Acta Physiologiae Plantarum

, Volume 30, Issue 5, pp 581–593

Betalain production in plant in vitro systems

Authors

  • Vasil Georgiev
    • Department of Microbial Biosynthesis and Biotechnologies, Laboratory in PlovdivThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences
  • Mladenka Ilieva
    • Department of Microbial Biosynthesis and Biotechnologies, Laboratory in PlovdivThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences
  • Thomas Bley
    • Institute of Food Technology and Bioprocess EngineeringDresden University of Technology
    • Department of Microbial Biosynthesis and Biotechnologies, Laboratory in PlovdivThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences
Review

DOI: 10.1007/s11738-008-0170-6

Cite this article as:
Georgiev, V., Ilieva, M., Bley, T. et al. Acta Physiol Plant (2008) 30: 581. doi:10.1007/s11738-008-0170-6

Abstract

Betalains have been widely used as natural colorants for many centuries, but their attractiveness for use as colorants of foods (or drugs and cosmetics) has increased recently due to their reportedly high anti-oxidative, free radical scavenging activities and concerns about the use of various synthetic alternatives. The main commercial sources of betalains are powders and concentrates of red beet (Beta vulgaris) or cactus pear (Opuntia ficus-indica) extracts. However, in recent years the technical and commercial feasibility of various in vitro systems to produce them biotechnologically has been explored. These research activities have included assessments of novel approaches for cultivating plant cell or tissue cultures, and diverse bioreactor systems for increasing production levels of secondary metabolites. This paper reviews recent progress in plant in vitro systems for producing betalain pigments. In addition, the factors that could be manipulated, the bioreactor systems that could be used, and the strategies that could be applied to improve betalain production are discussed.

Keywords

BetalainsBiological activityBioreactorsPlant in vitro systemsProcess optimization

Abbreviations

2,4-D

2,4-Dichlorophenoxyacetic acid

B5

Gamborg’s B5 medium

BAP

6-Benzylaminopurine

DOPA

3,4-dihydroxy-l-phenylalanine

DPPH

1,1-Diphenyl-2-picrylhydrazyl

LDL

Low density lipoproteins

MS

Murashige and Skoog medium

ROS

Reactive oxygen species

Introduction

Since ancient times various pigments have been used for dyeing clothes, printing and painting (examples of their use can be seen in cave paintings, Egyptian pyramids and sacred relicts of the Aztecs) (Zrÿd and Christinet 2004; Kumar and Sinha 2004). Extracts from colored plants have also been used in many countries for coloring foods (Nilsson 1970; Vygodin et al. 1995; Downham and Collins 2000; Cai et al. 2001; Stintzing and Carle 2004), e.g. kefir, yogurt (Vygodin et al. 1995), pancakes and dishes (Nottingham 2004). Following the huge advances in chemical synthesis in the last century, many synthetic colorants were also used in foods. However, many of them were later found to cause significant ecological and/or human health problems (Downham and Collins 2000; Kumar and Sinha 2004). Therefore, increasingly strict controls on synthetic colorants have been imposed. For instance, since 1979 more than 700 synthetic colorants have been banned for use in food and cosmetics by the United States Food and Drug Administration (FDA) (Parkinson and Brown 1981), and nowadays the FDA permits the general use of only 36 (7 synthetic and 26 natural) colorants in foods and cosmetics (Parkinson and Brown 1981; Downham and Collins 2000). Similarly, in 1994 the European Union authorized the use of just 43 (17 synthetic, 13 natural and 13 “naturally identical”) colorants as food additives, in Directive 94/36/EC on “colors”, which were allocated “E” numbers (Downham and Collins 2000). This list is currently under review by the European Food Safety Authority, and on 20 July 2007, the European Commission unanimously approved a draft Regulation to suspend use of the Azo dye Red 2G (E128) as a food coloring. Nevertheless, despite these strict controls, many consumers prefer to pay more to buy products with “natural” compounds than products containing chemically produced ones (Sasson 1991).

Chemical structure and biological activity of betalains

Betalains comprise a class of nitrogen-containing plant pigments found in the cell sap of plants representing most families of the Caryophyllales (Achatocarpaceae, Aizoaceae, Amaranthaceae, Basellaceae, Cactaceae, Chenopodiaceae, Didiereaceae, Halophytaceae, Hectorellaceae, Nyctaginaceae, Phytolaccaceae, Portulacaceae and Stegnospermataceae). Only two families—the Caryophyllaceae and Molluginaceae—produce anthocyanins (Mabry 2001). Betalains are also found in some higher fungi, belonging to the genera Amanita and Hygrocybe (Zrÿd and Christinet 2004).

Historical aspects of the discovery of betalains have been discussed in several reviews (Piattelli 1976; Mabry 2001; Hilpert and Dreiding 2007). However, to briefly summarize, in 1918, Willstätter and Schudel described them as “Betacyane” and successfully isolated the main red pigment from red beet—“betanin” (Nilsson 1970; Hilpert and Dreiding 2007). A couple of decades later, Robinson described the relationships between the color of these “nitrogenous anthocyanins” and the presence of nitrogen (Mabry 2001), but did not clarify their chemical structure. This was one of the issues that prompted further investigations at Dreiding’s laboratory in Zürich, which led to the isolation of crystal betanin and its hydrolysis to betanidin (Hilpert and Dreiding 2007). Several years later, Mabry discovered that betanidin, a condensation product of betalamic acid (see Fig. 1) with cyclo-DOPA, was the main aglycone of all betacyanins (Mabry 2001). Piattelli (1976) investigated the yellow pigments [named “betaxanthins” by Dreiding’s group (Nilsson 1970; Hilpert and Dreiding 2007)], which are often present in mixtures together with betacyanins, and in 1964 they isolated the first yellow pigment, “indicaxanthin”, from prickly pear (O. ficus-indica) fruits. The betaxanthins have been proved to be condensation products of betalamic acid with amino acids or amines (Nilsson 1970; Piattelli 1976). Current knowledge of the chemistry and biochemistry of betalains and their biosynthesis is summarized in a number of reviews (Nilsson 1970; Piattelli 1976; Schliemann et al. 1999; Steiner et al. 1999; Strack et al. 2003; Zrÿd and Christinet 2004; Grotewold 2006; Stintzing and Carle 2008a). It has been established that in the plant kingdom there is mutual exclusivity with respect to betalains and anthocyanins (Mabry 2001), i.e. plants that accumulate betalains do not produce anthocyanins (Stintzing and Carle 2004; Moreno et al. 2008), probably due to their lack of the enzyme anthocyanidin synthase, which participates in the last step of the flavonoid pathway (Grotewold 2006). In the plants that respectively produce them, betacyanins and anthocyanins have several common functions, notably attracting pollinators and promoting seed dispersal by animals (Piattelli 1976), but in some plants they also have specific functions. For example, betacyanins may provide protection from the harmful effects of UV light in ice plants, which do not produce anthocyanins (Ibdah et al. 2002). Betalains provide insect-repelling signals in cactus thorns, and they can improve the resistance of underground plant parts to soil pathogens (Stintzing and Carle 2004). Betaxanthins exhibit visible green fluorescence and Gandia-Herrero et al. (2005a) have suggested that they play important roles as signals for attracting budgerigars, mantis shrimps, bees and bats in the pollination of various plants (Gandía-Herrero et al. 2005b). It has also been reported recently that betacyanins act as ROS scavengers, limiting damage caused by wounding and pathogen infiltration in plant tissues (Sepúlveda-Jiménez et al. 2004). Correlations have also been established between the expression of a glucosyltransferase biosynthesis gene in B. vulgaris leaves and betacyanin production (Sepúlveda-Jiménez et al. 2005). The cited authors found high levels of similarity (71.8%) between this gene and a gene in Dorotheanthus bellidiformis encoding betanidin 5-O-glucosyltransferase, the main enzyme responsible for the glucosylation of betanidin to betanin. A detailed review of the physiological functions of betalains in plants has been provided by Stintzing and Carle (2004). The degradation processes of betalains have been studied as well, as recent reports indicated basic pathways of decarboxylation of betacyanins (Wybranec 2005; Wybranec and Mizrahi 2005). Beside the presence of decarboxylated betalains in degradation products, they have been also isolated from plants and in vitro cultures, as the 2-descarboxy-betanidin has been identified as the major betacyanin in hairy root cultures of yellow beet (Kobayashi et al.2001).
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-008-0170-6/MediaObjects/11738_2008_170_Fig1_HTML.gif
Fig. 1

Chemical structures of 1 betalamic acid; 2 Cyclo-DOPA; 3 vulgaxanthin- I (the main yellow pigment in beet); 4 betanidin; 5 betanin (the main red pigment in beet)

Recent reports that betalains and betalain-containing plant extracts have high antioxidant capacities have significantly increased scientific interest in them (Cao et al. 1996; Escribano et al. 1998; Butera et al. 2002; Lee et al. 2002; Ou et al. 2002; Pavlov et al. 2002; Wettasinghe et al. 2002a, b; Cai et al. 2003; Galati et al. 2003; Stintzing et al. 2005; Campos et al. 2006; Esquivel et al. 2007; Moreno et al. 2008). However, it should be noted that some authors have attributed the high antioxidant activity of crude betalain-containing extracts to their high concentrations of flavonoids (Lee et al. 2002) and other phenolic compounds (Campos et al. 2006). Results obtained in our laboratory have partially corroborated this conclusion. Crude ethanol extracts of hairy root cultures of B. vulgaris cv. Detroit Dark Red containing 30.8 mg betaxanthins, 16.3 mg betacyanins and 944 mg total phenolic compounds per g dry extract were found to have ca. 6-fold stronger DPPH radical scavenging activity than semi-purified extracts from the parent plant containing 23.3 mg betaxanthins, 16.5 mg betacyanins and 47.2 mg total phenolic compounds per g dry extract (unpublished data). The discrepancies between the differences in levels of scavenging activity and both betalain and total phenolic contents could be due to some of the compounds present in crude extracts having synergistic effects. Similar synergic effects have been found to increase the protection from oxidative damage provided by extracts of red beet plants to cellular systems in vivo (Frank et al. 2005). Betalains reportedly have diverse, desirable activities (Lila 2004; Stintzing and Carle 2004), including anti-inflammatory (Allegra et al. 2005; Campos et al. 2006; Lee et al. 2006), hepatoprotective (Galati et al. 2005) and cancer chemo-preventative activities (Kapadia et al. 1996, 1997, 2003), and the ability to reduce oxidative stress (Tesoriere et al. 2004) and protect low density lipoproteins (LDLs) from oxidation (Tesoriere et al. 2004a). Recentely, Sreekanth et al. (2007) have reported that betanin induces apoptosis in human chronic mylloid leucemia cells. Furthermore, it should be noted that betalains appear to have neither hepatocarcinogenic (Schwartz and von Elbe 1983), nor mutagenic (von Elbe and Schwartz 1981) effects in mammalians.

The level of bioavailability of betalains is of great importance for evaluating their activities in vivo (Kanner et al. 2001). Studies of the renal excretion of betalains have shown that renal clearance is a minor pathway in their overall elimination (Netzel et al. 2005) and highlighted the need to measure not just the initial compounds supplied, but also their metabolites in plasma, urine and bile (Franket al. 2005). The stability and antioxidative properties of ethanol extracts of betalains from hairy root cultures of B. vulgaris in simulated human gastrointestinal tract conditions have also been investigated (Pavlov et al. 2005). It has been established that at the end of the ileum betaxanthins are more stable than betacyanins, and their DPPH radical scavenging activity has declined ca. 2-fold (the main losses occurring under gastric conditions). Hence, betalains are likely to be highly suitable natural colorants for preparing healthy foods and their consumption is likely to increase.

Production by plant in vitro systems

Currently, the main sources of betalain pigments are powders and/or concentrates from red beet, and there are well-established techniques for producing them from these sources (Vygodin et al. 1995; Schoefs 2004; Sajilata and Singhal 2006; Chethana et al. 2007; Stintzing and Carle 2008b). Attempts to synthesize them chemically have not been promising, because of the multiple steps involved in the synthesis of betalamic acid and the consequently low yields (Gandía-Herrero et al. 2006). However, techniques for obtaining betalain concentrates from Opuntia ficus-indica, O. stricta, Hylocereuss polyrhizus, Myrtillocactus geometrizans and some Amaranthaceae plants (Moßhammer et al. 2005; Castellar et al. 2006; Moreno et al. 2008) have also been reported, and as described bellow, in vitro production processes have also been explored.

Plant cell and tissue cultures are attractive alternative sources of bioactive plant substances, including betalain pigments (Rao et al. 2002; Vanisree et al. 2004). The biotechnological production of food colorants using plant in vitro cultures offers several advantages over the conventional cultivation of whole plants, notably the ability to maintain aseptic, controlled conditions, regardless of variations in the climate or soil properties (Vanisree et al. 2004). The ability to define and control production conditions helps ensure continuous supplies of easily extractable products with uniform quality and yield, independently of geographic location and minimizing risks of political interference (Rao et al. 2002). Moreover, colorants produced in this way are classified as “natural” rather than “natural identical”, which increases their desirability for customers (Hancock 1997). Although the most widely used plant in vitro systems for producing bioactive substances are suspensions, hairy root cultures obtained by Agrobacterium rhizogenes-mediated transformation (Nilsson and Olsson 1997; Altamura 2004) have recently been recognized as prospective alternatives, providing new opportunities for large-scale production of these metabolites (Flores and Medina-Bolívar 1995; Shanks and Morgan 1999; Guillon et al. 2006; Georgiev et al. 2007). The main disadvantages of these in vitro cultures are the presence of bacterial plasmid DNA in their genomes and the need to use complex bioreactor systems for their large-scale cultivation. The potential utility of plant cell suspensions and hairy root cultures for synthesizing betalains, and various related issues, have been reviewed by several authors recently (Leathers et al. 1992; Jimenez-Aparicio and Gutierrez-Lopez 1999; Mukundan et al. 1999), and examples of plant in vitro systems developed for the production of betalain pigments are presented in Table 1. Clearly, a number of in vitro systems could be used to produce betaxanthin- or betacyanin-type pigments and various strategies for enhancing their production could be applied.
Table 1

Plant in vitro systems for producing betalain pigments

Species

In vitro culture

Pigment

Content

References

B. vulgaris var. Boltardy

Hairy root

Betacyanins

Betaxanthins

0.7 mg g−1 FW

1.3 mg g−1 FW

Hamill et al. (1986)

B. vulgaris L. cv. Detroit Dark Red

Hairy root

Betanin

Vulgaxanthin-I

6.1 mg g−1 DW

9.3 mg g−1 DW

Taya et al. (1992)

B. vulgaris L. cv. Detroit Dark Red

Hairy root

Betalains

2.9 mg g−1 DW

Weathers and Zobel (1992)

B. vulgaris var. lutea

Hairy root

Portulaxanthin-II

Vulgaxanthin-I

Hempel and Böhm (1997)

B.vulgaris var. Mahyco Red

Hairy root

Betalains

7.34 mg g−1 DW

Mukundan et al. (1998)

B. vulgaris L. cv. Detroit Dark Red

Hairy root

Betacyanins

18.5 mg g−1 DW

Shin et al. (2002)

B. vulgaris var. Ruby Queen

Hairy root

Betalains

11.4 mg g−1 FW

Thimmaraju et al. (2003)

B. vulgaris L. cv. Detroit Dark Red

Hairy root

Betacyanins

Betaxanthins

Total betalains

10 mg g−1 DW

14.7 mg g−1 DW

24.7 mg g−1 DW

Pavlov et al. (2003)

B. vulgaris var. Bikores Monogerm

Callus cultures

  

Girod and Zryd (1991)

Yellow phenotype

Vulgaxanthin-II

1.813 μmol g−1 DW

Miraxanthin-V

1.177 μmol g−1 DW

Total betaxanthins

4.278 μmol g−1 DW

Orange phenotype

Vulgaxanthin-II

5.519 μmol g−1 DW

Miraxanthin-V

3.061 μmol g−1 DW

Total betaxanthins

12.210 μmol g−1 DW

Red phenotype

Betanin

8.688 μmol g−1 DW

Isobetanin

0.721 μmol g−1 DW

Total betacyanins

11.222 μmol g−1 DW

Violet phenotype

Betanin

22.629 μmol g−1 DW

Isobetanin

2.903 μmol g−1 DW

Total betacyanins

28.016 μmol g−1 DW

G. marocephala St.-Hil.

Callus culture

Betalains

Vieira et al. (1995)

Mammillaria candida

Callus culture

Modified betaxanthins

Santos-Díaz et al. (2005)

Portulaca sp. Jewel

Callus culture

Betalains

8.5 ng g−1 FW

Kishima et al. (1995)

Chenopodium rubrum L.

Cell suspension

Betacyanins (80 % Amaranthin)

55 μmol g−1 DW

Berlin et al. (1986)

C. album L.

Cell suspension

Betacyanins

Rudat and Göring (1995)

B. vulgaris

Cell suspension

Betanin

Iampranthin-II

2.6 μmol g−1 DW

5.1 μmol g−1 DW

Bokern et al. (1991)

B. vulgaris var. Bikores Monogerm

Cell suspension

  

Leathers et al. (1992)

Orange phenotype

Vulgaxanthin-I

10 mg g−1 DW

Violet phenotype

Betanin

28 mg g−1 DW

B. vulgaris var. Bikores Monogerm

Cell suspension

Betalains

12÷35 mg g−1 DW

Khlebnikov et al. (1995)

B. vulgaris var. Bikores Monogerm

Cell suspension

Betalains

4.2 mg g−1 DW

Rodríguez-Monroy et al. (1994)

B. vulgarisL. cv. Detroit Dark Red

Cell suspension

Betacyanins

7.9 mg g−1 DW

Akita et al. (2000)

Portulaca sp. Jewel

Cell suspension

Betacyanins

5.3 mg g−1 FW

Bhuiyan and Adachi (2003)

Phytolacca americana L.

P. acinosa Roxb.

Cell suspension

Betanidin 5-0-[(5′-0-E-feruloyl)-2′-0-β-d-apiofuranosyl]- β-d-glucopyranoside

Iampranthin-II

Schliemann et al. (1996)

Portulaca grandiflora Hook

Cell suspension

Betaxanthins

Böhm et al. (1991)

P. americana L.

Cell suspension

Betacyanins

Sakuta et al. (1991)

Strategies for exploiting metabolic potential

To exploit plant in vitro systems at commercially viable levels, it is necessary to improve and maximize the initial productivity of the cultures. Since basic yields have usually been quite low, several strategies for enhancing them in different types of in vitro systems have been developed. Diverse physical and chemical factors have also been found to affect betalain biosynthesis in cell suspensions, callus, hairy root and tissue cultures.

Selection of highly productive cell lines

The selection and maintenance of high-yielding lines is one of the most important steps for the development of plant in vitro systems. Callus cultures induced from different plant species usually consist of yellow (betaxanthin-rich) and/or red (betacyanin-rich) clusters. For example, Akita et al. (2000) have initiated calluses from B. vulgaris cv. Detroit Dark Red consisting of yellow and red clusters, and from B. vulgaris cv. Extra Early Flat Egyptian consisting of violet clusters. The latter were chosen for establishing suspension cultures for betacyanin production. Subcultivation of selected red areas of calluses from Chenopodium rubrum resulted in the establishment of suspension cultures producing higher levels of amaranthin (80% of 55 μmol g−1 DW total betacyanin contents) than the whole plants (30% of total betacyanin contents) (Berlin et al. 1986). High betaxanthin-producing cell lines have also been established via 5 years of selection of yellow-colored parts of Portulaca grandiflora calli with an orange phenotype (selected from red–violet callus culture) (Böhm et al. 1991). In addition, secondary callogenesis of B. vulgaris cv. Bikores Monogerm, on media with various ratios of auxin (2,4-D) and cytokinin (BAP), has yielded calli with green, yellow, orange, red and violet phenotypes (Girod and Zryd 1991), and suspension cultures from the violet callus showed markedly higher betacyanin content (28.0 μmol g−1 DW) than those found in the whole plant—21.18 μmol g−1 DW (Leathers et al. 1992).

Choosing appropriate cultivars is important for obtaining highly productive hairy root lines, since both transformation-associated and intra-specific variations may occur (as observed in morphology, growth rates and branching in different lines of B. vulgaris cv. Detroit Dark Red, for instance) (Taya et al. 1992). It has been reported that hairy root lines obtained from four different cultivars of B. vulgaris (cvs. Detroit Dark Red, Egypt, Bordo and Detroit 2) accumulated widely differing levels of betalains: 13.2, 5.0, 3.1 and 3.7 mg g−1 DW, respectively (Pavlov et al. 2002).

Optimization of inoculum conditions

The age of inocula and the durations of their subcultivation periods strongly influence the growth and product formation rates of hairy root cultures. For example, reductions in the viability of hairy root cultures of B. vulgaris cv. Detroit Dark Red caused by extending their subcultivation period led to reductions in the numbers of growing points and prolongation of the culture’s lag phase (Takahashi et al. 2001). Furthermore, when inoculum was cultivated in liquid media for 14 days, the betalain contents of hairy root cultures of B. vulgaris cv. Detroit Dark Red increased to 42 mg flask−1: 14% more than when inoculum of the same age grown on solid medium was used (Pavlov et al. 2003).

Optimization of nutrient medium composition

Phosphate ions

Investigations on the physiology of B. vulgaris cv. Detroit Dark Red hairy root cultures have shown that they consume large amounts of phosphate ions. For instance, reductions in the concentration of these ions from initial levels of 120 to 10–15 mg L−1 after the start of the exponential phase of cultivation have been observed (Pavlov et al. 2005a). However, Taya et al. (1994) found that cultivating hairy root cultures of B. vulgaris cv. Detroit Dark Red in phosphate-free medium resulted in total betalain contents rising to 19 g L−1: 4.8 times higher than the amounts accumulated during the cultivation of hairy roots in a standard MS medium. In another study with B. vulgaris hairy root cultures, it was found that phosphate limitation affected betacyanin formation more than betaxanthin formation (inducing 7.8- and 3.5-fold increases, respectively) (Mukundan et al. 1999).

Nitrate ions

The concentration of nitrate ions has been found to affect the derivation and stabilization of B. vulgaris cv. Bikores Monogerm cell lines exhibiting different color phenotypes (Leathers et al. 1992). In addition, in a study by Akita et al. reducing the nitrogen concentration from 60 to 30 mM L−1 in combination with some changes in microelements, increased betacyanin production by red beet cell suspensions up to 40 mg L−1 day−1 (Akita et al. 2002). However, in another study, the concentration of nitrogen was found to have no significant effect on betalain accumulation by hairy root cultures of B. vulgaris cv. Detroit Dark Red (Taya et al. 1994). It has also been reported that ammonium ions are utilized more rapidly than nitrate ions during the cultivation of B. vulgaris L. hairy roots, but no significant amounts of either ammonium or nitrate ions are consumed in the first 5 days of their cultivation (Shin et al. 2003). In another cultivation system (hairy roots of B. vulgaris cv. Detroit Dark Red), ammonium ions were consumed rapidly until the third day of cultivation and concentration of nitrate ions reached about 350 mg L−1 (from 2,443 mg L−1 initial concentration) and remains constant until the end of cultivation (Pavlov et al. 2005a).

Microelements

Few data are available in the scientific literature about the influence of microelements on betalain production. Increase of Fe2+ concentration in LS media to 2 mM (20-fold higher concentration than that in the standard LS medium) has been found markedly to increase the betacyanin content without any affect on cell growth of B. vulgaris cv. Extra Early Flat Egyptian cell suspension (Akita et al. 2000). The absence of zinc from cultivation medium leaded to the increase of betacyanin content (up to 25 mg g−1 DW on day 21) compared to the cultivation in standard medium but slowed down the cell growth (Akita et al. 2001). Removing of boron, iodine, manganese and molybdenum was found to decrease both the cell growth and betacyanin content, whereas absence of copper and cobalt did not show any negative effect on red beet cell suspension. These effects of microelements have been confirmed in three different type B. vulgaris L. cell suspension (Akita et al. 2002). The authors also suggested an optimal HB (revised LS) medium for betacyanin production (Akita et al. 2002). On the other hand, study of the effects of copper, manganese, iron, molybdenum, zinc and cobalt concentration in Gamborg B5 media on the betalain production by B. vulgaris cv. Crosby Egyptian cell suspension, showed that higher concentrations of all microelements except manganese had positive effect on betalain production. Fivefold increase of cobalt concentration was found to provoke the highest betalain accumulation (up to 17.75 mg g−1 DW) which was 60% higher than the reached using standard B5 medium (Trejo-Tapia et al. 2001). Investigation on betalain accumulation in hairy root culture of B. vulgaris cv. Detroit Dark Red showed that the culture was more sensitive to changes in macroelements of MS medium while the changes of microelements composition almost had no effect on betalain production (Taya et al. 1994). Shin et al. (2003) found that B. vulgaris L. hairy root started to uptake manganese, and zinc from day 6 of cultivation and totally exhausted them at the end of culture period in contrast to iron and copper which was very slowly utilized by hairy root culture. Copper ions are considered to be cofactors rather than nutrients and have been shown to have different effects in different culture systems (Leathers et al. 1992). Hence, the optimization on microelements composition significantly influence on betalain production by different in vitro systems, especially in cell suspension. However, the optimal concentrations are specificity of the used in vitro systems (lines) and they must be optimized for every production system.

Carbon source

The type and concentration of carbon source supplied can also affect betalain biosynthesis, in various ways. B. vulgaris cell lines with a violet phenotype are obtained on medium containing 10 g L−1 sucrose, while green, orange and red phenotypes are obtained on media with 30 g L−1 sucrose (Mukundan et al. 1999). In addition, cell suspensions of B. vulgaris cv. Crosby Egyptian cultivated on B5 medium supplemented with 10 g L−1 sucrose have been found to produce twice as much betalain (48 mg g−1 DW) as cultures supplied with other carbon sources (Rodríguez-Monroy et al. 1994). Sucrose was also utilized more rapidly than maltose and glucose by hairy root cultures of B. vulgaris cv. Ruby Queen. Other hexoses, like fructose, lactose, xylose and galactose totally suppressed the growth and betalain production of the culture (Bhagyalakshmi et al. 2004).

Growth regulators and precursors

In hairy root cultures cultivated in hormone-free media the types of pigments produced are usually similar to those of the cultivar from which they have been established (Mukundan et al. 1999), but by changing the levels and/or ratios of growth regulators, it is possible to enhance the level of betacyanins or betaxanthins in plant in vitro systems. For example, adding 5 μM 2,4-D to MS medium used to grow suspension cultures of Phytolacca americana L. increased the betacyanin content of the cells 2.52-fold (Sakuta et al. 1991), and the biosynthesis of betacyanins in suspension culture of B. vulgaris cv. Detroit Dark Red also increased when auxins were added (Akita et al. 2000). In contrast, cytokinins have an antagonistic effect on callus cultures of Mammillaria candida; their addition to MS medium reduces betalain production (Santos-Díaz et al. 2005). On the other hand, it has been reported that red callus cultures of C. rubrum L. are able to grow on growth regulator-free MS medium for several months without reducing their betalain contents (Berlin et al. 1986). The effects of growth regulators on betacyanin biosynthesis have been discussed in two recent reviews (Jimenez-Aparicio and Gutierrez-Lopez 1999; Mukundan et al. 1999).

Feeding hairy root cultures with amino acids should theoretically stimulate the production of corresponding betaxanthins (Hempel and Böhm 1997), but recently it has been reported that feeding B. vulgaris cv. Lutea hairy root culture with S-glutamate does not lead to the formation of the corresponding Vulgaxanthin II, indicating that levels of the amino acids are not the only factors influencing their reactions with betalamic acid (Böhm and Mäck 2004). Adding DOPA to the nutrient medium does not increase the betactyanin contents in cell suspensions of C. rubrum L., but adding tyrosine after the fifth day of cultivation increases them between 50 and 100% (Berlin et al. 1986).

Light

Light affects betalain biosynthesis in all types of plant in vitro systems. Leaf disks from B. vulgaris (Wohlpart and Black 1973) and seedlings of Amaranthus caudatus L. cv. Pendula (Bianco-Colomas 1980) grown in vitro reportedly accumulate more betacyanins and amaranthin, respectively, when they are cultivated in light rather than in the dark. When green callus culture of B. vulgaris cv. Bikores Monogerm was exposed to light, it started to form red-colored segments (Girod and Zryd 1987).

It should be noted that in some cases the effects of light depends on its wavelength. For example, white light enhances the accumulation of betacyanins by nodal segments of Alternanthera brasiliana L. cv. Kuntze more strongly than UV-A light (Silva et al. 2005). Betalain biosynthesis can be induced by exposure to blue and UV light in callus cultures of Portulaca sp. Jewel (Kishima et al. 1995). It has also been suggested that betacyanin formation in suspension cultures of Chenopodium album L. is regulated by a mechanism that can be induced by UV-light (Rudat and Göring 1995). Maximum levels of betalains are accumulated by hairy root cultures of B. vulgaris L. when they are grown under continuous illumination (Mukundan et al. 1999). Moreover, when hairy roots of B. vulgaris cv. Detroit Dark Red are grown under a combination of blue and far red radiation, the accumulation of both betacyanins and betaxanthins is enhanced (Shin et al. 2003a).

Elicitation

It has been reported that in many cases adding appropriate elicitors can significantly increase the yield of secondary metabolites (Zhao et al. 2005). Such elicitation is recognized as one of the most promising strategies for enhancing betalain production, partly because knowledge of their physiological functions and roles in plant cell defence systems can be exploited. Methyl jasmonate and β-glucan have been found to increase betacyanin levels in suspension cultures of Portulaca sp. cv. Jewel 2.6- and 1.8-fold, respectively (Bhuiyan and Adachi 2003). Treating hairy root cultures of B. vulgaris cv. Ruby Queen with methyl jasmonate also resulted in a 1.4-fold increase in betalain contents (to 36.13 mg g−1 DW) (Suresh et al. 2004), and adding 5 μM of copper sulfate to hairy root cultures of B. vulgaris L. has been found to enhance their accumulation of betalains 2-fold (Mukundan et al. 1999). The influence of a wide range of biotic and abiotic elicitors on betalain biosynthesis by B. vulgaris L. hairy root cultures have been studied and the reported data identified Ca2+ as the strongest abiotic elicitor (inducing a 2.92-fold increase in betalain content) (Savitha et al. 2006).

Permeabilization, release and recovery

A possible strategy for enhancing the yield of betalains is to permeabilize the plant cells non-destructively, allowing them to secrete the pigments into the culture medium. A number of approaches could be applied for this purpose. For instance, hairy root cultures of B. vulgaris cv. Detroit Dark Red released betalains into the medium under oxygen limitation (Taya et al. 1992), and it has been found that the secretion of betalain pigments by B. vulgaris hairy root cultures can be controlled by adjusting the concentration of dissolved oxygen (Uozumi 2004). Combinations of oxygen stress and various chemical agents, including Tween-80, Triton X-100 and CTAB, have also been used to induce B. vulgaris cv. Ruby Queen betalain cultures to secrete up to 45, 70, and 90%, respectively, of their total pigment contents into the culture medium (Thimmaraju 2003). Temperature stress can also be used to induce betalain secretion; in a study by DiIorio et al. increasing the cultivation temperature to 42°C for 45 min resulted in the secretion of 15% of the betalains accumulated by B. vulgaris cv. Detroit Dark Red hairy root cultures, and had minimal effects on their viability (DiIorio 1993). Similarly, exposing hairy root cultures of B. vulgaris cv. Mahyco Red to low pH for a short time resulted in the release of 36.8% of their total betalain contents, with no loss in their viability (Mukundan et al. 1998). Sonication with ultrasound (1.02 MHz) has also been successfully used to release betalains from suspension culture of B. vulgaris cvs. Boltardy (Hunter and Kilby 1999) and Ruby Queen hairy roots (Thimmaraju 2003a). However, before this approach could be routinely applied for betalain production the stability of betalains secreted into the culture medium has to be investigated and appropriate cultivation systems, ensuring their storage, must be developed.

Bioreactor cultivation

Many different types of bioreactor systems for cultivating plant cultures in vitro are available, and choosing the most appropriate for specific applications is of great importance (Eibl and Eibl 2002). Therefore, the ideal type of in vitro system for producing betalains from any kind of plant cells or tissues needs to be carefully investigated on a case-by-case basis.

Suspension cultures of B. vulgaris cv. Bikores Monogerm have been cultivated in both 5 and 50 L fluidized bed bioreactors (Fig. 2.1 and 2), yielding 14–17 mg betalains g−1 day−1 (close to yields obtained in shake flasks; 13–24 mg g−1 day−1) (Khlebnikov et al. 1995). The advantage of these systems is that low concentrations of initial inoculum can be used (1–3% of cell FW), which is important for the further industrialization of the process. In contrast, cultivation of B. vulgaris cv. Crosby Egyptian in a stirred tank bioreactor (Fig. 2.4) resulted in 62.5% lower betalain contents than cultivation in shake flasks (Rodríguez-Monroy and Galindo 1999). It was also found that the former cell culture secreted high amounts of polysaccharides and proteins, which changed the rheological enviroment, due to sub-lethal damage caused by hydrodynamic stress in the bioreactor. To overcome this problem, the cell suspension was cultivated in a 10 L airlift bioreactor (Fig. 2.3), in which the levels of secreted proteins were considerably lower, but betalain production levels remained low (Sánchez 2002). B. vulgaris cv. Red Ball cell suspension cultures have been cultivated in a specially constructed electrophoretic tubular membrane reactor (Fig. 2.6) designed for the simultaneous growth and permeabilization of the cells to release betalains. It was shown that the cells grew well in this apparatus, and released betalains without losing their viability (Yang et al. 2003).

Hairy roots can be cultivated in vitro in various bioreactors with appropriate features (Eibl and Eibl 2002). For instance, transformed B. vulgaris cv. Detroit Dark Red hairy roots grown in stirred tanks can develop tolerance to shear stress (Fig. 2.4) (Hitaka et al. 2000), yielding a growth index of 13.3 and betalain contents of 10.06 mg g−1 DW (Georgiev et al. 2006). The main problems limiting the industrial application of hairy root cultures seem to be the difficulties involved in inoculating the cultivation systems. In an attempt to solve these problems, we have tested a fed-batch process with five feeding cycles, which delivered a ca. 10% increase in betalain contents, compared to an otherwise similar batch process. Hairy root cultures of B. vulgaris cv. Detroit Dark Red have also been cultivated in a nutrient mist bioreactor (Fig. 2.13) with a 1.8 L culture chamber, in which a 5 min ON/6 min OFF mist cycle was reportedly optimal for biomass accumulation (DiIorio et al. 1992). After a week in this bioreactor system the betalain contents of the culture amounted to 3.3 mg g−1 DW; significantly higher than in a shake flask −2.9 mg g−1 DW (Weathers and Zobel 1992). In addition, the suitability of various types of airlift bioreactors (cone, balloon, bulb, drum and column; Fig. 2.7–11) for cultivating hairy roots of B. vulgaris cv. Detroit Dark Red has been investigated, and the cone type appeared to be the best (27 mg g−1 DW betacyanins) (Shin et al. 2002). Cultivating the same culture in a 1 L balloon type airlift bioreactor under a combination of blue plus far red light in a 1:1 ratio increased the levels of betacyanins and betaxanthins produced to 20.8 and 11.3 mg g−1 DW, respectively, compared to 17.5 and 4.0 mg g−1 DW, respectively, when cultivated in the same type of bioreactor without illumination (Shin et al. 2003). Cultivation of B. vulgaris cv. Detroit Dark Red hairy roots in a column bioreactor in which the level of dissolved oxygen was controlled and the medium was circulated through an absorbent column (Fig. 2.12) packed with SP207 Sepabeads to recover betalains also yielded more betanin than shake flask cultivation (16.3 mg retained in the roots, and 8.0 mg recovered from the column) (Kino-Oka et al. 1995). Recently, B. vulgaris cv. Detroit Dark Red hairy root cultures have also been cultivated in the RITA® temporary immersion system (CIRAD Ltd., France; Fig. 2.5), and betalain contents up to 18.8 mg g−1 DW (9.6 mg g−1 DW betacyanins and 9.2 mg g−1 DW betaxanthins) have been obtained using 15 min flooding and 60 min stand-by periods: comparable to levels recorded in shake flasks (Pavlov and Bley 2006). An important advance in bioreactor technology for betalain production was the introduction of online growth control systems, based on the relationship between increases in biomass and reductions in conductivity (Ramakrishnan et al. 1999; Pavlov et al. 2005a; Pavlov and Bley 2006).

On the base of the reviewed advances, as well as our point of view for the further steps of the optimization process we propose an integrated approach for maximizing the production of betalains by plant in vitro systems (Fig. 3). It should be emphasized that no rigorous multivariate analyses have been performed as yet on the optimal nutrient contents of media for betalain production by plant in vitro systems. Such optimization could significantly improve betalain yields by adjusting the concentrations and ratios of the nutrients to optimally promote relevant aspects of the secondary metabolism of the cultures.
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Fig. 2

Bioreactor systems used for betalain production: 1 and 2 fluidized bed bioreactors; 3 airlift bioreactor; 4 stirred tank; 5 RITA® system; 6 electrophoretic tubular membrane reactor; 711 cone, balloon, bulb, drum and column airlift bioreactors, respectively; 12 column bioreactor with control of dissolved oxygen and absorbent column; 13 mist bioreactor

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Fig. 3

Integrated approach for improving betalain production by plant in vitro systems

Conclusions and prospects

During the past decade research activities in many disciplines, such as phytochemistry, food sciences, biotechnology, medicine, etc., broadened the hitherto narrow view on betalains. The chalenge to bringing together knowledge from all these different areas is considered to be most fruitful (Stintzing and Carle 2007). The coloring properties and desirable biological activities of betalains have aroused strong scientific interest in the in vitro production of these important food colorants. Although no large-scale processes have been developed yet, several highly productive plant in vitro systems, including cell suspensions and hairy root cultures, have been reported. Moreover, applying non-conventional methods, such as elicitation, cell permeabilization, product recovery and exposure to radiation of selected wavelengths (either singly or in combination) could significantly increase betalain yields. In addition, the choice of available bioreactors has been found to significantly influence important cultivation and yield parameters. However, while expensive, state-of-the-art bioreactors may provide significantly better performance than other options in some cases, use of low-cost bioreactors, such as the RITA system, plastic-lined reactors or Wave bioreactors, could markedly reduce costs, and thus be more profitable in others. These considerations clearly indicate that large-scale production of betalains is technically and commercially feasible. Furthermore, there are no problems concerning the application of betalains produced by hairy root cultures in food products.

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2008