NATURAL FOOD COLOURS OF BIOLOGICAL SOURCES

II. NATURAL FOOD COLOURS OF BIOLOGICAL SOURCES

A. Pigments from Plant Sources

The plant kingdom, with its multitude of colors, generates vast interest among many re- searchers and is most widely studied as a major source of food colorants. Flavonoids, carotenoids, and chlorophyll are the major contributors to the natural colors of most plants, with betalines and curcumin playing a minor yet significant role.

1. Flavonoids Anthocyanin, chalcone, and flavones belong to a group of compounds collectively known

as flavonoids.

a. Anthocyanin. Anthocyanins are basically glycosides of anthocyanidins (aglycones). The six major anthocyanidins are illustrated in Fig. 1. The sugar moieties are usually attached to the anthocyanidins via the 3-hydroxyl or 5-hydroxyl positions and to a lesser extent the 7-hydroxyl position. The anthocyanin sugars may be simple sugars—the most common being glucose, galactose, rhamnose, and arabinose—or complex sugars such as rutinose and sambubiose (2). These sugar moieties may be acylated, the most common of which being phenolic acids such as coumaric acid and caffeic acids, and to a lesser extent p-hydroxybenzoic, malonic, and acetic acids (2,3).

Anthocyanins are the most established food colorants and may be found in a wide variety of edible plant materials, such as the skin of red apples, plums, and grapes, in Anthocyanins are the most established food colorants and may be found in a wide variety of edible plant materials, such as the skin of red apples, plums, and grapes, in

The major source of anthocyanins is still the grape skin. It is, therefore, not surprising that nearly all the commercially available anthocyanins, known under the generic name of enocyanina, are obtained from the grape skin and other byproducts of the vine industry. Currently, Europe (in particular, Italy, France, and Germany) is the main producer of commercial anthocyanin using grape skins, producing about 50 tons annually. This is followed closely by the United States.

Application of anthocyanins in food is restricted due to their ability to participate in a number of reactions, resulting in its decolorization. These include reactions with ascorbic acids, oxygen, hydrogen peroxide, and sulfur dioxide to form colorless com- pounds; formation of complexes with metal ions and proteins; and hydrolysis of the sugar moieties to form unstable anthocyanidins. Anthocyanins are also sensitive to pH, being more stable at low pH (4). In addition, the anthocyanin colors vary with changes in the pH: at pH 1 and below, the anthocyanin pigment gives an intense red but becomes colorless or purple when the pH is increased to between 4 and 6. Meanwhile, the pigment turns a deep blue when the pH is between 7 and 8. Further increase in pH sees the anthocyanin pigment turning from blue to green and then to yellow. Such variation in color has been attributed to structural transformation in response to changes in pH, as illustrated in Fig.

2 (8). On the other hand, anthocyanins, which are only soluble in water or polar organic solvent, are fairly stable to heat. Studies on the effect of temperature on anthocyanin have indicated that the stability is dependent on the structure of anthocyanin, with the sugar moiety playing a significant role (9,10).

The stability of anthocyanin in the lower pH range means that anthocyanins are best suited for use in food of low pH. Anthocyanins are currently being used to provide a natural red or blue coloring for foodstuffs. Successful application of the anthocyanins includes the coloring of canned fruit, fruit syrups, yogurt, and soft drinks. Commercial anthocyanins have also been used to intensify the color of wine.

In view of the considerable consumption of anthocyanin, toxicological as well as mutagenic studies of the pigment have been carried out. Timberlake (11) reviewed the studies that have been done and concluded that anthocyanin is neither toxic nor mutagenic. On the other hand, anthocyanins were found to have beneficial therapeutic properties and would, therefore, find increasing application in not just the food area but in the medical field as well.

b. Chalcone. The chalcones are water-soluble pigments ( Fig. 3 ) extracted from petals of safflower (Carthamas tinctorius). Red carthamin, safflor yellow A, and safflor yellow

B are the three chalcones that have been isolated and identified in the safflower extract. At the moment, chalcone does not have many applications in the food industry. This is

Figure 2 Structural transformation of anthocyanin.

conditions. However, it has been noted that chalcone color is relatively insensitive to pH changes, light, and microbial degradation. Other observations regarding changes in the color of chalcone indicate that it is affected by heating and contact with metal. Due to such limitations, applications of chalcone so far are restricted to foodstuffs such as noodles, yogurts, and fruit juices (e.g., pineapple juice).

2. Carotenoids Carotenoids are noted for their great diversity and distribution. They can be found not

only in plants (e.g., carrots, tomatoes, and capsicum) but also in bacteria, fungi, algae, and animals. To date over 500 carotenoids have been isolated and identified (12). The

general structure of the carotenoids comprises a C 40 hydrocarbon chain made up of eight isoprenoid units. Naturally occurring carotenoids exist mainly as the more stable trans general structure of the carotenoids comprises a C 40 hydrocarbon chain made up of eight isoprenoid units. Naturally occurring carotenoids exist mainly as the more stable trans

A minimum of seven conjugated double bonds in the tetraterpenoid molecule is required before the carotenoid compound may have perceivable color (13). The presence of numer- ous double bonds in the carotenoid molecule also causes the compound to be very prone to oxidation, especially in the presence of light, enzymes, metals, and lipid hydroperoxides (14). Such reactions are believed to promote trans–cis isomerisation. Generally, however, carotenoids are relatively stable over a wide pH range and are fat soluble.

Beta carotene is the most abundant carotenoid in nature, particularly in plant materi- als. It is the major coloring principle in carrot and as well as palm oil seed extracts. The extracts are oil soluble and impart a yellow color to foods; they find applications in dairy products, cakes, soup, and confectionery. It is also a known fact that beta carotene is a precursor of vitamin A while possessing antioxidation properties which may help in the prevention of cancer and other diseases. This has resulted in the incorporation of beta carotene in health products, such as functional or nutraceutical beverages, with increasing usage being predicted in the future.

Although present in a lesser amount than beta carotene, annatto, saffron, and garde- nia extracts are the more commonly used carotenoids for coloring foodstuffs. Paprika, tomato, carrot, and palm oil seed have also been utilized for the extraction of carotenoids. The carotenoids are used to provide orange and yellow colors in food, particularly in fat- based food products.

a. Annatto. Annatto is an orange-yellow colored carotenoid derived from the pericarp of the seeds belonging to the shrub Bixa orellana, which can be found in tropical countries such as Brazil, Mexico, Peru, Jamaica, and India. It is estimated that every year about 7000 tons of the annatto seed are used for the production of the food colorant worldwide, with the main market being the United States and Western Europe.

Unlike other carotenoids, annatto is quite stable to pH changes and exposure to air, but moderately stable to heat. It is, however, unstable when exposed to strong light. The annatto color precipitates in acidic conditions and is also decolorized by sodium dioxide, as in the case of anthocyanin. Annatto is basically a mixture of two compounds, bixin and norbixin. Bixin, which is the mono-methyl ester of a dicarboxylic carotenoid, is the major component in the mixture (Fig. 5). It is also a fat-soluble compound which is ex- tracted from annatto seeds with edible oil (14). Bixin, whose tinctorial strength is compara- ble to that of beta carotene, gives rise to an orange color which finds uses in dairy and fat-based food products, such as margarine, cheese, creams, and baked goods. The fat- soluble bixin is also used in conjunction with other food colorants to produce various shades of color. For example, it may be used with paprika oleoresin to give a redder shade in processed cheese. Alternatively, bixin may be combined with tumeric oleoresin to provide a much more yellow shade when required.

Alkaline hydrolysis of bixin yields the free acid norbixin (Fig. 5), which is also the minor component found in annatto. The water-soluble norbixin may also be extracted from the annatto seeds using aqueous alkali (14). Applications of norbixin include smoked fish, cheese, baked goods, meat products (e.g., frankfurter sausages), snack foods, and sugar confectionery.

b. Saffron. Saffron is one of the earliest food additives used by man. It is a water- soluble extract obtained from the stigma of the flowers of Crocus sativus. This plant is the major source for the commercial production of the pigment. The same pigment may also be obtained from the flowers of C. albifloris, C. lutens, Cedrela toona, Nyctasthes arbortristes , Verbascum phlomoides, and Gardenia jasminoides.

Figure 6 Structure of saffron pigments.

Many studies have been carried out on the stability of saffron (15). Unlike the an- natto extract, saffron extract is rather sensitive to pH changes and is prone to oxidation. It is, however, moderately resistant to heat. The saffron extract is made up of water-soluble crocin and fat-soluble crocetin. The major component of the saffron extract is crocin, which is the digentiobioside ester of crocetin (Fig. 6). Crocetin, like bixin, is a dicarboxylic carotenoid. In addition to crocin and crocetin, zeaxanthin, beta carotene and certain flavor- ing compounds (mainly picrocrocin and safranal) are also found to be present in saffron extract. The flavoring compounds impart a distinct spicy flavor, thus restricting the usage of saffron extract as a food colorant.

Generally, it takes about 140,000 stigma from the Crocus flowers to produce about

1 kg of saffron powder. Coupled with the high cost of production, it makes saffron one of the most expensive food colorants (at about US$1,000 per kilogram). In view of that, it is used sparingly not only as a colorant but as a spice as well. That is why it is usually added to foodstuffs, such as curry products, soups, meat, and certain confectionery goods, where a spicy flavor is desirable while at the same time to enhance the yellow color of these products.

c. Other Carotenoid Pigments. Lycopene is one of the numerous carotenoids that are being assessed for use as new food colorants. This carotenoid can be found in watermelon and red grapefruit and has an intense red color. However, tomato is the major source of this pigment. The apparent stability of lycopene in tomatoes during standard industrial tomato processing has prompted investigation into its potential as a new commercial natu- ral colorant (16). Studies have shown that lycopene is soluble in aqueous solution and to

a certain extent in nonpolar solution. Potential application of lycopene includes beverages, confectionery, boiled sweets, bread, and cakes. Paprika extract is an orange-red oil-soluble extract obtained from the red pepper Capsicum annum . The color is insensitive to light but stable at high temperature. The main carotenoids found in this extract are capsanthin and capsorubin as well as beta caro- tene ( Fig. 4 ). Like the saffron extract, it also contains some flavoring compounds which impart a characteristic pungency to the food. Paprika’s characteristic color has seen use in coloring sauces, confectionery, salad dressing, meat products, sausages, and baked goods.

3. Betalains Centrospermae, the plant order to which beet belongs, is the only group of plants known

to produce betalains. Betalains can be divided into two classes of pigments, namely, beta- cyanins and betaxanthin. Betacyanin refers to the red pigment that may be extracted from

Figure 7 Betanin, a major component of betacyanin, which is extracted from the red beet root.

(Fig. 7). Betaxanthin, on the other hand, refers to the yellow pigment obtained from the yellow beet root Beta vulgaris var. lutea. The major components found in this class of pigment are vulgaxanthine I and II (Fig. 8).

Due to the high level of betalains found in beetroot, the plant is deemed a valuable source of food colorant. Commercial production of betalains involves a countercurrent liquid/solid extraction process. This is followed by an aerobic fermentation, generally with Candida utilis, to remove the large amount of sugar present (17,18).

Much work has been done on both betanin and vulgaxanthine in order to determine their suitability as food colorants. The stability of betanin with respect to pH, temperature, light, and air was studied by von Elbe et al. (19). Studies on the degradation rates of vulgaxanthine I with regard to pH, temperature, and oxygen were also carried out by Singer et al. (16). Both studies established that betanin and vulgaxanthine are most stable between pH 4.0 and 6.0. Both pigments are quite sensitive to air and relatively heat labile. Betanin was also found to be sensitive to light, since in the presence of light the degrada- tion rate of the pigment was found to be increased by 15.6 ⫾ 0.5% (20). As a result, both betanin and vulgaxanthine can only be used in foodstuffs with a short shelf-life as well as in those food products that do not undergo prolonged heat treatment.

Figure 8 Vulgaxanthin I and II, major components of betaxanthin, which is extracted from the

Nevertheless, application studies of betanin in selected food have suggested the presence of a protective effect on betanin from light and oxygen. This was evident based on data collected regarding color change in sausages, protein-gel, and soy protein con- taining added betanin (21). The protective effect has been attributed to the protein system present in the food. As a result, betanin is used mainly in food products with a high protein content, such as poultry meat sausages, soya protein products, gelatin dessert, and dairy products like yogurt and ice cream.

4. Chlorophyll Chlorophyll is the green pigment found in all green plants as well as green alga. The

pigment is responsible for the photosynthetic process in plants. Chlorophylls a and b are the two main types of chlorophyll pigment found in nature. The former is a bluish green pigment, while the latter is yellowish green in color. In addition, related pigments known as bacteriochlorophylls are found in photosynthetic bacteria.

The chlorophyll is a porphyrin pigment, made up of four pyrrole rings joined to- gether via methine linkages (Fig. 9). There is also a magnesium atom within the center of the porphyrin structure, held in position by two covalent and two coordinate bonds. The magnesium can be easily released from the molecule through acid-catalyzed hydrolysis to give phaeophytin. However, its stability is increased when chlorophyll is subjected to hydrolysis in an alkaline condition. In addition to the magnesium atom, a 20-carbon mo- nounsaturated alcohol, phytol, is also associated with the porphyrin molecule. This phytol side chain is responsible for the hydrophobic nature of the chlorophyll. Removal of phytol through hydrolysis yields chlorophylide, which has increased solubility in polar solvents.

The self-renewing nature of the sources of chlorophyll has generated much commer- cial interest due to its economic value. Commercial production of chlorophyll for use as

a food colorant dates back to the 1920s. The current commercial output of chlorophyll stands at the estimated figure of 11 ⫻ 10 8 tons per year. The United Kingdom is thought

to be the largest producer, accounting for about one-third of the world’s output. Three- quarters of the plant materials that are used for chlorophyll extraction are of aquatic origin with the rest being terrestrial plants. However, most of the chlorophyll that is being used as a food colorant is obtained from land plants. Lucerne, alfalfa, and nettles are some of the popular plant materials being used. The chlorophyll pigments are usually extracted from dried plant materials using aqueous solvents, such as chlorinated hydrocarbons and

Figure 10 Commercial production of chlorophyll.

acetone (22). The resulting phaeophytin extract is then further processed to give a more stable copper complex. The general scheme for the commercial production of chlorophyll is as shown in Fig. 10.

Both the oil-soluble and water-soluble forms of chlorophyll are commercially avail- able in the form of the stable copper complex. Both forms of commercial chlorophyll are relatively stable toward light and heat. However, unlike the water-soluble chlorophyll, the oil-soluble form is not very stable in acids and alkalis. A major portion of the commercial chlorophyll is used in the food industry for coloring dairy products, edible oil, soups, chewing gum, and sugar confectionery. It is mainly added to fat-based food, particularly canned products, confectionery, and pet foods. The pharmaceutical and cosmetic industries also apply chlorophyll to some of their products.

5. Miscellaneous Plant Pigments Turmeric is a fluorescent yellow colored extract obtained from the root of the curcuma

plant, with Curcuma longa being the important commercial source. Traditional use of tumeric involves grinding the tuber into powder and adding it to the food as a spice rather than as a coloring agent. The tumeric extract actually comprises three pigments: curcumin, demethoxycurcumin, and bisdemethoxycurcumin. The major pigment is curcumin, which is insoluble in water. However, it has been reported that a water-soluble complex may

be obtained by reaction the pigment with metals such as zinc chloride (22). The major disadvantage of using tumeric or curcumin is that they impart a characteristic odor and sharp taste to the foodstuff to which they are applied. On top of that, curcumin is not stable in the presence of light and alkaline conditions. Consequently, uses of tumeric or curcumin extracts are rather restricted. However after a deodorization process, the odorless commercial extract generally finds application in food products such as soups, mustard, pickles, confectionery, and canned products. Its acid stability has also found application

B. Pigments from Microbial Sources

Microorganisms are known to produce a variety of pigments, namely, chlorophyll, carot- enoids, and some unique pigments, and are, therefore a promising source of food colorants. Use of microorganisms as a source of food colorants also has the added advantages of rapid growth and ease of control.

Of all the microorganisms, the Monascus pigments and algae are perhaps the most widely studied for their potential as a source of food colorants.

1. Monascus Pigments Monascus pigments have been used in the Orient, particularly China, Japan, Indonesia,

and the Philippines, to color food for centuries. Chinese red rice wines, red soybean cheese, pickled vegetables, fish, and salted meats are some examples of Oriental food colored red by the Monascus pigment. The main source of the Monascus pigments is the fungus Mon- ascus purpureus , and the traditional method of pigment production involves the growth of the fungus on solid medium such as steamed rice (23). The resulting mass is then dried and ground to powder to be used as a colorant.

However, Lin (24) observed that Monascus sp. was able to produce the pigment in

a submerged culture. Rice powder (24) and tapioca starch (25) were found to be the suit- able carbon source for maximum pigment production with the optimum ratio of carbon source to nitrogen source being 5.33 : 7.11 (24). Since then, many studies have been de- voted to the optimization of cultural conditions for maximum pigment yield, particularly the method of submerged fermentation of the fungus (26–29). Novel methods of pigment production using roller bottles and solid–liquid state fermentation were also suggested.

The Monascus pigments are essentially a mixture of pigments whose major compo- nents are the orange monascorubrin and rubropunctatin, the red monascorubramine and rubropunctamine, as well as the yellow monascin and ankaflavin. Their structures are shown in Fig. 11 .

The native Monascus pigments generally exist in the orange-yellow insoluble form, which is bound to the membrane of the fungal mycelial. Extraction of the pigments would involve the use of organic solvents, such as methylene chloride and methanol, thereby making the pigments undesirable for use in food. Attempts in producing water-soluble pigments involve the reaction of the extracted pigments with water-soluble proteins to form the water-soluble red pigments. Solubilization of the Monascus pigments has been attributed to the formation of a complex that involves the substitution of an oxygen atom by the nitrogen of the amino group belonging to the water-soluble protein through a ring opening and a Schiffs rearrangement reaction, as shown in Fig. 11. Moll et al. (31) recom- mended the use of chitosan to produce a water-soluble red colorant, as it is not metabolized by man and is abundant in nature (31). A method for the direct aqueous extraction of water-soluble red Monascus pigments has also been developed by Chen (32). The Mon- ascus pigments are relatively stable to heat treatment and are able to withstand the auto- clave process. The pigments are also stable in pH ranging from 3 to 10. The pigments may be freeze-dried or spray-dried and may be considered for use in protein-rich foods such as meat (33), sausages (34), processed seafoods, milk, and baked goods.

2. Algal Pigments Beside chlorophyll, of which Chlorella is one of the major producers, algae are also known

Figure 11 Structural variation of the different Monascus pigments.

pigments is produced mainly by the red algae (Rhodophyta), blue-green algae (Cyano- phyta), and the cryptomonad algae (Cryptophyta). The biliproteins may further be divided into two groups: the red phycoerythrins and the blue phycocyanins. The bilin portion of the biliprotein is made up of four pyrrole rings linked together forming an open chain. The tetrapyrrole is, in turn, bound to an apoprotein by means of one or two thioether linkages ( Fig. 12 ).

Both phycoerythrins and phycocyanins are soluble in water. Studies carried out by Arad et al. ( 35) showed that the biliproteins are more stable in pH ranging from 5 to 9 and tend to precipitate at lower pH. However, stability of the pigments at low pH could

be achieved by subjecting the pigments to hydrolytic reaction using proteolytic enzymes such as pronase (35). While pigments extracted from the normal algae are relatively sensi- tive to heat, pigments obtained from thermophilic algae are quite stable to heat.

At the moment, a blue colorant extracted from the blue-green algae Spirulina plat- ensis , known as ‘‘Lina blue,’’ is being produced commercially by the Japanese company Dainippon Ink and Chemicals Inc. The Spirulina, with a protein content of 60 to 70%, has been a part of the African and Mexican diets for centuries. With its increasing popular- ity, clinical studies have been carried out by Dainippon Ink and Chemicals Inc. The studies include acute toxicity and pharmacological studies, as well as chronic toxicity of Spirulina and effects of Spirulina on photoallergic sensitivity. So far, no adverse effects on Spirulina have been reported.

Uses of the colorant in chewing gum, soft drinks, alcoholic drinks, and fermented milk products such as yogurt, have been patented in Japan. Other applications of the biliprotein includes confectioneries, candied ices, and sherbets.

Figure 12 The biliproteins found in alga.

C. Pigments from Animal and Insect Sources

1. Cochineal Cochineal is an anthraquinone which forms part of a group of pigments known as the

quininoid pigments. It has been used for centuries as a red colorant, mainly for dyeing textiles. The cochineal pigment actually refers to various red pigments obtained from female coccid insect. Different species of coccid insect are associated with different cochi- neal colors. The most well known cochineal pigment is carminic acid which comes from the female Dactylopius coccus Costa, a parasite of the cactus plants belonging to the Opuntia and Nopalea genera, particularly N. cochenillifera. The latter is a native of Central and South America. Other cochineal-related pigments that are also extracted from insects are

1. Armenian red—obtained from the coccid insect Porphyrophyra hameli, which is found growing on the lower stems and roots of grasses found in wet and alkaline regions of Azarbaijan and Armenia.

2. Kermes—extracts of Kermes lilcis, an insect which grows on species of Quercus.

3. Polish cochineal—extracted from the insect Margarodes polonicus, which in- habits the roots of Scleranthus perennis, a plant native to Central and Eastern Europe.

4. Lac dyes (laccaic acid)—produced by the lac insect, otherwise known as Laccif- era lacca , which can be found in the trees Schleichera oleosa, Zizyphus maure-

Figure 13 Carminic acid.

tania , and Butea monosperma in the Indian subcontinent, China, and Malaysia. It has been used traditionally by the Chinese to color agar.

In most cases, the female insects are harvested by hand when they reach sexual maturity. The insects collected are then dried in the open air. Conventional extraction of the cochineal pigment involves the treatment of the dried bodies with hot water. However, treatment of the ground insect bodies with proteolytic enzymes in a suitable surfactant has been proposed. This is then followed by a purification step using ion-exchange chroma- tography. Better extraction yield of the pigment has been reported with this method (36).

The principal cochineal pigment that is being used industrially is carminic acid (Fig. 13). On its own, carminic acid has little intrinsic color at pH 7. However, it is able to complex with various metals to produce a bright red color. Aluminium is the metal usually used in the commercial preparation of the carminic acid complex. The resulting product is known as carmine. The intense red color of carmine makes it a popular coloring agent for jams, syrups, preserves, confectionery, and baked goods. By varying the ratio of car- minic acid to aluminium, one can also obtain a range of colors, ranging from pale ‘‘straw- berry’’ to near ‘‘black currant’’ (36). The soluble form of carmine is obtained through the treatment of the complex with ethanol, while the nonsoluble form is obtained by the addition of calcium salt to the final solution.

2. Heme Pigments Like chlorophyll, heme also possesses four pyrrole rings. However, it differs from chloro-

phyll in that the central magnesium atom has been replaced by and iron atom. Heme is most abundant throughout the animal kingdom where it tends to associate with proteins forming complexes, such as the myglobin in the muscle and the hemoglobin in the blood ( Fig. 14 ). It functions as an oxygen carrier within the animal bodies. When the central iron atom is oxidized, as in oxygenated blood, the complex is bright red in color. Upon heating, however, the oxygen atom which is loosely bound will be lost, giving rise to a brownish color, characteristic of cooked meat.

Various extraction methods of heme from the protein complexes have been reported. Generally, it involves the use of a mixture of organic solvent and acid, such as ether and acetic acid or ethyl acetate and acetic acid. A yield of up to 80% for bulk preparations of heme using a mixture of acetone and acetic acid has been reported (37). In order to preserve the red color in heme pigment, other ligands are used to substitute the less stable oxygen atom. Ligands suggested include imidazole, S-nitrosocysteine, carbon monoxide, various amino acids, and nitrite (38). Alternatively, the central iron atom in the heme

Figure 14 Structure of heme.

A preliminary toxicity study carried out on animals had indicated that the heme pigments are harmless. Nevertheless, the characteristic color of the heme pigments had restricted their use to food products in which the color of cooked meat is desired, for example, in sausages and meat analogs.