observed that furfural is able to replace acetaldehyde in the polymerization process when the wine is aging inside oak barrels (Es Safi et al., 2000). The formation of polymeric pigments and the color evolution of red wines can be assessed using a method developed by Somers and Evans (1997); it is a spectrophotometric method for rapid assessment of anthocyanin content, ionized anthocyanins, total phenolic content, and polymeric fraction of red wines. This method, together with thiolysis depolymerization, may allow researchers to characterize monomeric and polymeric fractions in red wines as well as to evaluate the formation of larger pigmented molecular structures, although this methodology may not be reproducible or specific. More advanced technologies such as high-performance liquid chromatography (HPLC) (Ky et  al., 2014), direct infusion electrospray ionization tandem mass spectrometry (ESI Q-TOF MS/MS) (Zhang and Zhu, 2015), nuclear magnetic resonance (NMR) and mass spectrometry (MS) for dimer and trimer identification (Mateus et al., 2002), or ultrahigh-performanceliquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPLC/Q-TOF) (De Rosso et al., 2015), among others, allow the identification and quantification of monomeric pigments as well as oligomer/polymer fractions, and they can even describe the molecular structure of new pigments for the very first time. Some of the oligomeric structures, such as ethyl-linked anthocyaninflavanol, anthocyanin-flavanol, and ethyl-linked pyranoanthocyaninsflavanol interactions are described hereafter.

Chapter 12  Anthocyanins as Natural Pigments in Beverages   399

12.3.1 Ethyl-Linked Anthocyanin-Flavanol The formation of anthocyanin-flavanol ethyl-linked oligomers happens through a condensation reaction between an anthocyanin and a molecule of either (+)-catechin or (−)-epicatechin to form the simplest dimeric form. Es-Safi et al. (1999a) observed that the flavanolethyl-flavanol dimerization happens faster with (−)-epicatechin regardless of pH values (from 2.2 to 4.0). The competitive reaction also happens between (−)-epicatechin and the anthocyanins producing the formation of both flavanol-ethyl-flavanol and flavanol-ethylanthocyanin dimers (Es-Safi et al., 1999b). Flavanol-ethyl-flavanol dimers are less stable than flavanolethyl-anthocyanin dimers and this last molecule can be even formed when the anthocyanin is in its hemiketal form without having the positive charge from the flavylium ion (Es-Safi and Cheynier, 2004). Regarding the degree of polymerization achieved, Es-Safi et al. (1999b) have found that a polymer moiety stops the polymerization process when two anthocyanin molecules are located at both ends of the oligomer. Experimental fermentations of red grape musts (cv. Tempranillo), where a strain of Schizosaccharomyces pombe was used, showed the interaction between malvidin-3-glucoside with (+)-catechin (Fig. 12.5B) and with the dimer procyanidin B2 (Fig. 12.5C) to produce this type of ethyl-linked oligomers; both experiments are compared to a control trial (Fig. 12.5A). The high-performance liquid chromatography, used to separate red wine pigments, had to be adjusted to avoid the overlapping of peaks for better identification and quantification. The peaks formed during experimental fermentation were identified as follows: malvidin-3-glucoside-ethyl-catechin enantiomers with molecular ion [M]+ (m/z) 809 and fragment ions (m/z) 657, 331 when adding (+)-catechin; and malvidin-3-glucoside-ethyl-procyanidinB2 with molecular ion [M]+ (m/z) 1097 and fragment ion (m/z) 519 when procyanidin B2 was added. In further studies, Escott et al. (2018) showed that the species Lachancea thermotolerans lead to producing even higher concentrations of oligomers and polymeric pigments, especially when used in sequential fermentation with the species S. pombe. Besides the simplest anthocyanin-flavanol-ethyl-linked pigments already mentioned, larger oligomers have been documented by Asenstorfer et al. (2001). These authors worked on a methodology to isolate oligomers from a 4-year-old Shiraz red wine and from grape marc; they were able to identify malvin-3-glucoside, nonacylated, acetylated, or p-coumaroylated derivatives as well as oligomeric pigments. The oligomers described by Asenstorfer et al. (2001) comprised one of the above-mentioned anthocyanins with up to two units of catechin in red wine, while oligomers found in grape marc extracts had up to four units of catechin linked by vinyl bonds.

mAU

Control

250 200 150 100 50 0

(A)

0

mAU

2.5

5

7.5

10

12.5

15

17.5 min

OH

(+)-Catechin

OMe

OH

O

250

OH

HO

O+

HO

OH

OMe

200 OGlu OH

150

2

1 100 50 0

(B)

0

2.5

5

7.5

10

12.5

mAU

15

17.5 min

15

17.5 min

HO

Procyanidin B2

OH O HO

250 HO

200

CH3 OH

OH O HO

OMe OH

CH3

O+

OMe OGlu

150

HO

OH

3

OH

4

100 50 0

(C)

0

2.5

5

7.5

10

12.5

Retention time

Fig. 12.5  High-performance liquid chromatography (HPLC) chromatogram of three fermentation trials: (A) Control, (B) (+)-catechin, and (C) procyanidin B2, to show the appearance of oligomeric pigment peaks. The observed peaks were identified as: (1) and (2) malvidin-3glucoside-ethyl-catechin enantiomers; (3) and (4) malvidin-3-glucoside-ethyl-procyanidin B2.

Chapter 12  Anthocyanins as Natural Pigments in Beverages   401

12.3.2 Anthocyanin-Flavanol The reaction to produce anthocyanin-flavanol dimers undergoes a slower mechanism than the one previously described for ethyl-linked units. Eglinton et al. (2004) demonstrated that these oligomers could be formed before and after fermentation at a slower rate than in the presence of fermenting yeasts, as well as in a lower production yield associated with the lack of yeast metabolites. In the presence of acetaldehyde and constant SO2 levels, the condensation reaction for the production of ethyl-linked polymeric pigments is enhanced. Nonetheless, in the absence of yeast metabolites, the simplest form of interaction between anthocyanins and flavanols occurs with catechin or epicatechin units. Remy et al. (2000) documented the formation of anthocyanintannin and tannin-anthocyanin interactions. The dimer formed between a malvidin-3-glucoside molecule and (+)-catechin with molecular ion (m/z) 781 in negative ion mode (mass spectrometry) corresponds to either the copigmentation or the direct linkage of them. In model solutions, the condensation of dimer procyanidin B2 with anthocyanins could also take place in the absence of acetaldehyde (Dallas et al., 1996); nonetheless, the reaction is faster when acetaldehyde is present in solution. Dallas et al. (1996) also showed that malvidin-3-glucoside reacted slower than other anthocyanins such as cyanidin-3-glucoside and petunidin-3-O-glucoside in the oligomers formation mechanism. Malvidin-3-glucoside is the larger anthocyanin found in red grapes, and therefore red grape varieties with larger amounts of these other anthocyanins may also yield a higher concentration of oligomeric or polymeric pigments. Although the interaction between anthocyanin and flavanol molecules may be through the nucleophilic attack of the positions C6 and C8 in the ring of the anthocyanin moiety in the hydrated hemiketal form, followed by a dehydration step to form the flavylium ion, there may be other types of nucleophilic interaction involving these molecules and yielding colorless molecules known as A-type dimers (Salas et  al., 2003). The A-type oligomers comprise both interflavonoid bonds, 4β-8 (known as B-type and the most common one) and a second bond 2β-7. The copigmentation path followed for the production of flavanolanthocyanin dimers is through the formation of the carbocation in the flavan-3-ol molecule yielding an electrophilic moiety that reacts with the anthocyanin acting as a nucleophile (Salas et al., 2004). This dimer is colorless, as well as the dimer anthocyanin-flavanol freshly formed, but a dehydration step after the condensation produces the flavylium ion with its characteristic red color at acidic conditions. Dallas et  al. (1996) also demonstrated that the degradation rate of ethyl-linked anthocyanin flavanol pigments is higher than the one

402  Chapter 12  Anthocyanins as Natural Pigments in Beverages

observed in condensed anthocyanin-flavanol dimers; thus, the prevalence of such molecules might be expected to be greater if they happen to be formed in wines.

12.3.3 Ethyl-Linked VinylpyranoanthocyaninFlavanol (Portisins) Portisins are pigments with a maximum absorption wavelength larger than the one of anthocyanins; it is around 570 nm (He et  al., 2012), which may be the reason why Mateus et al. (2003) described these molecules as blue-purple anthocyanin-derived pigments. According to these researchers, the position C10 of a pyranoanthocyanin moiety reacts with the vinyl group of 8-vinyl-flavanol adducts. The reaction occurring between one pyranoanthocyanin moiety and a flavan-3-ol is, as in many other pigment formation reactions, mediated by an acetaldehyde molecule (Mateus et al., 2004a). The molecules produced from the reaction of vitisin type A pyranoanthocyanin with vinylflavanols are the so-called Portisins A, whereas portisins formed through the nucleophilic attack of hydroxycinnamic acids (Oliveira et  al., 2007) or viniylphenols (Mateus et  al., 2006) in the same C10 position of the vitisin second pyran ring derive in pigments named Portisins B. The structure of these molecules can be seen in Fig. 12.6. These molecules were first found in Port wine, and therefore they got their name from it. These pigments are usually found in aged wines since anthocyanin-pyruvic acid adducts, precursors of portisins, are more abundant in wine as the anthocyanins content decreases over time.

12.3.4 Stability of Polymeric Pigments Polymeric pigments are not more stable than their anthocyanin precursors due to their greater molecular weight. Pigments are more stable to color change by variations in pH values if position 4 (C4) from the pyran cycle has been protected during formation of ethyl-linked anthocyanin oligomeric derivatives or through the formation of pyranoanthocyanins (Cheynier et  al., 2006). Nonprotected position C4 may go under nucleophilic attack by SO2 (Fig. 12.1) producing colorless molecules, or anthocyanins’ bleaching in red wines. Larger molecular weight polymeric pigments may not mean greater stability as it might be related to tannin astringency. Cheynier (2005) mentioned that this organoleptic parameter increases when having larger molecules in solution; therefore, polymeric tannins will increase their astringency power in function of their increasing molecular weight. Astringency reduction of polymeric tannins might be linked to the cleavage tannin molecules to

R1

Anthocyanin

R2 O

R6

+

R3

R5

O

R6

O H3C

or

H

H3C

R3 OR4

O

COOH

Pyruvic acid

OH OH

+

O

Flavanol

H

R1

Acetaldehyde

R2

OH

OR4

R2 O

+

O

R3

OH

OH

R5

O

R6

OH

+

Flavanol

R2'

R6

(B)

O

R2

+ R3 OR4

R5

+ H3C

O

OR4'

R1' O

H

Acetaldehyde

+

R5'

H3C OH

HO

OH

O

"

R1

O

R6

R2

CH3

R2

R3 OR4

+

R3 OR4

+

O

R6

R1 CH3

CH3

OH

HO

R3'

Anthocyanin H

Acetaldehyde

OH

(C)

H3C

OH OH

R1

O

O

(E)

R5

HO

OH

R3

O OH

HO

HO

+

OR4

OH

OH

OH

R2

HO

HO

OH

R1

OH

O

OR4

O HO

R3

OH

OH

O

(A)

O

R6 R1

HO

+

(D)

OH

R5

OH

R5

Fig. 12.6  Different types of oligomeric (polymeric) pigments found in aged red wines: (A) Dimer anthocyanin-flavanol, (B) dimer anthocyanin-ethyl-flavanol, (C) trimer anthocyanin-flavanol-flavanol, (D) trimer anthocyanin-flavanol-anthocyanin, and (E) dimer pyranoanthocyanin-ethyl-flavanol (portisin A type).

Chapter 12  Anthocyanins as Natural Pigments in Beverages   403

H3C

H

Acetaldehyde

O

+

R6

O H3C

OH OH

R2

+

O

Acetaldehyde

HO

R1

Pyranoanthocyanin

OR4

404  Chapter 12  Anthocyanins as Natural Pigments in Beverages

anthocyanin units and other processes such as interactions with polysaccharides and proteins from wine. Fruits such as blackberry differ in anthocyanin composition from red grapes; therefore, the stability of the polymeric pigments produced from one another may differ as well. In the case of blackberry, the main anthocyanins found are cyanidin-3-glucoside and cyanidin3-O-rutinoside (Rommel et  al., 1992) and, as already mentioned by Dallas et al. (1996), the reactivity of these pigments may be higher than the one observed in malvidin-3-glucoside. The stability of polymeric pigments from these anthocyanins against degradation factors may be expected to be different as well.

12.4 Copigmentation The first evidence of copigmentation was observed when the color of flowers was studied and some inconsistencies were detected, for example, that red roses (Rosa L.) and cornflower (Centaurea cyanus) contain the same type of anthocyanins, mainly cyanidin derivatives (Willstätter and Everest, 1913); however, they show a blue color. This blue pigment was called protocyanidin. The blue color was formerly explained by the pH effect. The molecular structure of protocyanin was elucidated later and this has demonstrated that the blue color is produced by a tetra-metal (Fe3+, Mg2+, 2Ca2+) complex pigment containing anthocyanins, metals, and flavone glycosides (Takeda, 2006). The color of anthocyanins can be changed when they are associated with some organic molecules such as flavonoids or alkaloids and metals. Copigmentation is the modification in UV-visible adsorption of anthocyanins when they are located close to other molecules, called copigments. Copigmentation does not produce chemical bonds; it is only chemical proximity between the anthocyanin and copigment. There are several ways of copigmentation depending on the nature of the copigment. When the copigment molecule is an organic molecule (e.g., alkaloid or flavonoid), it is called intermolecular copigmentation (Fig.  12.7A). The uncolored molecule interacts with the chromogenic pyrylium ring affecting the electron density and distribution, which produces modifications in its absorption spectra. A special kind of intermolecular copigmentation is self-association (Fig. 12.7B); in this case, the copigment is the same anthocyanin that is being copigmented. This process normally occurs at a high concentration of anthocyanins and can be measured by a nonlinear behavior in color density; in spite of being at high concentrations, the color is more intense than can be expected. The last possibility is called metal complexation (Fig.  12.7C). The copigment is a metallic cation that also modifies the absorption spectra of the pyrylium ring by affecting

Chapter 12  Anthocyanins as Natural Pigments in Beverages   405

Fig. 12.7  Types of copigmentation. (A) Intermolecular copigmentation, (B) self-association, and (C) metal complexation.

the distribution of the unlocalized electrons. Intense effects can be reached with high positively charged alkaline earth metals and poor metals (+2-+3). There is other specific copigmentation that can occur in some special anthocyanins with a long and branched structure in which the nonanthocyanin moiety can work as a copigment. This mechanism is called intramolecular copigmentation. The copigment fragment is usually a hydroxycinnamic acid. So, in fact, it is an internal copigmentation in which the same molecule is pigment and copigment in a model known as “sandwich stacking.” Copigmentation is a very remarkable property in anthocyanins because it strongly affects color intensity and tonality. It is typically a bathochromic effect with a shift in the visible maximum of absorption at higher wavelengths (red bluish colors) and a hyperchromic effect increasing color intensity. Bathochromic and hyperchromic effects can be produced by either organic or metallic copigments (Fig. 12.8). The more intense and bluish color of the solution (Fig. 12.8A) is due to the nature and concentration of the copigment agent and it is observed in the visible spectra (Fig. 12.8B).

406  Chapter 12  Anthocyanins as Natural Pigments in Beverages

Fig. 12.8  Metallic (Al+3) and organic (caffeine) intermolecular copigmentation. Anthocyanin concentration 100 mg/L. Effect of copigmentation in color (A) and visible absorption spectra (B) of anthocyanins.

In the food industry, copigmentation could be very helpful because most of the anthocyanin colors range from red-orange to red bluish but copigmentation lets increase the chromatic range to blue and violet colors. These colors are currently in very great demand in the food industry. Development of stable copigments that could be compatible with foods but also stable in food systems can help to get better colors and different tonalities.

12.5 Pyranoanthocyanins Pyranoanthocyanins are natural pigments whose characteristic, chemical wisdom is to have a second heterocycle in its anthocyanin molecular structure (Vivar-Quintana et al., 1999); this second heterocycle is formed through the covalent bonding of fermentative metabolites, such as acetaldehyde, pyruvate, acetoin, acetone, etc., between the position C4 and the hydroxyl group located at position C5 (Morata et  al., 2003). The formation of pyranoanthocyanins in wines is enhanced by the presence of these fermentative metabolites, especially pyruvic acid, acetaldehyde, and vinylphenols from HCDC activity. Pyranoanthocyanins are more stable than normal anthocyanins because they have a double pyrylium; therefore, the number

Chapter 12  Anthocyanins as Natural Pigments in Beverages   407

of resonant forms is double, giving more resistance to antioxidant degradation. The formation of pyranoanthocyanins may also be strongly related to the fermentative yeasts used during wine production; different Saccharomyces cerevisiae strains (Morata et  al., 2006) and non- Saccharomyces yeasts such as S. pombe (Morata et al., 2012) have been shown to have positive HCDC enzymatic activity; therefore, the production of vinyl pyranoanthocyanins is favored. There is more evidence of yeasts promoting the formation of stable pigments such as pyranoanthocyanins and polymeric adducts as mentioned by Morata et al. (2016). For these authors, the proper selection of the fermentative yeast strain is important for the red wine color stabilization, and therefore for the production of more stable colored molecules. In wines produced with fruits, such as salal and aronia, the color stabilization apparently comes from the diglycoside pentose nature of the anthocyanins present in salal fruit (McDougall et  al., 2016), different from the monoglycoside structure of most Vitis vinifera anthocyanins. Pyranoanthocyanins have also been found in sources other than wine and grape pomace; these sources include fruits, vegetables, and flowers. Pyranoanthocyanins, different from the pyranoanthocyanins formed during wine production and wine aging, were found in acidified extracts of red onions (Fossen and Andersen, 2003), in the acidified extracts of strawberries (Andersen et al., 2004), and in rose petal (Rosa hybrid) extracts (Fukui et al., 2002). Aglycone 5-carboxypyranocyanidin and aglycone 5-carboxypyranopetunidin were observed in the red onion extracts; aglycone 5-carboxypyranopelargonidin was found in strawberry extracts and rosacyanin B (condensate from cyanidin and gallic acid reaction) was identified in rose petals, respectively. Unfortunately, the amount of some pyranoanthocyanins naturally forming during wine fermentation and red wine aging does not make possible the isolation in large amounts for further structural characterization and carrying on stability studies (Cruz et al., 2008). It seems that the chemical synthesis of stable pyranoanthocyanins from the reaction of anthocyanins with fermentative metabolites, vinylflavanols, vinylphenolic compounds, hydroxycinnamic acids, and further precursors may play an important role in the production of stable pigments for food colorants manufacturing. Some of the most common pyranoanthocyanin pigments found in red wine mainly will be described in this section in function of their molecular structure.

12.5.1 Vitisins Vitisin-type pigments, a form of pyranoanthocyanins, are produced during grape must fermentation or during wine aging (Amić et al., 2000) from the interaction between anthocyanins and fermentative metabolites acetaldehyde and pyruvic acid. Addition of these

408  Chapter 12  Anthocyanins as Natural Pigments in Beverages

fermentative metabolites to experimental red wines lead to the formation of a higher concentration of both vitisin A and vitisin B (Morata et  al., 2006). The most common vitisin-type pigments, vitisin A and vitisin B, are described hereafter.

12.5.1.1  Vitisin A Vitisin A is formed by the condensation of the most abundant anthocyanin in V. vinifera grapes (malvidin-3-glucoside) with pyruvic acid (Fig. 12.9). The location of a carboxyl group on the second pyran ring is the reason why they are also called 5-carboxypyranoanthocyanins (Rentzsch et al., 2007). Although malvidin-3-glucoside is the most abundant anthocyanin in red grapes, any anthocyanin can react with metabolic pyruvate released from the yeast cytoplasm during fermentation; exocytoplasmic pyruvic acid concentration is higher during the first stages of fermentation (fourth day) and becomes scarce over time as nutrients diminish (Morata et  al., 2003). Some yeast species may increase the production of pyruvic acid during fermentation, like S. pombe strains (Morata et  al., 2012), which, as a consequence, reduced urea formation (Benito et al., 2012) and enhanced the formation of vitisin A (Morata et al., 2012).

12.5.1.2  Vitisin B Vitisin B is the reaction product between malvidin-3-glucoside and metabolic acetaldehyde (Rentzsch et al., 2007). The concentration of acetaldehyde, a fermentative metabolite, is higher than pyruvic acid at the end of the fermentation, and therefore the formation of vitisin B tends to be higher at this stage of the fermentation (Morata et al., 2003). Vitisin B does not have a carboxyl group on the second pyran ring like vitisin A (see Fig. 12.9) and can be considered the simplest pyranoanthocyanin structure existing. This vitisin has an orange shade (hue), which is most of the time associated with wine aging (Oliveira et al., 2009a,b), and it is a consequence of its maximum absorbance wavelength at ca. 490 nm (He et al., 2012). The amount of acetaldehyde, as well as other fermentative metabolites, differs from yeast genera to yeast strains. During co-­fermentation of red grape must, non-Saccharomyces yeasts of the genera Hanseniaspora (Hanseniaspora vineae and Hanseniaspora clermontiae) co-fermenting with S. cerevisiae increased the production of metabolic acetaldehyde compared to the pure culture fermentations of either S. cerevisiae or non-Saccharomyces yeasts, and, as a consequence of this, the concentration of vitisin B was higher as well (Medina et al., 2016). In another experimental work, Morata et al. (2016) also showed the difference in the production of m ­ etabolic a­ cetaldehyde

Vitisins A

OCH 3 H OH

H O

O

H H

COOH

OH

O

Malvidin-3- O-(6-O-acetyl)-glucoside

COOH

O

HO

O

O

COOH

H H

O

HO

OH CH 2OH

O

H H

OH CH 2OH

OH

C C H H

p-Coumaroylvitisin A Malvidin-3-O-(6-O-p-coumaroyl)-glucoside-pyruvic acid

OH

OH

O

HO

HO O

O

Fig. 12.9  Vitisin-derived pigments.

O

OCH 3 H OH

OCH 3

+

O

HO

OH

H

Vitisin B Malvidin-3-O-glucoside-vinyl adduct

OH

C O H2

+

HO O

OCH 3

OCH 3 H OH

O

H H

OH

H 3C

Malvidin Malvidin-3- O-glucoside

+

H

OCH 3 H OH

H

OH

OH

HO O

OCH 3

O HO O

OH CH 2OH

H

+

OCH 3

O

H H

Acetilvitisin B Malvidin-3-O-(6-O-acetyl)-glucoside-vinyl adduct

Acetaldehyde

OH

O

Vitisin A Malvidin-3-O-glucoside-pyruvic acid

HO

OH O C O CH CH 3 H2

O

O

(yeast metabolite)

HOOC

H H

OCH 3 H OH

H

OCH 3

OCH 3 H OH

O

HO O

OH O C O CH CH 3 H2

(yeast metabolite)

H 3C

O

H H

O

H O

OCH 3 H OH H H

OH O C O H2

+

HO O C C H H

Malvidin-3- O-(6-O-p-coumaroyl)-glucoside

OH

O

H

OCH 3 H OH

O H H

OH O C O H2

C C H H

p-Coumaroylvitisin B Malvidin-3-O-(6-O-p-coumaroyl)-glucoside-vinyl adduct

OH

Chapter 12  Anthocyanins as Natural Pigments in Beverages   409

Pyruvic acid

+

HO O

O

+

HO

OCH 3 H OH

H

OH

OH

H

+

HO O

C O CH CH 3 H2

OCH 3

O

O

HO

Acetylvitisin A Malvidin-3-O-(6-O-p-acetyl)-glucoside-pyruvic acid

HO

OH

OH

+

HO O

OCH 3

OCH 3

OH O

(pyranoanthocyanins)

(Vitis vinífera L.)

OCH 3

HO

Vitisins B

Anthocyanins

(pyranoanthocyanins)

410  Chapter 12  Anthocyanins as Natural Pigments in Beverages

from six strains of S. cerevisiae yielding to different amounts of vitisin B as well as other vinyl-linked oligomeric pigments. In terms of stability in aqueous solutions, Oliveira et al. (2009a,b) have observed that vitisin B or pyranomalvidin-3-glucoside is more soluble and seems to have less self-aggregation than the coumaroyl derivative. This effect was identified in the acylated glycone moiety as the pyranomalvidin-3-glucoside coumaroyl derivative precipitated at some pH values.

12.5.2 Methylpyranoanthocyanins This type of pyranoanthocyanins is produced by the reaction of an anthocyanin with acetoacetic acid, produced during red grape must fermentation, through a cycloaddition mechanism, similar to the formation of vitisins A and B (He et al., 2006). These pigments were discovered during the extraction of anthocyanins from black currant seeds with an aqueous acetone solution (Lu et  al., 2000). The color of these pigments goes from yellow to orange since their maximum absorbance wavelength is ca. 478 nm. The behavior of methylpyranomalvidin-3-glucoside in aqueous solution at different pH values has been evaluated by Oliveira et al. (2011b). The authors observed that the methylpyranoanthocyanin has four equilibrium forms producing changes in color from orange to blue-violet as pH increases; above pH 11.5 (basic pH), the full unprotonated structure shows a yellow color as the methyl group is no longer protonated. This dianionic structure is prone to reacting toward electrophilic moieties, such as carboxypyranoanthocyanins, in order to form larger structures. Guzmán-Figueroa et  al. (2016) synthesized methylpyranoanthocyanins using natural anthocyanins extracted from rosella (Hibiscus sabdariffa L.); these anthocyanins are delphinidin-3-sambubioside and cyanidin-3-sambubioside. The methylpyranoanthocyanins were produced from the reaction of the anthocyanins with acetone and 2butanone. These pigments have a hypsochromic shift from the original anthocyanins since the maximum absorption wavelength is ca. 475–478 nm. Oliveira et  al. (2011a) have even synthesized more complex pigments from the reaction of methylpyranoanthocyanin with aldehydes, more precisely sinapaldehyde. The new pigment is called ­pyranomalvidin-budadienylidene-sinapyl and it has a maximum absorption wavelength at 568 nm; therefore, the color of this pigment is bluish.

12.5.3 Vinylphenolic Pyranoanthocyanin Vinylphenol pyranoanthocyanins, or pinotins, are formed through the reaction of pyranoanthocyanins with hydroxycinnamic acids or 4-vinyl phenols (Quaglieri et  al., 2017). These pigments were found

Chapter 12  Anthocyanins as Natural Pigments in Beverages   411

in greater amounts in Pinotage wines due to the higher concentration of caffeic acid in the Pinot Noir variety (Schwarz et  al., 2004); therefore, they were named pinotins. In this case, pinotin A is formed during wine aging from the direct reaction of malvidin-3-glucoside with caffeic acid. Besides this reaction, the production of vinylpyranoanthocyanins may be also linked to the enzymatic activity found in yeast strains having HCDC activity. Sequential fermentations using Torulaspora delbrueckii and S. cerevisiae lead to having less vinylpyranoanthocyanins than pure culture fermentations with S. cerevisiae, as shown by Loira et  al. (2014), and this is due to the fact that the T. delbrueckii strains did not show HCDC activity in this particular case; therefore, after the inoculation of S. cerevisiae, the production of vinylphenols did not increase significantly. Pichia guillermondii is another example of yeasts with positive HCDC activity; this yeast species has even more HCDC activity than S. cerevisiae, as mentioned by Benito et al. (2011), and, as a consequence, the production of vinylphenolic pyranoanthocyanins might be enhanced. An interesting color change effect has been observed in these pigments from red to blue when frozen. The physical-chemical phenomenon happens when the vinylpyranoanthocyanin in solution is frozen and electronic and vibrational effects take place according to Carvalho et  al. (2010). This is, nonetheless, a reversible effect that disappears when the frozen solution is again melted, which is apparently pH independent. Fig. 12.10 shows the condensation of malvidin-3-glucoside with vinylphenols obtained after the enzymatic decarboxylation of hydroxycinnamic acids (Mateus et al., 2006). Fig. 12.11 shows, in a similar way to the previous figure, the formation of pinotins from the reaction with hydroxycinnamic acids (Oliveira et  al., 2007); this reaction is yeastmediated through HCDC activity.

12.5.4 Ethyl-Linked Flavanol-Vinylpyranoanthocyanin Portisin A, as previously described in Section  12.3.3, is formed through the condensation of electrophilic pyranoanthocyanins with nucleophilic molecules such as flavanols (De Freitas and Mateus, 2011). These types of pigments are mainly found in aged Porto wine, which is the reason why they have that name. Different from other pyranoanthocyanins with an orange-red shade, portisin A has a bluish color in acidic conditions; this effect is attributed to a bathochromic shift toward higher absorption wavelengths (Mateus et al., 2005). Although their formation occurs naturally during wine aging, Mateus et al. (2004b) synthetized portisin A at 35°C by mixing vitisin

412  Chapter 12  Anthocyanins as Natural Pigments in Beverages

OCH 3 OH

δ+

+

O

HO

HO O OH

OCH 3 H OH

H O

H H

OCH 3

δ-

OH O

HO

+

OH CH 2OH

+

HO O O

OH

Malvidin-3- O-glucoside

OCH 3 H OH

H O

H H

OH CH 2OH

4-Vinylphenol OH

Malvidin-3-O-glucoside-4-vinylphenol adduct

Fig. 12.10  Formation of vinylphenolic pyranoanthocyanins.

Malvidin-3-O-glucoside (grape)

OMe OH

+

HO

O

OMe O-Glucose OMe

OH

OH O

OH

(fermentation) R1

R2 OH

Hydroxycinnamic acid (grape)

+

HO

Yeast hydroxycinnamate decarboxylase +

O

OMe O-Glucose

O R1

R2 OH

Vinylphenol R1

R2

OH Vinylphenolic pyranoanthocyanin (wine)

R1= —OH; R2=—H; Malvidin-3-O-glucoside-4-vinylcatechol Caffeic acid: Malvidin-3-O-glucoside-4-vinylphenol p-Coumaric acid: R1= —H; R2= —H; Ferulic acid: R1= —OCH3; R2=—H; Malvidin-3-O-glucoside-4-vinylguaiacol

Fig. 12.11  Mechanism of formation of vinylphenolic pyranoanthocyanins through the decarboxylation of hydroxycinnamic acids during fermentation. The name of the pigment will depend on the substituent groups.

Chapter 12  Anthocyanins as Natural Pigments in Beverages   413

A (a malvidin-3-glucoside-pyruvic acid derivative) with (+)-catechin in ethanol solution (20% v/v) and acetaldehyde in molar ratio 1:2 with respect to the (+)-catechin. The reaction has, as intermediate, the formation of 8-vinylcatechin, which further reacts with the vitisin A moiety. This reaction may be favored in Porto wines since the addition of wine spirits in the production of Porto wines (Mateus et al., 2005) increases the levels of acetaldehyde and, thus, the potential of forming flavanol-vinylpyranoanthocyanins. The intermediate 8-vinylcatechin, on the other hand, is highly unstable and can also react with itself to produce vinylcatechin dimers through the formation of a new dihydropyran ring (Cruz et al., 2009). As a summary of this chapter, Fig. 12.12 shows the different types of pyranoanthocyanins found in either young and aged red wines.

12.5.5 Stability of Pyranoanthocyanins Romero and Bakker (2000) have seen, through experimental design in wine model solutions, that vitisin A-type pigments are formed faster at lower temperature in the presence of pyruvate, while, on the other hand, higher temperatures slowed down the formation reaction kinetics. Once the vitisins are formed, a similar effect was observed; they tend to be less stable at higher temperatures (>32°C) when simulating high-temperature storage conditions for wines (Baranowski and Nagel, 1983). The formation of pyranoanthocyanins is susceptible to pH variations and SO2 used as bacteriostatic during wine production. In this matter, Morata et al. (2006) have found different behaviors in strains from Saccharomyces genera; pH values and SO2 modify the amount of vitisins formed when using S. cerevisiae while Saccharomyces uvarum did not show the same pattern. Regarding the stability of vitisins once formed, it can be mentioned that these compounds are more stable to a wider pH values range (up to pH 7) than anthocyanins and they are also more resistant to bleaching by SO2 (Bakker and Timberlake, 1997) due to the saturation in carbon 4 (position C4) of the pyran ring. The fact that vitisins may not undergo hydration reactions at higher pH values may also explain the retention of color in vitisins at higher pH values and their stability against monomeric anthocyanins (Oliveira et al., 2014). Anthocyanins need more acidic conditions to be in flavylium form and, therefore, to have color. The Fig. 12.13 shows how position C4 in anthocyanins is protected against nucleophilic attack by SO2 once the pyranoanthocyanin is formed. Pyranoanthocyanins are also more stable than normal anthocyanins because they have a double pyrylium; therefore, the number of resonant forms is double, giving more resistance to antioxidant degradation.

(F) (B)

(E)

(C) (D)

Fig. 12.12  Summary of pyranoanthocyanins and their precursor molecules: (A) vitisin A, (B) vitisin B, (C) methylpyranoanthocyanins, (D) vinylphenolic pyranoanthocyanins, (E) pinotins, and (F) portisins.

414  Chapter 12  Anthocyanins as Natural Pigments in Beverages

(A)

Chapter 12  Anthocyanins as Natural Pigments in Beverages   415

OCH 3

OCH 3

OH

OH +

O

HO

HO O

H

OH

O H H

O

HO

OCH 3 H OH

HO O

+

C

OH CH 2OH

OH

OCH 3 H OH

H

OH CH 2OH

O H H

Malvidin Malvidin-3-O-glucoside

OCH 3 OH O

HO

SO2

+

O H

H O OH2

H2SO3

O

H O

S

S

O

+

HO O

+

H

OH

O

H

OCH 3 H OH

H

OSO 2 H

O H H

OH CH 2OH

Uncolored molecule OCH 3 OH +

O

HO

HO O O R

H

OCH 3 H OH

O H H

OH CH 2O

R'

Vitisin/pyranoanthocyanin

Fig. 12.13  Stability of pyranoanthocyanins against SO2 attack. Pyranoanthocyanins cannot be bleached by SO2 because of the saturation in carbon 4.

Hydroxyphenyl pyranoanthocyanins, condensed from the reaction of anthocyanins and decarboxylated hydroxycinnamic acids, are prone to agglomerating and thus having absorption bands wavelengths at λ > 600 nm at pH values above four (Vallverdú-Queralt et al., 2016). In the presence of Al3+ and Fe3+, catechyl derivatives do not aggregate any longer. These molecules, similar to other pyranoanthocyanin pigments, are more stable than monomeric anthocyanins. Vitisin A can also retain its color during copigmentation with Al3+ when adding AlCl3 (Tománková et al., 2016), showing almost no change in hue at different pH values. The stability of pyranoanthocyanins could also be extended to the presence of metallic ions in solution. On the other hand, the addition of AlCl3 to malvidin-3-glucoside produced a shift in the maximum absorption due to a bathochromic effect, and the variations in pH also modified the hue from intense red at low pH to blue-purple shades at higher values. Other stable forms of pyranoanthocyanins not described in this chapter are amino-derived pyranoanthocyanin pigments. These pigments are chemically synthesized through the reaction of anthocyanins with p-dimethyl amino cinnamic acid (Schwarz and Winterhalter, 2003) and specifically from the reaction between malvidin-3-glucoside,

416  Chapter 12  Anthocyanins as Natural Pigments in Beverages

carboxypyranomalvidin-3-glucoside, and methylpyranomalvidin-3glucoside with 4-(dimethylamino)-cinnamic acid in the first two cases and 4-(dimethylamino)-cinnamaldehyde in the third one according to Oliveira et al. (2017). The reaction products are blue- violet stable pigments due to a bathochromic shift of approximately 40 nm. These pigments are apparently stable in a wide pH range with values from 1 to 12. According to Schwarz and Winterhalter (2003) the aminoderived pyranoanthocyanins had maximum absorbance at 545–555 nm at pH 2, while, in other research work regarding these stable pigments synthesis, Chassaing et al. (2015) have even reported maximum absorbance observed up to 628 and 640 nm for molecules with amino groups in their pyranoanthocyanin building block structure.

12.6  Applications in Beverages The use of anthocyanins (grape skin extract) as a food additive is regulated by the Codex Alimentarius. This food additive belongs to the functional class color and can be used in a wide variety of food products. Regarding beverage products, food category 14 in Codex Alimentarius for nonalcoholic and alcoholic beverages excluding liquid dairy products, anthocyanins could be used and are used in: waterbased flavored drinks, including “sport,” “energy,” or “electrolyte” drinks and particulated drinks, cider and Perry, wines (other than grape), distilled spirituous beverages containing more than 15% alcohol, and aromatized alcoholic beverages (e.g., beer, wine, and spirituous cooler-type beverages, low alcoholic refreshers) with a maximum dose of 300 mg/kg (FAO—Food and Agricultural Organization of the United Nations, 2017). These natural pigments could also be used in other food liquid products such as: flavored fluid milk drinks (100 mg/ kg); edible ices, including sherbet and sorbet (100 mg/kg), etc. For food beverages, it must be considered that anthocyanins are unstable pigments in food systems translated into tonality and color intensity evolution along time. Moreover, physicochemical parameters in food systems, such as pH, oxidation, presence of metals and other reactive substances, discoloring preservatives like SO2 and so on, strongly affect color evolution of anthocyanins. Fig. 12.14A–C shows color evolution in liquid yogurt during a typical 28 days self-life. It can also be observed how color can be modulated when grape anthocyanins are treated with acetaldehyde and pyruvate to promote the formation of pyranoanthocyanin derivatives (respectively, vitisin B and vitisin A) or caffeine to promote intermolecular copigmentation. The stability of anthocyanin in food products has been evaluated for several anthocyanin sources and in a wide variety of beverage products. Shalgam (or Şalgam suyu) is a Turkish beverage produced

Chapter 12  Anthocyanins as Natural Pigments in Beverages   417

Fig. 12.14  Color evolution in liquid yogurt dyed with grape natural anthocyanins and acetaldehyde (A), pyruvate (P), and caffeine (Caf). Days 1 (A), 14 (B), and 28 (C).

from the lactic fermentation of black carrot juice (Daucus carota var. L.) with a characteristic red-violet color; even though shalgam is a beverage that contains anthocyanins naturally, meaning it has no added anthocyanins as colorants, experiments carried out on this product at different storage temperatures allow beverage developers to better understand the behavior of these natural pigments in food products. The outcome of that evaluation, as expected, has shown that low storage temperatures help preserve the color of natural pigments over longer periods of time (Turker et  al., 2004). These results reinforce the fact that the temperature-controlled supply chain should be strictly kept at any time possibly to assure the quality on product properties besides ensuring food safety. The authors of this experimental work have also observed that acylated monomeric anthocyanins had better stability to temperature changes compared to nonacylated monomeric molecules. As previously mentioned throughout this chapter, the nature of the anthocyanins may have an impact on the stability of the color ­under different storage conditions; this supposes the use of

418  Chapter 12  Anthocyanins as Natural Pigments in Beverages

pigments in function of the matrix they will be added to, in order for the pigments to perform as expected. Another study showed that red sweet potato and purple corn from the Andean region had similarities, in anthocyanin composition, to those found in purple carrot and red grapes, respectively. Red sweet potato has more acylated anthocyanins, as purple carrot does, while, on the other hand, purple corn is rich in nonacylated anthocyanins like red grapes (Cevallos-Casals and Cisneros-Zevallos, 2004). Nonetheless, red sweet potato showed greater color stability than purple carrot and purple corn had more stability than grape anthocyanins; anthocyanins from this last fruit had the lowest stability over time (span of 138 days) and pH range evaluated (from 0.9 to 4). Sweet potato anthocyanins also showed higher stability toward temperature and light. The stability of natural anthocyanin and copigmented anthocyanin, obtained from jambolan fruits (Syzygium cumini) or black plum originally found in Southeast Asia and the Indian subcontinent, was evaluated in model beverage colored media (Sari et al., 2012). Storage temperature was once again an important parameter for color stability of naturally colored beverages; the disaccharides obtained from jambolan fruits showed greater stability at low and room storage temperatures. The stabilization of anthocyanins through copigmentation was done using different acids such as sinapic, caffeic, and ferulic acids as well as rosemary polyphenolic extracts; the observed effects on color properties of copigmented anthocyanins were an increase in color intensity as well as a bathochromic shift of color shade toward bluish tonality, expressed as getting a higher maximum absorption wavelength. The copigmented anthocyanins had greater free radical scavenging activity compared to the noncopigmented natural anthocyanins showing, due to this, a better performance of such pigments when used as colorants in beverage-like products. Jambolan anthocyanins differ from those anthocyanins present in V. vinifera grapes in the number of sugars bonded to their C6C3C6 chemical flavonoid structure; the same five anthocyanins found in red grapes (delphinidin, cyanidin, petunidin, peonidin, and malvidin) are found in jambolan fruits as well but having two sugar moieties instead of having just one. Although the copigmentation approach may be translated into more stable natural pigments for food colorant production, their impact on food product sensory properties is to be evaluated so as to assure quality and safety. The use of edible nanoparticles of chitosan could be a strategy to increase anthocyanin stability in beverages, delaying as well the gastrointestinal degradation of them (He et  al., 2017). There are more studies regarding the use of biopolymers as natural pigment stabilizers. Fernandes et al. (2014) reported the use of pectin as a stabilizer for anthocyanin. The interactions between ionic carbohydrates and

Chapter 12  Anthocyanins as Natural Pigments in Beverages   419

anthocyanin flavylium ions appear stronger under acidic conditions but are also present within the hemiketal form. Arabic gum has also been shown to improve the stability of purple carrot anthocyanins in beverage model solutions (Chung et al., 2016); in this case, there has been some evidence showing interaction between glycoproteins from the gum and the anthocyanin molecules used as colorants through hydrogen bonds. This interaction was observed at concentrations of 1.5% by weight of Arabic gum; at higher doses, the gum seems to reduce its efficacy due to conformational changes in its structure. Proteins have also been shown to help stabilize anthocyanins in solution (Chung et al., 2015; He et al., 2016). Chung et al. (2015) saw that whey protein was helping anthocyanins to keep the color stable through the formation of complexes by hydrogen bonds. This type of interaction made anthocyanins more stable in beverage-like solutions containing l-ascorbic acid (vitamin C). According to He et al. (2016), after using preheated casein and whey proteins from milk, the proteins reduced the thermal degradation in the same time that they increased their photo stability. The stabilization observed is also through the formation of hydrogen bonding between the protein structure and the anthocyanins. Encapsulation techniques could also be used not only to preserve anthocyanin color but also to extend the use of anthocyanins as food colorants. There are different encapsulation techniques such as spray drying, lyophilization, thermal gelation, ionic gelation, and inclusion complexation (Cavalcanti et  al., 2011). The encapsulation materials may comprise the use of one of the following substances: maltodextrin, β-cyclodextrin, pullulans, glucans, Arabic gum, curdlan, sodium alginate, and pectin. The use of encapsulation techniques to stabilize anthocyanins may extend the food product shelf life since the most common degradation that anthocyanins are subjected to, especially a wide range of pH conditions, could be prevented or delayed in a specific food matrix.

12.7 Conclusions Anthocyanins have really good properties to be used as food and beverage pigments because of their innocuous nature and positive repercussion on health. They also express enough color variability to be used in a wide range of color palette. The stability of anthocyanins in foods and beverages can be improved by using anthocyanin derivatives as polymeric anthocyanins or copigments. The use of suitable copigments increases color variability in foods in the same time that it also increases color intensity.

420  Chapter 12  Anthocyanins as Natural Pigments in Beverages

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Further Reading Lee, J.H., Lee, H.-J., Choung, M.-G., 2011. Anthocyanin compositions and biological activities from the red petals of Korean edible rose (Rosa hybrida cv. Noblered). Food Chem. 129, 272–278.