Natural Plant Colourants

Carotenoids

Carotenoids are compounds constituted by eight isoprenoid units (ip). The ip units are joined in a head-to-tail pattern, but the order is inverted at the molecule center (Delgaldo and Lopez, 2003).  Carotenoids are naturally occurring tetraterpene pigments widely distributed through out the living world (Zdzlaslaw, 2002). Lycopene (C₄₀ H₅₆) is considered the first colored carotenoid in the biosynthesis of many other natural carotenoids and it has a linear structure (Delgaldo and Lopez, 2003). Lycopene is the carotenoid that gives the red color to tomatoes and watermelons. Moreover, it is also common to find acyclic, cyclic, and shortened carotenoids, among others. Carotenoids have very important biological activities and their use as food and feed today is recommended largely due to their vitamin A and antioxidant activities important in maintaining life (Delgaldo and Lopez, 2003; Zdzislaw, 2002). Due to their ability to quench singlet oxygen and trap peroxyl radicals, carotenoids have been described as excellent antioxidants. In addition, an inverse association between the ingestion of carotenoid-containing fruits and vegetables and the risk of certain forms of chronic diseases has been suggested (Østerlie and Lerfall, 2005). Carotenoids of the higher plants are found in roots, stems, leaves, flowers and fruits giving them the red, yellow, and orange colors. In higher plants, carotenoids are found in plastids (Delgaldo and Lopez, 2003). They are in the chloroplast of photosynthetic tissues and they are found in chromoplasts. The majority of the carotenoids found in plant tissues is β-carotene (Zdzislaw, 2002). Zdzislaw (2002) defines β-carotene as an asymmetrical molecule of 40 carbon atoms, consisting of 8 isoprene units having 11 conjugated double bonds and 2 β-ionone rings. Animals derive carotenoid pigments by consumption of carotenoid-containing plant materials. The name carotenoid has been derived from the major pigment of the carrot (Zdzislaw, 2002).

 

Fig 7: Structural Formulae of different types of carotenes (Zdzislaw, 2002).

The color of carotenoid pigments is as a result of the presence of a system of conjugated double bonds. A minimum of seven conjugated double bonds is required for the yellow color to appear. The increase of double bonds results into a shift of the major adsorption bands to the longer wavelengths, and the hue of carotenoids become more red (Zdzislaw, 2002). Because of the highly conjugated double-bond system, carotenoids show ultraviolet and visible absorption spectrum characteristics. According to Zdzislaw (2002), for most carotenoids, three peaks or two peaks and a shoulder, are absorbed in the range of 400–500 nm. The absorption maxima and molecular extinction values are significantly affected by the solvent used.

In unprocessed plants, usually all-trans double-bond configurations occur, but cis isomers of each carotenoid are also possible. Processing and storage cause isomerization of carotenoids in foods and affect the color. The deepest color is due to compounds with all-trans configurations. So, increasing the number of cis bonds results into gradual lightening of the color. This is because cis isomers absorb less strongly than the all-trans isomer, and peak at 330–340 nm (Zdzislaw, 2002).

Due to the presence of asymmetric carbon atoms, many carotenoids have chiral centers. However, natural carotenoids exist only in one of the possible enantiomeric forms, because the biosynthesis is enantiomere selective.

Carotenoid classification.

By their structural elements

-Carotenes; carotenoids constituted by carbon and hydrogen e.g. α-carotene, β-carotene and β-cryptoxanthin.

-Xanthophylls; constituted by carbon, hydrogen and additionally oxygen. For example lutein, zeaxanthin, violaxanthin, neoxanthin, and flucoxanthin.

By their functionality.

-Primary; carotenoids required for the photosynthetic process e.g. β-carotene, lutein, zeaxanthin, violaxanthin, neoxanthin, and antheraxanthin.

-Secondary; their presence is not directly related with plant survival.

Carotenoids as food colors

Natural carotenoid extracts have been used as food colorants for centuries. The most commonly used natural carotenoid extracts for foodstuffs are annatto, paprika, and saffron. Many others sources, including alfalfa, carrot, and tomato, citrus peel, and palm oil, are also utilized (Delgado and Lopez, 2003). In food industries, carotenoids are utilized mainly to color alcoholic beverages, soft drinks, fresh meat, cheese, cereals, salad dressings, canned fruits and vegetables, chewing gum, yoghurt, ice-cream, milk, margarine, pasta, canned fish, bread, potato derived products, sauces, fruit juices, instant soups, oil, butter, etc.

Tomatoes (Lycopersicon esculentum)

Tomatoes have a high content of carotenoids with lycopene being the main compound making up 80 to 90% of the total carotenoid, followed by β-carotene (Delgado and Lopez, 2003). The lycopene content in tomatoes increases with ripeness. New tomato varieties with high and improved content have been developed and lycopene products have begun to be commercialized (Zdzislaw, 2002). However, according to Delgado and Lopez (2003), other modifications are required to use tomato extract as colorant because of its strong flavor. Adding lycopene from natural sources to minced meat could lead to a meat product with different taste, better color and with a well, documented health benefit and a new functional food would be developed and may be natural lycopene could replace nitrite added minced meat(Østerlie and Lerfall, 2005).

Processing and stability

In processing or storage of colored food, carotenoids are sensitive to treatments. High and short times are preferred conditions during processing of carotenoid-containing foods ( Fennema 1996; DeMan, 1999; Delgado and Lopez, 2003). Drying (uncontrolled) leads to carotenoid degradation due to the presence of free radicals. In tomatoes, the dehydration process leads to isomerization but it has been observed that osmotic treatment does not affect the isomeric profile of lycopene (Delgado, and Lopez, 2003). Freezing causes little changes in carotene content. Blanching increases carotenoid content relative to the raw tissue due to the inactivation of lipoxygenase which is known to catalyze oxidative decomposition of carotenoids. Carotenoids are extremely lipophilic compounds that are almost insoluble in water. In aqueous surroundings they tend to form aggregates or adhere to surfaces (Zdzislaw, 2002; Fennema 1996; DeMan, 1999; Delgado and Lopez, 2003)

Anthocyanins

Anthocyanins are flavonoids with a characteristic C₆ C₃C₆ carbon skeleton making them the most commonly distributed pigment group in plants kingdom (Deman, 1999; Fennema, 1996; Zdzislaw, 2002).

Anthocyanins occur in all higher plants, mostly in flowers and fruits but also in leaves, stems, and roots. In these parts they are found predominantly in outer cell layer and account for a wide range of colors including blue, purple, pink, orange, red, magenta and violet in the plant kingdom (http://www.food-info.net/uk/colour/anthocyanin.htm; DeMan, 1999). In food plants, the main sources of anthocyanins are berries, such as blackberries, grapes, blueberries, and some vegetables, such as egg-plants (aubergine), red cabbage and avocado. Other sources include oranges, elderberry, olives, red onion, fig, sweet potato, mango, Hibiscus spp (musayi plant) and purple corn. They play a definite role in attracting animals in pollination and seed dispersal. Accordng to Zdzislaw (2002), they may also have a role in the mechanism of plant resistance to insect attack.

The structure of anthocyanins is based on a C₁₅ skeleton consisting of a chromane ring bearing a second aromatic ring. The cyclic structures are arranged in the pattern C-6-C-3-C-6. Anthocyanins structure is complemented by one or more sugar molecules bonded at different hydroxylated position of the basic structure (Delgado and Lopez, 2003).

                                                                                   

All anthocyanins are based on a single basic core structure, the flavyllium ion (Zdzislaw, 2002). 

Fig 8; Flavylium cation (Zdzislaw, 2002).

These side groups can be a hydrogen atom (H), a hydroxide (OH) or a methoxy-group (OCH) depending on the considered pigment. According to Zdzislaw (2002), from about 20 known naturally occurring anthocyanidins, only 6 occur most frequently in plants and these include: pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin.

Classification

Anthocyanins are glycosides of polyhydroxy and polymetoxy derivatives of 2-phenylbenzopyrylium or flavylium cation. They show high diversity in nature but all are based on a reduced number of basic anthocyanidin structure. Anthocyanin diversity is associated with the number of sugars found in nature but glycosylated anthocyanins are formed with glucose, rhamnose, xylose, galactose, arabinose, and fructose (Deman, 1999; Fennema, 1996; Zdzislaw, 2002).

The chemical combinations of these sugars with organic acids to produce acylated anthocyanins also increase diversity. Differences between individual anthocyanins are: the number of hydroxyl groups in the molecule; the degree of methylation of the hydroxyl groups; the nature, number, and position of glycosylation; and the nature and number of aromatic or aliphatic acids attached to the glucosyl residue.

Substitution of the hydroxyl and methoxyl groups affects the color of the anthocyanins. Color is also affected by the number of hydroxyl and methoxyl groups. If more hydroxyl groups are present, then the color goes towards bluish shade, and redness is increased if more methoxyl groups are present (http://en.wikipedia.org/wiki/Anthocyanin; Zdzislaw, 2002)

Chemically anthocyanins are subdivided into the; sugar-free anthocyanidine aglycons and anthocyanin glycosides.

Anthocyanins as food colors.

The anthocyanins constitute a large family of differently colored compounds and occur in countless mixtures in practically all parts of higher plants. They are of great economic importance as fruit pigments and thus are used to color fruit juices, jams, wines, some beverages, canned fruit, fruit syrups, yogurt, and other products. They are used as food additive with European Union number E163. In the USA, the grape color extracts were approved for use in non beverage foods, where as grape skin extract (enocyanin) is permitted in beverages. (DeMan, 1999; Delgado and Lopez, 2003)

Processing and stability

Anthocyanins are water soluble strong colors and have been used to color food since historical times (Delgado and Lopez 2003). Extracts of berries have been used to color drinks, pastries and other foods. However, some drawbacks in the use of anthocyanins as food colors exist.

Anthocyanins are water soluble and pH dependent which restricts their use. For example the color of red cabbage is enhanced with the addition of vinegar or other acid. On the other hand, when cooked in aluminum pans which cause a more alkaline environment, the color changes to purple and blue (http://www.food-info.net/uk/colour/anthocyanin.htm).

The color is also susceptible towards temperature, oxygen, UV-light and different co-factors. Temperatures destroy the flavylium ion, and thus cause loss of color. Temperature also causes maillard reactions, in which the sugar residues in the anthocyanins may be involved. Light may have a similar effect (Delgado and Lopez 2003; Zdzislaw 2002). Oxygen may destroy the anthocyanins, as do other oxidizing reagents, such as peroxides and vitamin C. Many other components in plants and foods may interact with the anthocyanins either destroying, changing or increasing the color. Quinones in apples for example, enhance the degradation of anthocyanins whereas the addition of sugar to strawberries stabilizes the color. (Delgado and Lopez, 2003; http://www.food-info.net/uk/colour/anthocyanin.htm)

All these factors limit the use of anthocyanins in foods. Some loss of color during storage may be prevented by storing at low temperatures, in dark containers or under oxygen-free packaging (Deman, 1999; Fennema, 1996; Zdzislaw, 2002; Delgado and Lopez 2003).

In practice the pure colors are very hard to obtain and most often (crude) extracts are used as food colors. Grape peel (E163 (i)), and black currant extract (E163 (iii)) are the most widely used anthocyanin mixtures in foods. (http://www.food-info.net/uk/colour/anthocyanin.htm)

Betalains

Betalains are immonium derivatives of betalamic acid, with a general formula based on the protonated 1, 2, 4, 7, 7-pentasubstituted 1, 7- diazaheptamethin system (Delgado and Lopez, 2003). Betalains occur in centrospermae, mainly in red beets, but also in some cactus fruits and mushrooms (Zdzislaw, 2002; Deman, 1996). Zdzislaw, (2002) reported that they consist of red-violet betacyanins (λmax ~ 540 nm) and yellow betaxanthins (λmax ~ 480 nm). The major betacyanin is betanin, glucoside of betanidin, which accounts for 75–95% of the total pigments of beets. The other red pigments are isobetanin (C-15 epimer of betanin), prebetanin, and isoprebetanin. According to Deman (1996), the latter two are sulfate monoesters of betanin and isobetanin, respectively. Unlike anthocyanins, betanins cannot be hydrolyzed to aglycone by acid hydrolysis without degradation (Zdzislaw, 2002). The major yellow pigments are vulgaxanthin I and II. High betalain content in beet root, on average 1% of the total solids, makes this vegetable a valuable source of the food colorant (Deman, 1996; Zdzislaw, 2002).

Fig 9: Molecular structures of betanin and isobetanin and the major yellow pigments, vulgaxanthin I and II (Zdzislaw, 2002).

Betalains are found in different plant organs, and are accumulated in cell vacuoles, mainly in epidermal and sub epidermal tissues (Delgado and Lopez, 2003). Some accumulate in plant stalks such as in the roots of red beet. Betalains are also present in the higher fungi Amanita, Hygrocybe, and Hygrosporus (Zdzislaw, 2002).

Classification:

Betalains are commonly classified into two based on their structural characteristics;

(i)                 Betacyanins (red-purple)

(ii)               Betaxanthins (yellow)

Each group of pigment is characterized by specific R₁-N-R₂ moieties.  R₁ and R₂ groups can be hydrogen, aromatic group or another substituent. Betalain color is attributable to the resonating double bonds. A large number of betaxanthins can be formed with the same dihydropyridine moiety, by conjugation with several amine compounds such as amino acids. The diversity of betacyanins is associated with the combination of the basic structures (betanidin and isobetanidin) with different glycosyl and acyl groups attached by the hydroxyl groups at positions 5 and 6. The most common glycosyl moiety is glucose and the most common acyl groups are sulfuric, malonic, citric and caffeic acids.

Betalains as food color.

According to Delgado and Lopez (2003), betalains have been in use as food colorants at least since the return of the 20^th century. Diaz et al., (2006) reported that there is a growing propensity to substitute synthetic colorants with natural pigments in the food industry. This is further confirmed by the red beet betacyanins being approved for use as a food additive in the United States of America (No. 1600), and in the European Union (E-162). Commercially, the red beet betacyanins are exempt from batch certification (http://www.food-info.net/uk/colour).

Processing and stability

The most-studied betalains are found in red beets (Beta vulgaris) in which the main betacyanins are betanin and isobetanin (Delgado and Lopez, 2003). Betalains stability is affected by temperature, pH, oxygen, light, and aqueous activity (Diaz et al., 2006). According to Delgado and Lopez (2003), enzyme degradation is also an important factor that must be considered when a betalain pigment product is to be processed.

Zdzislaw (2000), reviewed that color stability of betanin solution is strongly influenced by pH and heating. Betanins are stable at pH values of 4–6, but thermostability is greatest between pH values of 4 and 5. As a result of betanin degradation cyclo-DOPA and betalamic acid are formed. This reaction is reversible.

Fig 10: Degradation of betanin (Zdzislaw, 2002)

Light and air have a degrading effect on betanin according to Zdzislaw (2002). These effects are cumulative, but some protection may be offered by antioxidants such as ascorbic acid. Small amounts of metallic ions increase the rate of betanin degradation. Therefore a chelating agent can stabilize the color. Many protein systems present in food products also have some protective effect (Zdzislaw, 2002).

Freeze-drying is the best method to dry pigments which are sensitive to high temperatures (Diaz et al., 2006).

On market, Beetroot red (E 162) is available as liquid beetroot concentrate and as beetroot concentrate powders. According to Zdzislaw(2002) and Delgado and Lopez(2003), it is suitable for products of relatively short shelf life, which do not undergo as severe heat treatment such as meat and Soya protein products, ice cream, and gelatin desserts.

Note that all references used in all postings related to the topic of sausages, meat and meat product colorings will be posted in the last article about this topic

About the author
Mr. Sempiri Geoffery, the author of this article graduated from Makerere University with a Bsc In Food Science and Technology Degree in January, 2011.