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2.4 N-Heterocyclic Pigments: Betalains

Florian C. Stintzing and Reinhold Carle

CONTENTS

2.4.1 Classification and Biosynthesis ...................................................................872.4.2 Physical and Chemical Properties ...............................................................892.4.3 Production of Betalain Colors and Coloring Foodstuffs ............................902.4.4 Stability of Betalain Preparations in Colored Foods ..................................922.4.5 Legislation....................................................................................................92References................................................................................................................93

2.4.1 CLASSIFICATION AND BIOSYNTHESIS

The term betalains (Latin: beta = beet) was coined in 1968 by Mabry and Dreidingfor the yellow and red N-heterocyclic pigments from cactus pear and red beet, whichuntil then had been erroneously called flavocyanins (betaxanthins, Greek: xanthos= yellow) and nitrogenous anthocyanins (betacyanins, Greek: kyaneos = blue),respectively.1 In 1963 and 1964, betanin and indicaxanthin were the first betacyaninand betaxanthin structurally elucidated from red beet (Beta vulgaris L.) and cactuspear fruits (Opuntia ficus indica [L.] Mill.), respectively.2,3 One year later, the termbetalamic acid was proposed for their mutual biosynthetic precursor.4

Betalains represent immonium derivatives of betalamic acid and are subdividedinto the red-violet betacyanins and the yellow-orange betaxanthins. It is most intrigu-ing that anthocyanins and betalains mutually exclude each other — they have neverbeen found together in the same plant.5,6 Moreover, Vogt provided evidence thatbetalains appeared after the anthocyanins on an evolutionary scale.7 These factsindicate that anthocyanins and betalains replace each other with respect to theirparticular functions in the plant tissue such as pollinator attraction, upholding theantioxidant potential, and shielding against noxious ultraviolet (UV) light.8,9 Whileboth pigment classes share the shikimate pathway (Figure 2.4.1), differentiationcommences at the arogenate level, leading to either tyrosine (betalains) or phenyla-lanine (anthocyanins).

Whereas the anthocyanins bear hues from orange (pelargonidin) to red (cyanidin)to blue (delphinidin), the betalains may be subdivided into distinct yellow-orange(betaxanthins) and red-violet structures (betacyanins). On the other hand, betalainic

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pertiesFIGURE 2.4.1 Biosynthetic routes leading to betalains and anthocyanins.10–14

Shikimate pathway

Chorismate

Prephenate

Arogenate PhenylalanineTyrosine

Dopa

Dopaquinone

Cyclo-Dopa

4, 5-seco-Dopa

Stizolobic acid Betalamic acid

Muscaaurin-II

Dopamine

Dopamine quinone

2,3-seco-Dopa

Muscapurpurinicacid

Muscapurpurin

Muscaflavin

Hygroaurin

Amino compound

2-Descarboxy-cyclo-dopa

Cyclo-Dopa-glycoside

2-Descarboxy-betanidinBetanidin

2-Descarboxy-betacyaninBetacyanin

SaccharideSaccharide

Acylated Betacyanin Acylated2-Descarboxy-betacyanin

Acid

Betaxanthin,Muscaaurin III-VII

Cinnamic acid p-Coumaricacid

p-Coumaroyl-CoA

CoA

Chalcone

3 Malonyl-CoA

Flavanone

Dihydroflavonol

Leucoanthocyanidin

3-Hydroxy-anthocyanidin

Anthocyanin

AcylatedAnthocyanin

Saccharide

Acid

Amino compound

Muscaaurin-I

Ibotenic acid

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N-Heterocyclic Pigments: Betalains 89

plants may contain leucoanthocyanidins and proanthocyanidins, but lack the enzy-matic provision to generate 3-hydroxy-anthocyanidins.15–19

The betalain pathway splits into three main routes, the first leading to cyclo-dopa and its glucoside. After scission of dopa, either 4,5-secodopa or 2,3-secodopais generated, the latter procuring the betalamic acid isomer muscaflavin typical offungi belonging to the Hygrophorus, Hygrocybe and Amanita genera.10,12 The path-way typical of higher plants is continued via the 4,5-secodopa structure mainlyyielding betalamic acid, the key precursor for most betacyanins and betaxanthins.For deeper insights into betalain biosynthesis and its regulation, the reader mayconsult two excellent reviews published recently.13,20 However, it is not ultimatelyunderstood whether condensation of amino acids or amines with betalamic acid toyield betaxanthins proceeds spontaneously and/or is controlled by enzymaticaction.21–24 For betacyanins, current research is directed to clarify whether cyclo-dopa is glycosylated before or after condensation with betalamic acid.25–28 Further-more, it has not yet been unveiled how specific color patterns in betalainic plantsare achieved and by which mechanisms these may be controlled.13 The endogenousenzymes in particular may regulate betalain turnover in vivo and these have receivedscarce attention.20

2.4.2 PHYSICAL AND CHEMICAL PROPERTIES

Betalains are vacuolar plant pigments. Hence their hydrophilic nature is compre-hensible. Although they are slightly soluble in ethanol and methanol, water is thebest suited solvent both for stability and solubility reasons. In contrast to the antho-cyanins, the betalains are even more polar as can be demonstrated by shorter retentiontimes in RP-HPLC and lower solubilities in alcoholic solutions.29 The varyingpolarities may also be beneficially used to separate anthocyanins from betalains onan RP-18 solid-phase extraction cartridge (Stintzing, unpublished data).

In addition, the extinction coefficients of anthocyanins are smaller than thosefor betalains, i.e., 11,300 to 29,000 L/mol*cm have been reported for the mostcommon anthocyanin 3-glucosides, while for betaxanthins and betacyanins,48,000 L/mol*cm and 60,000 L/mol*cm values, respectively, have been published,translating into higher coloring capacities of the latter.30–32 Some chemical tests bywhich anthocyanins may be differentiated from betalains have been compiled byDelgado-Vargas and co-workers.33 The color changes upon acidification or alcaliza-tion are most characteristic.

While the betalains visibly change to a canary yellow tint at alkaline pH throughrelease of betalamic acid, color strength and brilliance will increase for anthocyaninsat a pH value below 3 and rapidly decrease at low acid and neutral pH. As reportedearlier, betalains are sensitive to the presence of metals, sulfur dioxide, light expo-sure, high water activity, enzymatic action, pH and elevated temperatures.9,34,35 Thebetaxanthins are most stable at pH 5.5 to 7.36,37 Betacyanins are considered to exhibitoptimum stability at pH 5 to 6, while betalamic acid remains intact at pH 9.38–40

Betalain degradation under adverse pH conditions has not been thoroughly investi-gated with respect to resulting degradation products.33–35,41 In contrast, detailed



90 Food Colorants: Chemical and Functional Properties

investigations of the effect of thermal exposure on betalain stability have only veryrecently been of renewed research interest.42–51

2.4.3 PRODUCTION OF BETALAIN COLORS AND COLORING FOODSTUFFS

Since biotechnological production of betalain colors is still economically unfeasible,the main focus of the food industry is directed toward the exploitation of food cropsfor pigment extraction.52–57 Economic considerations include high initial color yield,optimization of the extraction process, and high color stability during processing.Furthermore, a good hygienic status, neutrality in taste and smell, and legal require-ments need to be considered.

Red Beet — Red beet juices, concentrates and powders are the typical applica-tions for coloring purposes authorized in Europe and North America and still rep-resent the sole betalainic source used commercially.58 Red beet application wassupported by research activities conducted by Von Elbe and co-workers starting inthe early 1970s. Usually, whole unpeeled beets are processed; more than 30% coloris lost by removal of the peels.59,60 This is noteworthy because the greatest polyphe-noloxidase activity, which is deleterious to both betacyanins and betaxanthins, islocated in the peel. Previous blanching presents a tool to inactivate unfavorableenzymatic action.61–63 In theory, the oxidizing and hydroxylating activities ofpolyphenol oxidase action require monophenolic or diphenolic structures rarelyfound in betaxanthins and betacyanins and only after previous hydrolysis by β-glucosidase activity.64–68 Hence, for enzymatic betalain degradation, a concertedaction of glucoside-cleaving enzymes, polyphenoloxidases and peroxidases isrequired.69–73 In general, small beets are favored because they accumulate higherbetalain concentrations.59,74,75

Tissue comminution is usually performed by milling, followed by acidificationof the resulting mash through the addition of citric acid until reaching pH 4. Loweringthe pH will preclude the action of polyphenol oxidases while peroxidases may stillbe active until the filtered juice is heated to a temperature exceeding 75°C.76,77 Throughacidification, a lower thermal load, i.e., pasteurization instead of sterilization, sufficesto secure microbial stability. Thus, pigments will be less severely affected.

In addition to chamber filtration, ultrafiltration has been successfully applied.78,79

More recent approaches using pulsed electric fields for pigment extraction have notyet entered industrial practice.80,81 Besides technologically oriented work focusedon stability aspects during processing, an extensive breeding program initiated inthe 1980s resulted in a color yield improvement of 200%.74,82–85 High pigment–lowsolid beets were suggested for food coloring purposes.86 Notably, comparativelylittle research has been dedicated to exploiting alternative food sources. This is evenmore surprising since red beet preparations are afflicted with adverse flavors ofgeosmin and methoxy-pyrazine derivatives, high nitrate levels, and the risk of carry-over of earth-bound germs.9,35 On the other hand, red beet is economically satisfac-tory because annual crop yields of 50 to 70 tons per hectare with 40 to 200 mgbetanin/100 g have not yet been reached by any other betalainic crop.




Most importantly, a clear differentiation between color preparations derived fromnatural sources requiring E number declarations (i.e., E 162 for beet red) and coloringfoodstuffs derived from typical food commodities should be made (Figure 2.4.2).For the latter, only physical unselective extraction based on oil or water followedby concentration through heating is allowed, following the recent trend for clean-labelled food.9,87

The so-obtained fruit or vegetable extracts may be applied quantum satis tosupport the particular color characteristics of foods. They are characterized by thetypical smells and tastes of the color crop sources. These colored extracts are labeledas ingredients, e.g., red beet extract. On the other hand, if the color extract isfermented with yeasts or molds to remove sugars for achieving a higher tinctorialstrength after five- to seven-fold concentration until reaching 65°Bx, the resultingproduct is considered a natural colorant.88–91 The same applies to coloring prepara-tions that have previously been denitrified.92–95

During fermentation, the betacyanins turned out to be more stable than thebetaxanthins, which is assumed to be due to their thermal stability rather thandifferent tendencies of pigments toward microbial degradation.96 Besides these bio-logical tools, beet extracts may also be purified by column chromatographic tech-niques. After removal of sugars, salts, and phenolics, the nature-derived color prep-aration will, however, require E number labeling.97

Amaranth — A great potential has been forecasted for grain and leaf amaranthboth for nutritional and ornamental purposes.98–101

The dye of amaranth leaves (Ama-

FIGURE 2.4.2 Production scheme of a coloring foodstuff (left) and a food colorant (right)from red beet.

Raw juice

Red beet

Pasteurisation

Juice/colouring foodstuff

Raw juice

Red beet

Pasteurisation

FermentationPasteurisationCentrifugationConcentration

EthanolConcentration

Concentrate/colouring foodstuff

Concentrate/food colorant E 162

Washing Grinding Mash enzymation Pressing Acidification Decantation

Washing Grinding Mash enzymation Pressing Acidification Decantation

Juice/colouring foodstuff




ranthaceae) is used as a food color in Bolivia and northwestern Argentina foralcoholic beverages, in Mexico and the southwest United States as a colorant formaize dough, and in Ecuador for different kinds of foods.99,100 In China, amaranthis authorized for the production of a natural dye.102 In a breeding and selectionprogram initiated in China in 1996, 388 genotypes of 37 species from 8 genera werestudied.103 Cultivated species exhibited higher color contents than wild species, andtotal betacyanin contents ranged from 46 to 199 mg/100g fresh plant material.104,105

Due to the high number of acylated betacyanin structures, a satisfactory stabilitywas achieved in spray-dried preparations.104,106 Because of the wider adaptability ofamaranth plants compared to red beets, the former were proposed as new sourcesof natural food colorants. On the other hand, saponins amounting to 0.1% of drymatter and dopamine contents of about 6 mg/g fresh weight need to be carefullyconsidered prior to their use in foods.99,107,108

Cactus Pear — Since 1998, cactus pear fruits (Opuntia sp.) are the focus ofthe production of betalain-based color preparations to extend the narrow hue rangeof red beet preparations.109–111 The chromatic properties of Opuntia ficus-indica cv.‘Rossa’ were found to be comparable to those of red beet.111 Together with orangefruits, different shades should be achievable. Therefore, a process for the productionof a yellow-orange cactus pear juice from O. ficus-indica cv. ‘Gialla’ has beenestablished which was extended to spray-dried powders and coloring concentrateson a semi-industrial scale.112,113 Processing of a red-purple cactus pear at laboratoryscale has been reported while a deep-red colored concentrate from O. stricta wasfound to be competitive to cochineal, red beet and commercial anthocyanicextracts.114,115 Although current pigment yields range from 15 to 80 mg/100 g fruit,new hybrids are promising higher yields up to 100 mg/100 g.111,116 It is thus expectedthat increased breeding efforts will strengthen the position of cactus pears as sourcesof purple, red, and yellow-orange hues and their use will have a bright future.

2.4.4 STABILITY OF BETALAIN PREPARATIONS IN COLORED FOODS

A small number of studies have dealt with betalain stability in colored food.33,117,118

In most cases, purified or nonpurified pigment solutions and juices were investi-gated with respect to their color stability in the presence of heat, light, varyingaw, metal ions, and oxygen. In general, betalains are considered to be most stableat near neutral conditions in foods that are devoid of sulfites, protected fromoxygen and light, and stored for short times at cooling temperatures. Hence,typical food commodities colored with betalains include dairy products, fruitfillings for bakery products, relishes, various instant products, confectionary, meatsubstitutes, and sausages.33,35

2.4.5 LEGISLATION

Synthetic colors are important from a regulatory point of view, but they lack con-sumer acceptance. They are increasingly rejected and considered unwholesome. In




some cases, they were shown to be toxic and were consequently prohibited asoccurred in 1977 when food authorizations for six azo compounds were withdrawnin Germany.119,120 In addition, artificial colors may cause adverse physiologicalreactions that again served to restrict their use.119–122 Therefore, systematic searchesfor natural substitutes have become important concerns both for industry and aca-demia.123 The acceptance of natural and nature-derived alternatives is being promotedpsychologically by portraying them as healthy and of good quality.

Food coloring is restricted by law to prevent misuse that may lead to deceptionof consumers related to reduced value or usability. For this purpose, the EuropeanUnion implemented food colorant guidelines in 1994 based on the understandingthat food coloration presents a technological need. While European Parliament andCouncil Directive 94/36/EC lists colors and their uses in food, the European Com-mission Directive 95/45/EC contains specific purity criteria for colors in foodstuffs,e.g., a maximal lead content of 20 ppm.58,124,125

Since national food legislation within the European Union varies and differencesalso exist with regard to United States, Asian, and South American legislation, coloringof foods, drugs, and cosmetics is indeed a most crucial issue for manufacturers.126

In Europe, a coloring foodstuff, although not legally defined, is considered aplant product used to color food, e.g., a concentrate or powder from beet, carrot orelderberry. Since the latter is exclusively obtained by means of physical processes,it is considered a food based on its characteristic ingredients and flavor. If, however,a selective extraction process is applied, the resulting product is regarded as acolorant and needs labeling with an EU number, e.g., E 162 for beet red, E 163 foranthocyanins.87 In this respect, a coloring foodstuff classified as food may be usedinternationally, although specific labeling requirements will apply.

The United States Code of Federal Regulations (CFR) 21.73.250/260 covers fruitand vegetable use for coloring (http://www.cfsan.fda.gov/~dms/col-toc.html; http://www.cfsan.fda.gov/~dms/opa-col2.html). The Joint Food and Agricultural Organi-zation (FAO)/World Health Organization (WHO) Expert Committee on Food Addi-tives Guidelines FAO 52/1 and FAO 52/2 are analogous to 94/36/EC and may beinstrumental for food manufacturers dealing with food coloring. Current updates forinternational trade agreements may be retrieved at http://www.codexalimentarius.net.

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http://www.cfsan.fda.gov



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http://www.codexalimentarius.net





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