**Biotechnological Production of Carotenoids and Their**

**Applications in Food and Pharmaceutical Products**

Ligia A. C. Cardoso, Susan G. Karp,

Francielo Vendruscolo, Karen Y. F. Kanno,

Liliana I. C. Zoz and Júlio C. Carvalho

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67725

#### **Abstract**

Pigments can be divided into four categories: natural, nature-identical, synthetic, and inorganic colors. Artificial colorants are the most used in food and pharmaceutical industries because of their advantages related to color range, price, resistance to oxygen degradation, and solubility. However, many natural pigments present health-promoting activities that make them an interesting option for human use and consumption. Natural colorants are derived from sources such as plants, insects, and microorganisms. Carotenoids are natural pigments with important biological activities, such as antioxidant and pro-vitamin A activity, that can be either extracted from plants and algae or synthesized by various microorganisms, including bacteria, yeasts, filamentous fungi, and microalgae. Advantages of microbial production include the ability of microorganisms to use a wide variety of low cost substrates, the better control of cultivation, and the minimized production time. After fermentation, carotenoids are usually recovered by cell disruption, solvent extraction, and concentration. Subsequent purification steps are followed depending on the application. The most prominent industrial applications of carotenoids, considering their health benefits, are in the food, feed, and pharmaceutical industries.

**Keywords:** biotechnology, natural pigments, microbial carotenoids, downstream, industrial applications

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1. Introduction**

Color has a great influence on the appearance, processing, and acceptance of food products, textiles and pharmaceutical products. The first quality impact by which consumers make the decision to purchase a product is its visual appearance.

Food colorants can be divided into four categories: natural, nature-identical, synthetic, and inorganic colors [1]. The production of synthetic coloring agents and other chemicals used as food additives is under increasing pressure due to a renewed interest in the use of natural products and the strong interest in minimizing the use of chemical processes [2]. Since the number of permitted synthetic colorants has decreased because of undesirable toxic effects including mutagenicity and potential carcinogenicity, interest focuses on the development of food grade pigments from natural sources [3–5].

Natural pigments are derived from sources such as plants, insects, and microorganisms. Algae and microalgae, bacteria, fungi, and yeasts are organisms commonly found in nature that can produce natural pigments in different color spectra, such as violacein, phycocyanin, monascins, flavins, quinones, and carotenoids.

Carotenoids represent one of the most important groups of natural pigments, they are responsible for the yellow, orange, red, and purple colors in a wide variety of plants, animals, and microorganisms [6]. They are lipid-soluble, commercially, and biotechnologically significant pigments produced from various organisms such as plants [7], algae and microalgae [8–12], bacteria [13–15], fungi [16–20], and yeasts [4, 21–25].

Pigments from natural sources have been obtained since long time ago, and their attractiveness has increased due to the toxicity problems caused by the synthetic pigments [26–28]. Carotenoids are obtained industrially by chemical synthesis or extraction from plants or algae; however, there has been an increasing interest in biotechnological processes for carotenoids production [29]. The pigments from microbial sources are a good alternative to obtain natural colorants for industrial uses.

The biotechnological production of carotenoids has advantages related to the diversity of microorganisms in nature, versatility in the use of substrates and agro-industrial wastes and the possibility to control operating conditions such as pH, temperature, dissolved oxygen, and light intensity; also, biomass from other bioprocesses can be submitted to the extraction of carotenoids. The production of microbial carotenoids has become a potential alternative for the replacement of artificial pigments, even with technological, economic, and legislation limitations.

Studies have demonstrated that carotenoids play an essential role for the maintenance of living bodies. In plants, carotenoids play an important role in photosynthesis, acting as light-harvesting pigments and protectors against photo-oxidation. In foods, carotenoids confer yellow, orange, or red color, serve as precursors of aroma compounds, and, as natural antioxidants, may help to extend the shelf-life [30, 31]. In humans, carotenoids have been associated with the reduction of the risk of developing chronic diseases such as cancer, cardiovascular diseases, high levels of cholesterol, cataract, and macular degeneration, aside from the pro-vitamin A activity of some of these compounds [31–33]. This is important because in the developed world, as life expectancy increases and the birth rate declines, the demand for solutions focusing on longevity and life quality increases too. The number of people aged >60 years is expected to account for approximately one-fifth of the world's population by 2050 [34].

### **2. Biotechnological production of carotenoids**

#### **2.1. Carotenoids diversity**

**1. Introduction**

126 Carotenoids

Color has a great influence on the appearance, processing, and acceptance of food products, textiles and pharmaceutical products. The first quality impact by which consumers make the

Food colorants can be divided into four categories: natural, nature-identical, synthetic, and inorganic colors [1]. The production of synthetic coloring agents and other chemicals used as food additives is under increasing pressure due to a renewed interest in the use of natural products and the strong interest in minimizing the use of chemical processes [2]. Since the number of permitted synthetic colorants has decreased because of undesirable toxic effects including mutagenicity and potential carcinogenicity, interest focuses on the development of

Natural pigments are derived from sources such as plants, insects, and microorganisms. Algae and microalgae, bacteria, fungi, and yeasts are organisms commonly found in nature that can produce natural pigments in different color spectra, such as violacein, phycocyanin,

Carotenoids represent one of the most important groups of natural pigments, they are responsible for the yellow, orange, red, and purple colors in a wide variety of plants, animals, and microorganisms [6]. They are lipid-soluble, commercially, and biotechnologically significant pigments produced from various organisms such as plants [7], algae and microalgae [8–12],

Pigments from natural sources have been obtained since long time ago, and their attractiveness has increased due to the toxicity problems caused by the synthetic pigments [26–28]. Carotenoids are obtained industrially by chemical synthesis or extraction from plants or algae; however, there has been an increasing interest in biotechnological processes for carotenoids production [29]. The pigments from microbial sources are a good alternative to obtain

The biotechnological production of carotenoids has advantages related to the diversity of microorganisms in nature, versatility in the use of substrates and agro-industrial wastes and the possibility to control operating conditions such as pH, temperature, dissolved oxygen, and light intensity; also, biomass from other bioprocesses can be submitted to the extraction of carotenoids. The production of microbial carotenoids has become a potential alternative for the replacement

Studies have demonstrated that carotenoids play an essential role for the maintenance of living bodies. In plants, carotenoids play an important role in photosynthesis, acting as light-harvesting pigments and protectors against photo-oxidation. In foods, carotenoids confer yellow, orange, or red color, serve as precursors of aroma compounds, and, as natural antioxidants, may help to extend the shelf-life [30, 31]. In humans, carotenoids have been associated with the reduction of the risk of developing chronic diseases such as cancer, cardiovascular diseases, high levels of cholesterol, cataract, and macular degeneration, aside from the pro-vitamin A activity of some of these compounds [31–33]. This is important because in the developed world,

of artificial pigments, even with technological, economic, and legislation limitations.

decision to purchase a product is its visual appearance.

food grade pigments from natural sources [3–5].

monascins, flavins, quinones, and carotenoids.

bacteria [13–15], fungi [16–20], and yeasts [4, 21–25].

natural colorants for industrial uses.

Carotenoids are lipid-soluble pigments, colored from yellow to red, with a basic structure consisting in a tetraterpene with a series of conjugated double bonds. They can have only carbon and hydrogen in their structures or have one or more oxygen atoms, being classified as xanthophylls. The majority of carotenoids are C40 terpenoids, which act as membrane-protective antioxidants scavenging O<sup>2</sup> and peroxyl radicals [35].

There are more than 700 types of carotenoids described and only about 50 are precursors of vitamin A. Carotenoids can reduce risks for degenerative diseases such as cancer, cardiovascular diseases, macular degeneration, and cataract. The biological activities, specially the antioxidant properties, depend on their chemical structure: number of conjugated double bonds, structural end-groups, and oxygen-containing substituents [36].

Carotenoids occur in photosynthetic systems of higher plants, algae, and phototrophic bacteria. In plants, carotenoids are embedded in the membranes of chloroplasts and chromoplasts. The colors of these pigments are masked by chlorophyll, but they contribute to the bright colors of many flowers and fruits [37].

Nonphotosynthetic organisms, as some bacteria and fungi, present carotenoids as protectors against photo-oxidative damage, a way of protection in growth conditions with light and abundant air. The main carotenoids produced by fungi are β-carotene, torulene, torularhodin, and astaxanthin [38]. Bacteria have been reported as producers of cantaxanthin mainly. The microalgae are producers of lutein, β-carotene, and astaxanthin [35].

Animals usually present carotenoids provenient from their diet. Marine animals that feed on algae or on products rich in carotenoids may exhibit the coloration of these pigments, as the salmon fish. The color of the feathers of some birds also comes from a diet rich in carotenoids, as flamingos [39].

The industrial production of carotenoids by plants is dependent on the season and geographic variability, and these cannot always be controlled. The chemical synthesis of carotenoids generates wastes that can cause damage to the environment and resistance by the consumers. Because of this, the biotechnological resources are becoming more interesting. The microbial production of carotenoids can be performed using low-cost substrates or substrates that are residues from industrial processes, like molasses, resulting in lower costs of production [40]. All conditions of this kind of production can be controlled and optimized, especially knowing the metabolic route of each microorganism utilized.

Carotenoids are intracellular products, and a process to increase their accessibility at the downstream stage is necessary. The techniques most used combine physical and chemical methods like maceration and contact with organic solvents [4].

#### **2.2. Main carotenoid biosynthesis pathways**

Carotenoids are usually produced from the building blocks geranyl geranyl diphosphate (GGPP) and farnesyl diphosphate (FPP), like other secondary metabolites such as sesquiterpenoids and steroids. The most common pathway is the condensation of 2 GGPP units into prephytoene diphosphate and then to phytoene, a 40-carbon polyunsaturated precursor which is colorless. This precursor is converted into lycopene and then into several derived carotenoids such as β-carotene and oxidized derivatives such as lutein. The condensation of two units of FPP leads to 30-carbon precursors that are converted to steroids or apocarotenoids such as staphyloxanthin [41, 42]. Apocarotenoids can also be produced by oxidative cleavage of carotenoids. **Figure 1** presents a simplified carotenoid biosynthesis pathway.

Most carotenoids present maximal absorption in the violet to green region of the visible spectrum, so these substances appear as red to yellow pigments. **Table 1** shows the carotenoids with permitted food use according to the Food and Drug Administration (FDA) and the Food and Agriculture Organization (FAO).

**Figure 1.** Biosynthesis pathways of common carotenoids. Source: Adapted from Ref. [38] with permission.


Sources: Compiled from the FDA Color Additive Status List [http://www.fda.gov/ForIndustry/ColorAdditives/ ColorAdditiveInventories/ucm106626.htm] and from the Combined Compendium of Food Additive Specifications [ftp://ftp.fao.org/docrep/fao/009/a0691e/a0691e00a.pdf].

**Table 1.** Carotenoids and carotenoid-rich products used as food color additives.

#### **2.3. Carotenoid sources**

**2.2. Main carotenoid biosynthesis pathways**

and Agriculture Organization (FAO).

sis pathway.

128 Carotenoids

Carotenoids are usually produced from the building blocks geranyl geranyl diphosphate (GGPP) and farnesyl diphosphate (FPP), like other secondary metabolites such as sesquiterpenoids and steroids. The most common pathway is the condensation of 2 GGPP units into prephytoene diphosphate and then to phytoene, a 40-carbon polyunsaturated precursor which is colorless. This precursor is converted into lycopene and then into several derived carotenoids such as β-carotene and oxidized derivatives such as lutein. The condensation of two units of FPP leads to 30-carbon precursors that are converted to steroids or apocarotenoids such as staphyloxanthin [41, 42]. Apocarotenoids can also be produced by oxidative cleavage of carotenoids. **Figure 1** presents a simplified carotenoid biosynthe-

Most carotenoids present maximal absorption in the violet to green region of the visible spectrum, so these substances appear as red to yellow pigments. **Table 1** shows the carotenoids with permitted food use according to the Food and Drug Administration (FDA) and the Food

**Figure 1.** Biosynthesis pathways of common carotenoids. Source: Adapted from Ref. [38] with permission.

The most common sources for natural carotenoids for food and cosmetic use are plants, although microorganism biomass is becoming more common as a source for these substances. **Table 2** illustrates some commercial sources for microorganism-based carotenoids.


\*Except where specified, these are mineral-based media. Recipes may be found at UTEX, SAG, or CCMP collections web sites.

\*\*Milligrams of carotenoids per gram of biomass.

\*\*\*Estimated. The original reference reports 28.1 mg/L carotenoids.

Xmax—maximum biomass concentration; Pmax—maximum carotenoids concentration; μX—biomass production rate. Source: Adapted from Ref. [58].

**Table 2.** Main sources for concentrated carotenoids.

#### **2.4. General downstream operations for carotenoid production**

**Microorganism Molecule Culture medium\* Xmax**

Canthaxanthin Astaxanthin β-carotene

β-carotene Lutein

Astaxanthin Canthaxanthin

\*\*\*Estimated. The original reference reports 28.1 mg/L carotenoids.

\*\*Milligrams of carotenoids per gram of biomass.

**Table 2.** Main sources for concentrated carotenoids.

Source: Adapted from Ref. [58].

*Blakeslea trispora* (fungus)

*Sporobolomyces roseus*

*Rhodotorula glutinis*

*Dietzia natronolimnaea* (bacterium)

*Phaffia rhodozyma* (yeast)

*Sporobolomyces ruberrimus* (yeast)

*Chlorella zofingiensis* (microalga)

*Coelastrella striolata* (microalga)

*Coccomyxa onubensis* (microalga)

*Haematococcus pluvialis* (microalga)

*Dunaliella salina* (microalga)

*Haematococcus pluvialis*

*Muriellopsis* sp. (microalga)

*Haematococcus pluvialis* (wild-type) *Haematococcus pluvialis* (mutant)

*Paracoccus carotinifaciens* (bacterium)

web sites.

(yeast)

130 Carotenoids

(yeast)

**(g/L)**

β-carotene Corn steep liquor 20 800 40 0.022 [43]

β-carotene Reconstituted whey 4.71 2.58 0.55 – [40]

β-carotene Potato extract 5.70 1.08 0.19 – [40]

Canthaxanthin Whey 3.29 2.87 0.87 0.020 [45]

Astaxanthin Cassava residues 8.6 2.98 0.35 0.060 [46]

Torularhodine Technical glycerol 30 3.7 0.12 0.040 [47]

Astaxanthin BBM with glucose 10.2 – 1 0.031 [48]

BBM 2.7 – 47.5

K9 1.6 – 2.88

Astaxanthin BBM 2.2 – 13.5 – [51]

β-carotene f2 – – 14\*\*\* 0.55 [53]

Astaxanthin Standard 3 – 12–15 0.56 [54]

Lutein Arnon, modified 5.37 – 6.51 0.17–0.23 [55]

2.25

\*Except where specified, these are mineral-based media. Recipes may be found at UTEX, SAG, or CCMP collections

Xmax—maximum biomass concentration; Pmax—maximum carotenoids concentration; μX—biomass production rate.

– –

*Chlorella zofingiensis* Astaxanthin Bristol, modified 10 – 1.25 0.043 [52]

Astaxanthin NIES medium 1.6

Glucose and peptone based

*Blakeslea trispora* β-carotene Whey 8 1360 170 0.023 [44]

**Pmax (mg/L)** **Conc. (mg/g)\*\***

1.5 7

6.48

47.62 54.78

– – 25–40 – [57]

0.07 0.08 [56]

0.30 [49]

0.50 [50]

**μx (h−1)** **References**

Carotenoids are nonpolar molecules that accumulate intracellularly in plant tissues and microorganisms. Therefore, the production usually consists in a biomass pretreatment that may accelerate the dissolution of these substances, followed by a solid-liquid extraction (leaching) with a suitable, low-polarity solvent. The resulting solution can be a final product, can be desolventized, and can be further purified, depending on the use intended for the extract. **Figure 2** illustrates the main steps in the production of carotenoids.

The first step in carotenoid production is the pretreatment of the raw biomass, usually by drying and milling. Drying is convenient because it reduces the weight of the material to be processed, facilitates the access for solvents to the biomass, and reduces contaminants that could be extracted in water micelles with the solvent. The milling step is also important because it increases the surface area of the biomass matrix, facilitating contact with the solvent. In the case of tough-walled organisms, chemical or mechanical cell disruption may be done prior to drying. Fine milling of the dry biomass is less common.

**Figure 2.** Main steps in carotenoids production.

The dry biomass is then extracted using a nonpolar solvent such as hexane or a vegetable oil, for the dissolution of carotenoids. A higher polarity solvent such as acetone can be used for the extraction of xanthophylls. In both cases, lipids are extracted in the mix. This extraction is an equilibrium operation; therefore, the final concentration in the solvent affects the extraction efficiency. Following extraction, the solution containing carotenoids must be concentrated and desolventized. This is why low boiling point solvents, which are easy to evaporate, are more common extractants than oils.

The carotenoids in the concentrated extract may be purified or not, depending on the intended use. For example, β-carotene that will be used as a vitamin A precursor must be purified, while paprika oleoresin is a mixture of carotenoids used mainly as a color and flavor additive and needs no further purification. In general, for carotenoids used as color additives, it is enough to concentrate the extract because (1) the tinctorial strength of the molecules is large—therefore, the additive is added at a low concentration to the formulated product and (2) the sources used are generally regarded as safe (GRAS), and the molecules extracted with the carotenoid are harmless in the concentrations used.

In the case of purified carotenoids, the operations to be used—adsorption, chromatography, crystallization, etc.—depend largely on the properties of the target molecule and the contaminants in the mixture, such as melting point, polarity, solubility, etc. All sorts of nonpolar compounds are extracted with the solvent, such as neutral and slightly polar lipids, steroids, and waxes. The differences in the properties of the carotenoid and the contaminants will be explored in the purification strategy.

Following extraction and purification, the carotenoid must be formulated for further application. This formulation will also depend on the intended use. The formulation may be as simple as adding an antioxidant such as butylated hydroxytoluene (BHT) or butylated hydroxyanisole (BHA) to the extract or may be more complex, such as emulsifying the carotenoid as an oil-in-water product for use in polar matrixes such as juices.

#### **3. Industrial application of carotenoids as additives in food, feed and pharmaceutical products**

Because of the rising of health concerns by consumers, the demand for carotenoids as natural coloring products is growing. Beta-carotene, astaxanthin, canthaxanthin, lycopen, and lutein are the most required and valuable carotenoids, and they are currently used by the food, feed, and cosmetic industries (**Table 3**). The use of carotenoids is regulated by the legislation of each country that specifies the source, purity, product, and quantities of the colorant that can be used [59].

According to BBC Research [65], the carotenoid global market in 2014 was of US\$ 1.5 billion, this value is increasing year by year and is expected to reach US\$ 1.8 billion in 2019, with an annual growth rate of 3.9%. Beta-carotene, the carotenoid of highest value, had a global market of US\$ 233 million in 2010, which is expected to reach US\$ 309 million by 2018. Astaxanthin, due to its powerful antioxidant activity, is the third carotenoid in terms of high added value, with a global market size of US\$ 225 million, estimated to increase to US\$ 253 million by 2018.

Biotechnological Production of Carotenoids and Their Applications in Food and Pharmaceutical Products http://dx.doi.org/10.5772/67725 133


**Table 3.** Carotenoids' colors, applications and biological activities.

The dry biomass is then extracted using a nonpolar solvent such as hexane or a vegetable oil, for the dissolution of carotenoids. A higher polarity solvent such as acetone can be used for the extraction of xanthophylls. In both cases, lipids are extracted in the mix. This extraction is an equilibrium operation; therefore, the final concentration in the solvent affects the extraction efficiency. Following extraction, the solution containing carotenoids must be concentrated and desolventized. This is why low boiling point solvents, which are easy to evaporate,

The carotenoids in the concentrated extract may be purified or not, depending on the intended use. For example, β-carotene that will be used as a vitamin A precursor must be purified, while paprika oleoresin is a mixture of carotenoids used mainly as a color and flavor additive and needs no further purification. In general, for carotenoids used as color additives, it is enough to concentrate the extract because (1) the tinctorial strength of the molecules is large—therefore, the additive is added at a low concentration to the formulated product and (2) the sources used are generally regarded as safe (GRAS), and the molecules extracted with

In the case of purified carotenoids, the operations to be used—adsorption, chromatography, crystallization, etc.—depend largely on the properties of the target molecule and the contaminants in the mixture, such as melting point, polarity, solubility, etc. All sorts of nonpolar compounds are extracted with the solvent, such as neutral and slightly polar lipids, steroids, and waxes. The differences in the properties of the carotenoid and the contaminants will be

Following extraction and purification, the carotenoid must be formulated for further application. This formulation will also depend on the intended use. The formulation may be as simple as adding an antioxidant such as butylated hydroxytoluene (BHT) or butylated hydroxyanisole (BHA) to the extract or may be more complex, such as emulsifying the carotenoid as an

**3. Industrial application of carotenoids as additives in food, feed and** 

Because of the rising of health concerns by consumers, the demand for carotenoids as natural coloring products is growing. Beta-carotene, astaxanthin, canthaxanthin, lycopen, and lutein are the most required and valuable carotenoids, and they are currently used by the food, feed, and cosmetic industries (**Table 3**). The use of carotenoids is regulated by the legislation of each country that specifies the source, purity, product, and quantities of the colorant that can be used [59]. According to BBC Research [65], the carotenoid global market in 2014 was of US\$ 1.5 billion, this value is increasing year by year and is expected to reach US\$ 1.8 billion in 2019, with an annual growth rate of 3.9%. Beta-carotene, the carotenoid of highest value, had a global market of US\$ 233 million in 2010, which is expected to reach US\$ 309 million by 2018. Astaxanthin, due to its powerful antioxidant activity, is the third carotenoid in terms of high added value, with a global market size of US\$ 225 million, estimated to increase to US\$ 253 million by 2018.

are more common extractants than oils.

132 Carotenoids

explored in the purification strategy.

**pharmaceutical products**

the carotenoid are harmless in the concentrations used.

oil-in-water product for use in polar matrixes such as juices.

#### **3.1. Importance and use of carotenoids in food products**

Commercial food products using carotenoids are expanding, and the greatest demand is in the Asian continent. The pigment is extracted from microalgae such as *Chlorella*, *Dunaliella*, *Haematococcus* [66, 67], from the cyanobacterium *Spirulina* [68], and from the fungus *Monascus* [69].

In Asia, the production red *koji* dates of hundreds of years and uses the fermentation of rice by *Monascus* to produce the typical reddish color. These red pigments are also used as food colorants for wine, red soy cheese, meat, and by-products of meat and fish [26]. The French cheese named *vieux-pan* contains the carotenoid produced by *Brevibacterium linens* due to its orange-red-brown color that improves the sensory quality of the product [70]. In Russia, infant formulas are enriched with natural pigments such as lutein, which is present in breast milk, in order to improve children's health [71].

Nutraceutical food products have also been applied in bakery products and pasta. In Japan, *Undaria pinnatifida* (wakame), an edible seaweed rich in fucoxanthin, is commercialized as an ingredient for pasta [72]. In India, a pasta containing fucoxanthin as an ingredient to improve its biofunctional and nutritional qualities was developed [73].

#### **3.2. Importance and applications of carotenoids in the pharmaceutical industry**

Besides the use of nutraceutical foods as a form of prevention and treatment of diseases, the administration of the bioactive compounds in their concentrated form is also a possibility for promoting health. The transport of carotenoids occurs from the intestinal mucosa to the blood vessels carried by lipoproteins [74]. Carotenoids functional properties are related to reactions such as oxidation, reduction, hydrogen abstraction, and addition in biological membranes, and their antioxidant power is fundamental for cell protection against free radicals and singlet oxygen formed in tissues [75].

Some carotenoids are precursors of vitamins, and they also present activities such as antiinflammatory, antioxidant, immunomodulatory, anticancer, for cardiovascular therapy and neurodegenerative diseases [76], and anti-obesity [77]. The carotenoids included as pro-vitamin A are β-carotene, α-carotene, and cryptoxanthin. Vitamin A is an essential nutrient for operation and maintenance of biological functions including vision, reproduction, and immunity [78]. Beta-carotene is present in blood and tissues, which is associated with antioxidant activity and concomitantly with other carotenoids or antioxidants can enhance their activity against free radicals. However, it can bring health risk at high doses [79].

Carotenoids, acting as antioxidants eliminating free radicals, can modulate the risk of developing chronic diseases by inhibiting reactions mediated by reactive oxygen species (ROS). Reactive species are produced during cellular metabolism as a defense to infectious and chemical agents that may cause damage to DNA, proteins, and tissues, contributing to the development of chronic diseases such as diabetes, Parkinson's, Alzheimer's, cardiovascular diseases, and cancer [80].

In addition to the antioxidant properties, carotenoids exhibit anti-inflammatory activities owing to the protective effects of phytochemicals such as lutein and astaxanthin. Astaxanthin has been shown to inhibit the production of pro-inflammatory mediators such as nitric oxide (NO) in macrophages, to increase the level of inflammatory cytokines, and to reduce oxidative stress. Neuroprotective effect, reduced neuroinflammation, improvement of insulin signals, and reduction of lipid levels were also verified [81].

Inhibition of cell proliferation of colon cancer cells by the use of *Neochloris oleoabundans* carotenoids was observed, enabling its use as a functional food additive or nutraceutical with potential for the prevention of colon cancer [82]. Beta-carotene, astaxanthin, and capsanthin demonstrated antiproliferative effects on leukemic K562 cells [83]. Studies indicated that the simultaneous use of different carotenoids was efficient against liver cancer. Patients were administered with β-cryptoxanthin-enriched mandarin orange juice and capsules of a carotenoids mixture-containing lutein, β-cryptoxanthin, lycopene, zeaxanthin, and fucoxanthin. Analyses of DNA array and protein-antibody array showed that the carotenoids interferred in the induction of genes such as p16 and p73 [84].

#### **4. Conclusion and final remarks**

There are many advantages related to the use of carotenoids instead of artificial pigments in food products and for pharmaceutical applications. Their biological properties such as antioxidant, anti-inflammatory, antitumoral, and pro-vitamin A activities contribute to the quality of the product and to the consumer's health. Among the production strategies, microbial synthesis is considered advantageous, and the downstream techniques usually involve cell disruption, solvent extraction, concentration, and purification, when necessary. Several researches have proved the beneficial effects of carotenoids on health, so they can meet the demand for solutions focusing on longevity and life quality.

### **Author details**

Some carotenoids are precursors of vitamins, and they also present activities such as antiinflammatory, antioxidant, immunomodulatory, anticancer, for cardiovascular therapy and neurodegenerative diseases [76], and anti-obesity [77]. The carotenoids included as pro-vitamin A are β-carotene, α-carotene, and cryptoxanthin. Vitamin A is an essential nutrient for operation and maintenance of biological functions including vision, reproduction, and immunity [78]. Beta-carotene is present in blood and tissues, which is associated with antioxidant activity and concomitantly with other carotenoids or antioxidants can enhance their activity against free radicals. However, it can bring health risk at high

Carotenoids, acting as antioxidants eliminating free radicals, can modulate the risk of developing chronic diseases by inhibiting reactions mediated by reactive oxygen species (ROS). Reactive species are produced during cellular metabolism as a defense to infectious and chemical agents that may cause damage to DNA, proteins, and tissues, contributing to the development of chronic diseases such as diabetes, Parkinson's, Alzheimer's, cardiovascular

In addition to the antioxidant properties, carotenoids exhibit anti-inflammatory activities owing to the protective effects of phytochemicals such as lutein and astaxanthin. Astaxanthin has been shown to inhibit the production of pro-inflammatory mediators such as nitric oxide (NO) in macrophages, to increase the level of inflammatory cytokines, and to reduce oxidative stress. Neuroprotective effect, reduced neuroinflammation, improvement of insulin signals,

Inhibition of cell proliferation of colon cancer cells by the use of *Neochloris oleoabundans* carotenoids was observed, enabling its use as a functional food additive or nutraceutical with potential for the prevention of colon cancer [82]. Beta-carotene, astaxanthin, and capsanthin demonstrated antiproliferative effects on leukemic K562 cells [83]. Studies indicated that the simultaneous use of different carotenoids was efficient against liver cancer. Patients were administered with β-cryptoxanthin-enriched mandarin orange juice and capsules of a carotenoids mixture-containing lutein, β-cryptoxanthin, lycopene, zeaxanthin, and fucoxanthin. Analyses of DNA array and protein-antibody array showed that the carotenoids interferred

There are many advantages related to the use of carotenoids instead of artificial pigments in food products and for pharmaceutical applications. Their biological properties such as antioxidant, anti-inflammatory, antitumoral, and pro-vitamin A activities contribute to the quality of the product and to the consumer's health. Among the production strategies, microbial synthesis is considered advantageous, and the downstream techniques usually involve cell disruption, solvent extraction, concentration, and purification, when necessary. Several researches have proved the beneficial effects of carotenoids on health, so they can meet the

doses [79].

134 Carotenoids

diseases, and cancer [80].

and reduction of lipid levels were also verified [81].

in the induction of genes such as p16 and p73 [84].

demand for solutions focusing on longevity and life quality.

**4. Conclusion and final remarks**

Ligia A. C. Cardoso1 \*, Susan G. Karp<sup>2</sup> , Francielo Vendruscolo<sup>3</sup> , Karen Y. F. Kanno1 , Liliana I. C. Zoz2 and Júlio C. Carvalho<sup>2</sup>


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## **Synthesis of Antioxidant Carotenoids in Microalgae in Response to Physiological Stress**

Cecilia Faraloni and Giuseppe Torzillo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67843

#### **Abstract**

Carotenoids act as potential antioxidants, quenching energy of excited singlet oxygen and scavenging free radicals. Among microalgae, *Haematococcus*, *Chlamydomonas*, *Chlorella*, *Dunaliella* and diatoms and dinoflagellates, such as *Phaeodactylum* and *Isochrysis*, are able to synthesize large amount of carotenoids. The main function of carotenoids consists in absorbing light to perform photosynthesis, and some of them are constitutively present in the cells (primary carotenoids). The main primary carotenoids usually found are neoxanthin, violaxanthin, lutein, and β-carotene. To preserve cells from oxidative damage, their production may be increased, while other carotenoids may be synthesized *de novo*. In particular, under stress conditions such as high light exposure, nutrient starvation, change in oxygen partial pressure, and high or low temperatures, microalgal metabolism is altered and photosynthetic activity may be reduced. In these conditions, photosynthetic electrons transport is reduced, and the intracellular reduction level increase may be associated with the formation of free radicals and species containing singlet oxygen. In order to prevent damage from photooxidation, microalgae are able to adopt strategies to contrast these dangerous oxidant molecules. One of the most active mechanisms is to synthesize large amount of carotenoids, which can act as antioxidants.

**Keywords:** carotenoids, microalgae, antioxidant, stress

#### **1. Introduction**

Carotenoids are a class of natural lipid-soluble pigments mainly found in plants, algae, and photosynthetic bacteria. They play a central role in photosynthesis, both as light-harvesting complexes and as photoprotectors. However, it is generally believed that they function as

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

passive photoprotectors (i.e., as a filter), reducing the amount of light that can reach the lightharvesting pigment complexes of photosystem II (PSII).

For their antioxidant properties, the role of carotenoids in human health has acquired importance in the recent years, mainly due to the attention toward the utilization of compounds obtained from natural sources.

Microalgae and cyanobacteria are photoautotrophic organisms that are exposed to high oxygen and radical stress in their natural environment, and consequently have developed several efficient protective systems against reactive oxygen species and free radicals [1]. They represent an almost untapped resource of natural antioxidants due to their enormous biodiversity, and the value of microalgae as a source of natural antioxidants is further enhanced by the relative ease of purification of target compounds [2].

Microalgae are capable, under stress conditions, of producing significant amounts of substances with high added value (antioxidant carotenoids, phenolic compounds, and polyunsaturated fatty acids), and for this reason, the study of the physiology of the growth of these microorganisms is of particular interest. In particular, carotenoids act by counteracting the effects of the damage caused by an excess of light and protecting the cells from oxidative damage.

Carotenoids are divided into two groups named primary and secondary carotenoids.

The primary carotenoids, such as the xanthophylls and β-carotene, are found in the chloroplast under standard conditions and are directly involved in performing photosynthesis for their role in the absorption of light energy. However, under stress conditions such as high light and nutrient deficiency, the provided energy may not be sustainable, and the content in primary carotenoids may increase, to dissipate the excess energy. Moreover, some photosynthetic microorganisms accumulate large amounts of secondary carotenoids in the cells, as a mechanism of photoprotection, in response to physiological stresses that induce the increase of reduction level inside the cells.

In particular, under high light stress conditions, the dissipation of the excess absorbed light energy occurs via the nonphotochemical quenching (NPQ) of chlorophyll fluorescence, a harmless nonradiative pathway of dissipation of energy. This defensive strategy involves the synthesis of antioxidant carotenoids, such as the secondary carotenoid astaxanthin, the pigment lutein, and the xanthophyll cycle pigments: violaxanthin, antheraxanhitn, and zeaxanthin [3–7]. Among the xanthophylls, also loroxanthin and fucoxanthin, mainly produced by marine strains such as *Phaeodactylum* and *Isochrysis*, have been found to be strong antioxidants.

Diatoms, such as *Phaeodactylum*, have a specific set of pigments with chlorophyll *c*, and they have an additive xanthophyll cycle, consisting in diadinoxanthin (Ddx), which can be deepoxidized to diatoxanthin (Ddx). These reactions lead to reduction of the singlet oxygen inside the cell, avoiding cellular damage. Among carotenoids, the ketocarotenoid astaxanthin has been shown to have a strong efficacy in quenching singlet oxygen.

Comparing the antioxidant activity of astaxanthin, β-carotene and the xanthophylls zeaxanthin and lutein with the one of alpha-tocopherol, a well-known noncarotenoid antioxidant, it is has been shown that these carotenoids are among the most powerful antioxidants [8].

Considering the role of carotenoids as quenchers of active oxygen species, they represent a very interesting natural source of antioxidant and antiaging substances.

Among photosynthetic microorganisms, the green unicellular microalga *Haematococcus pluvialis* is capable of producing a large amount of astaxanthin, a red pigment that starts to accumulate in the central part of the cell until the cell becomes entirely red. The other unicellular green microalga *Dunaliella salina* is well known for β-carotene production. In this microalga, the strong orange pigment is synthesized at one side of the cell, where it starts to accumulate in lipidic bodies, and then it continues to accumulate in the rest of the cell. Another big producer of antioxidant carotenoids is *Scenedesmus*, a colonial microalga able to produce large amounts of lutein, which makes the cells change their color from green to yellow.

Many studies on the physiology of microalgae have been carried out on the unicellular green alga *Chlamydomonas reinhardtii*. This microalga is considered a good model organism as it can be easily manipulated by means of genetic engineering; it has been the source of much information on photosynthetic responses to stress. Concerning the synthesis of carotenoids, particularly interesting were the studies on the xanthophyll cycle induction.

### **2. Physiology of the growth of microalgae**

passive photoprotectors (i.e., as a filter), reducing the amount of light that can reach the light-

For their antioxidant properties, the role of carotenoids in human health has acquired importance in the recent years, mainly due to the attention toward the utilization of compounds

Microalgae and cyanobacteria are photoautotrophic organisms that are exposed to high oxygen and radical stress in their natural environment, and consequently have developed several efficient protective systems against reactive oxygen species and free radicals [1]. They represent an almost untapped resource of natural antioxidants due to their enormous biodiversity, and the value of microalgae as a source of natural antioxidants is further enhanced by the

Microalgae are capable, under stress conditions, of producing significant amounts of substances with high added value (antioxidant carotenoids, phenolic compounds, and polyunsaturated fatty acids), and for this reason, the study of the physiology of the growth of these microorganisms is of particular interest. In particular, carotenoids act by counteracting the effects of the

The primary carotenoids, such as the xanthophylls and β-carotene, are found in the chloroplast under standard conditions and are directly involved in performing photosynthesis for their role in the absorption of light energy. However, under stress conditions such as high light and nutrient deficiency, the provided energy may not be sustainable, and the content in primary carotenoids may increase, to dissipate the excess energy. Moreover, some photosynthetic microorganisms accumulate large amounts of secondary carotenoids in the cells, as a mechanism of photoprotection, in response to physiological stresses that induce the increase

In particular, under high light stress conditions, the dissipation of the excess absorbed light energy occurs via the nonphotochemical quenching (NPQ) of chlorophyll fluorescence, a harmless nonradiative pathway of dissipation of energy. This defensive strategy involves the synthesis of antioxidant carotenoids, such as the secondary carotenoid astaxanthin, the pigment lutein, and the xanthophyll cycle pigments: violaxanthin, antheraxanhitn, and zeaxanthin [3–7]. Among the xanthophylls, also loroxanthin and fucoxanthin, mainly produced by marine strains such as *Phaeodactylum* and *Isochrysis*, have been found to be strong antioxidants. Diatoms, such as *Phaeodactylum*, have a specific set of pigments with chlorophyll *c*, and they have an additive xanthophyll cycle, consisting in diadinoxanthin (Ddx), which can be deepoxidized to diatoxanthin (Ddx). These reactions lead to reduction of the singlet oxygen inside the cell, avoiding cellular damage. Among carotenoids, the ketocarotenoid astaxanthin has

Comparing the antioxidant activity of astaxanthin, β-carotene and the xanthophylls zeaxanthin and lutein with the one of alpha-tocopherol, a well-known noncarotenoid antioxidant, it is has been shown that these carotenoids are among the most powerful antioxidants [8].

been shown to have a strong efficacy in quenching singlet oxygen.

damage caused by an excess of light and protecting the cells from oxidative damage. Carotenoids are divided into two groups named primary and secondary carotenoids.

harvesting pigment complexes of photosystem II (PSII).

relative ease of purification of target compounds [2].

obtained from natural sources.

144 Carotenoids

of reduction level inside the cells.

Photosynthetic microorganisms present a great variety of shape and size. Microalgae and cyanobacteria are distributed in a wide spectrum of habitat, having adapted their metabolism to complex and extreme environmental conditions (high salinity, extreme temperature, nutrient deficiency, and UV-radiation). To survive under such different harsh conditions, they have developed several strategies.

Each strain has its own optimal growth conditions, in regards to temperature, pH, salinity, light intensity, nutrient composition of the medium. Among these, one especially important parameter for photosynthetic microorganisms is light intensity.

The photosynthetic efficiency, i.e., transformation of light energy into chemical energy, is first and foremost limited by the fact that photosynthetic cells can only use light in the wavelength range from 400 to 700 nm so that only about 55% of incident solar light is useful to perform photosynthesis.

Moreover, it has to be considered that part of photosynthetic active radiation, about 10%, is reflected by the surface of the cells in the cultures; also, self-shading between cells further reduces the light utilization of each cell. Considering all these limitations, the percentage of light that can be used for photosynthesis is about 41%.

It is also important to consider some physiological limits of the photosynthetic apparatus, which makes it unable to utilize a light irradiation beyond a light intensity. Hence, about 20% of incident solar light is in excess, when it reaches the highest intensities in the central part of the day, and it is dissipated by heat and used to synthesize antioxidant pigments [9, 10].

In **Figure 1**, a typical light-curve response of *C. reinhardtii* is reported, comparing the electrons transport rate (ETR) of different strains with D1 protein mutation affecting photosynthetic performance with the wild type.

In this case, the photosynthetic activity is expressed as the capability to transfer electrons, but it could also be expressed as O2 evolution, or CO2 up-take. It is evident that different strains can have different behaviors at increasing light intensities, exhibiting different values of α, the slope of the first part of the curve, and different *I* k value, i.e., the saturation irradiance, given as an intercept between *α* and ETRmax. According to the light saturation value, the strains can react differently, having different sensitivity to high light stress, and accumulating different levels of photooxidative stress.

For this reason, imposing a light stress inducing the carotenoids synthesis, as well as other stress conditions, such as nutrient limitation-starvation and excessive low or high temperature, is a useful approach in order to accumulate antioxidant compounds, but it is not convenient in terms of culture productivity, as under these limiting conditions, the growth is strongly affected.

One of the main physiological parameters used to monitor stress is the measurement of the photosynthetic activity, by evaluating oxygen evolution and Chla fluorescence measurement. In the presence of stress, the photosynthetic activity usually decreases, and it can be a useful indication on the kind of stress occurring to the cells. In particular, when the photosynthetic apparatus is impaired, light cannot be used efficiently, an accumulation of electrons on the electrons transport chain occurs and cells need to dissipate this excess of energy.

**Figure 1.** Comparison of different light induction curves in *Chlamydomonas reinhardtii* wild type (WT) and D1 protein mutant strains (mutation affecting the photosynthetic activity) Mut1, Mut2, and Mut3.

In response to this overreductive cellular environment condition, microalgae are able to produce a great variety of secondary metabolites, with antioxidant properties, which are biologically active and which cannot be found in other organisms [11, 12].

In **Figure 1**, a typical light-curve response of *C. reinhardtii* is reported, comparing the electrons transport rate (ETR) of different strains with D1 protein mutation affecting photosynthetic

In this case, the photosynthetic activity is expressed as the capability to transfer electrons, but

can have different behaviors at increasing light intensities, exhibiting different values of α, the

as an intercept between *α* and ETRmax. According to the light saturation value, the strains can react differently, having different sensitivity to high light stress, and accumulating different

For this reason, imposing a light stress inducing the carotenoids synthesis, as well as other stress conditions, such as nutrient limitation-starvation and excessive low or high temperature, is a useful approach in order to accumulate antioxidant compounds, but it is not convenient in terms of culture productivity, as under these limiting conditions, the growth is

One of the main physiological parameters used to monitor stress is the measurement of the photosynthetic activity, by evaluating oxygen evolution and Chla fluorescence measurement. In the presence of stress, the photosynthetic activity usually decreases, and it can be a useful indication on the kind of stress occurring to the cells. In particular, when the photosynthetic apparatus is impaired, light cannot be used efficiently, an accumulation of electrons on the electrons transport chain occurs and cells need to dissipate this excess of

**Figure 1.** Comparison of different light induction curves in *Chlamydomonas reinhardtii* wild type (WT) and D1 protein

mutant strains (mutation affecting the photosynthetic activity) Mut1, Mut2, and Mut3.

k

up-take. It is evident that different strains

value, i.e., the saturation irradiance, given

evolution, or CO2

performance with the wild type.

146 Carotenoids

it could also be expressed as O2

levels of photooxidative stress.

strongly affected.

energy.

slope of the first part of the curve, and different *I*

Among them, antioxidant compounds are the one to have attracted major interest for health and pharmaceutical industry, for their strong efficiency in preventing or delaying the damages caused by free radicals. Several synthetic antioxidants such as butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT), α-tocopherol, and propyl gallate have been used for limiting the oxidative damage, but they are strongly suspected to be responsible for a variety of side effects, such as liver damage and carcinogenesis. For this reason, a strong interest has been focused on finding natural products acting as antioxidants, safe, and effective.

#### **3. Carotenoids: function and distribution in photosynthetic cells**

The main functions of carotenoids consist in light absorption, to perform photosynthesis, and photoprotection to preserve the photosynthetic apparatus from photodamage. A role for carotenoids in cell differentiation, cell cycle regulation, growth factors regulation, stimulation of immune systems, intracellular signaling, and modulation of different kinds of receptors has been suggested [13].

However, for their antioxidant properties, they act as quenchers of active oxygen species and physiological stress, such as high light exposure, nutrient limitation or starvation, UV exposure, temperature fluctuation, anaerobiosis, and induce the metabolic pathways for the synthesis of these compounds.

These molecules are constituted by a C40 hydrocarbon backbone liable to structural modifications. According to their structure, carotenoids may be distributed in different ways into the cell compartments. In particular, they can be found within the inner section of the lipid bilayer of cell membranes, only if they are strict hydrocarbons like β-carotene or lycopene, or they can protrude into an aqueous environment from the membrane surface with a hydrophilic portion if they contain oxygen atoms, which confer them a more polar structure [14, 15]. Xanthophylls, such as lutein, fucoxanthin, neoxanthin, and xanthophyll cycle pigments, are among these more hydrophilic carotenoids. The presence of such carotenoids into the membranes may influence the thickness, fluidity, or permeability of them so that they can influence the stability of the cell membrane conferring it resistance, for instance, to ROS.

#### **4. Photosynthetic and metabolic processes involved in the photoprotective responses in microalgae**

Damage occurs when the free radical encounters another molecule and seeks to find another electron to pair with. The unpaired electron of a free radical pulls an electron off of a neighboring molecule, causing the affected molecule to behave like a free radical itself.

A range of biochemical and biophysical techniques had provided a good understanding of the events that occur during absorption of the light energy, triggering the primary and secondary electron transfer processes leading to water oxidation. These electron transport pathways involve the redox state of the component of the electron transport chain, the plastoquinone (PQ) pool, which has been widely investigated, for its implication in the regulation of photosynthetic processes.

Under oxidative stress conditions, there is an accumulation of reducing power inside the cells, which increases the reduction of PQ-pool. For this reason, the redox level of PQ-pool play a crucial role in the induction of physiological responses to stress, and it is important also for the synthesis of carotenoids.

It has been shown that there is an involvement of the redox state of PQ pool in the distribution of light energy during photosystem II (PS II) and photosystem I (PS I), i.e., state transitions. State 2 transition is promoted by the reduction of the PQ-pool and consists in the transfer of the light harvesting complex associated with PSII (LHCII) to the PSI, whereas under State 1 transition, which occurs when the PQ-pool is oxidized, the LHCII is associated with the PSII [16, 17]

The degree of reduction of PQ pool is related to a switch between linear and cyclic electron flow. With an over-reduced PQ pool (State 2), the PSI cross section increases and a cyclic electron transport is promoted, by contrast under oxidative conditions (State 1), the cross section of PSII is decreased and linear electron transport can be observed [18–20]. This is one of the strategies that photosynthetic cells employ to reduce the impact of strong light intensity on the photosynthetic apparatus, and it is triggered by the PQ-pool overreduction, and it is commonly associated with induction of carotenoid synthesis. Indeed, under these conditions, the acidification of the thylakoid lumen occurs, and this can activate some enzymes involved in the carotenogenesis. For instance, the deepoxidation of violaxanthin to zeaxanthin, via antheraxanthin, is promoted by low pH in the thylakoid lumen [5, 21, 22].

The synthesis of these carotenoids is important for the cells not only because the deepoxidation is a quenching reaction but also because xanthophylls have the ability to donate electrons [23] and act as inhibitors of the process of oxidation even at relatively small concentrations. Antioxidants also act as radical scavengers and convert radicals to less reactive species.

#### **5. Stress-inducing the highest synthesis of antioxidant compounds**

Which are the main kinds of stress to induce the carotenoids synthesis?

All those kinds of stress reducing growth and photosynthetic efficiency so that the excess of energy not used for growth (i.e., converted into biomass) is accumulated as reducing power and generates free radicals. Some of the well-known microalgae high producers of carotenoids are reported in **Table 1**. For each microalga the main stress factor inducing the carotenoids synthesis is reported with the respective antioxidant pigment. The detailed explanation is reported below in the text.


**Table 1.** Microalgae high producers of antioxidant carotenoids and stress conditions inducing their synthesis.

#### **5.1. Light intensity**

A range of biochemical and biophysical techniques had provided a good understanding of the events that occur during absorption of the light energy, triggering the primary and secondary electron transfer processes leading to water oxidation. These electron transport pathways involve the redox state of the component of the electron transport chain, the plastoquinone (PQ) pool, which has been widely investigated, for its implication in the regulation

Under oxidative stress conditions, there is an accumulation of reducing power inside the cells, which increases the reduction of PQ-pool. For this reason, the redox level of PQ-pool play a crucial role in the induction of physiological responses to stress, and it is important also for

It has been shown that there is an involvement of the redox state of PQ pool in the distribution of light energy during photosystem II (PS II) and photosystem I (PS I), i.e., state transitions. State 2 transition is promoted by the reduction of the PQ-pool and consists in the transfer of the light harvesting complex associated with PSII (LHCII) to the PSI, whereas under State 1 transition, which occurs when the PQ-pool is oxidized, the LHCII is associated with the PSII

The degree of reduction of PQ pool is related to a switch between linear and cyclic electron flow. With an over-reduced PQ pool (State 2), the PSI cross section increases and a cyclic electron transport is promoted, by contrast under oxidative conditions (State 1), the cross section of PSII is decreased and linear electron transport can be observed [18–20]. This is one of the strategies that photosynthetic cells employ to reduce the impact of strong light intensity on the photosynthetic apparatus, and it is triggered by the PQ-pool overreduction, and it is commonly associated with induction of carotenoid synthesis. Indeed, under these conditions, the acidification of the thylakoid lumen occurs, and this can activate some enzymes involved in the carotenogenesis. For instance, the deepoxidation of violaxanthin to zeaxanthin, via anthe-

The synthesis of these carotenoids is important for the cells not only because the deepoxidation is a quenching reaction but also because xanthophylls have the ability to donate electrons [23] and act as inhibitors of the process of oxidation even at relatively small concentrations. Antioxidants also act as radical scavengers and convert radicals to less reactive species.

All those kinds of stress reducing growth and photosynthetic efficiency so that the excess of energy not used for growth (i.e., converted into biomass) is accumulated as reducing power and generates free radicals. Some of the well-known microalgae high producers of carotenoids are reported in **Table 1**. For each microalga the main stress factor inducing the carotenoids synthesis is reported with the respective antioxidant pigment. The detailed expla-

**5. Stress-inducing the highest synthesis of antioxidant compounds**

raxanthin, is promoted by low pH in the thylakoid lumen [5, 21, 22].

Which are the main kinds of stress to induce the carotenoids synthesis?

nation is reported below in the text.

of photosynthetic processes.

the synthesis of carotenoids.

[16, 17]

148 Carotenoids

In particular, the exposure to high light is one of the typical stresses that microalgae may experience under environmental conditions. Indeed, during the central part of the day, the light irradiance may reach and exceed 1800 μmol photons m−2 s−1.

A schematic explanation of the mechanism is reported in **Figure 2**.

Due to this accumulation of excess energy, leading to ROS formation, the synthesis of antioxidant carotenoids is induced in order to protect the cells from photodamage. Depending

**Figure 2.** Schematic explanation of induction of photoprotection by induction of carotenoids synthesis by high light stress.

on the kind of light and on the strain, the mechanism of induction may follow different metabolic pathways. For instance, in case of sudden exposure to high light intensity, the cells may react with the induction of the xanthophyll cycle, which is known to occur very quickly, within 15–30 min [24]. This phenomenon has been widely reported in the microalga *C. reinhardtii*, which is considered a model organism for physiological and biochemical study on photosynthesis, because it can be easily manipulated for genetic study, and it can grow very easily both under photoheterotrophic and autotrophic conditions [25]. For this microalga, the induction of zeaxanthin synthesis has been detected within 10 minutes of exposure to 800 μmol photons m−2 s−1, but a partial induction of violaxanthin de-epoxidation to antheraxanthin and then this one to zeaxanthin could be observed already at 300–350 μmol photons m−2 s−1 [26].

The induction of the xanthophyll cycle may affect also the synthesis of diatoxanthin by the de-epoxidation of diadinoxanthin, which represents an additional xanthophyll cycle in diatoms and dinoflagellates, such as *Phaeodactylum* and *Isochrysis*, respectively, among the main producers of this carotenoid. In *Phaeodactylum tricornutum,* a rapid diadinoxanthin to diatoxanthin conversion has been reported, within 15 min, during exposure to sunlight in outdoor cultures in tubular photobioreactors, with the highest diatoxanthin concentration reached in the central part of the day (highest light intensity) [27]. In addition, these microalgae are well known for the synthesis of fucoxanthin and important antioxidant carotenoid. Fucoxanthin is mainly naturally found in marine microalgae, associated with thylakoid membranes, and it works by transferring excitation energy to chlorophyll *a*, driving electrons to the electrons transport chain [28, 29]. Fucoxanthin is usually found to be 0.22–1.82% in the biomass of these microalgae, but it can reach much higher concentrations in *Isochrysis* cultured at proper light intensity, cell density, and mixing. In particular, it has been observed that in this microalga, the effect of self-shading and low light intensity induced an increase in total carotenoid concentration, probably due to the increase of photosystem number under low light, and consequently of the primary carotenoids.

Among the strongest antioxidant carotenoids, the pigment lutein can be overexpressed during high light exposure. It is a very interesting pigment, as it is constitutively present in most of photosynthetic cells, and its synthesis may increase under photooxidative stress. The microalga *Scenedesmus* produced high amounts of lutein (over 5 mg m−2 d−1) in a tubular photobioreactor outdoor, under 1900 μmol photons m−2 s−1 and at 35°C [30]. In this case, the combined effect of high light and high temperature induced the increase of lutein. Indeed, usually, the optimal temperature of growth for microalgal strains is around 25–28°C.

Another carotenoid that usually increases during high light exposure is β-carotene. It is a pigment constitutively present in the microalgal cells, which may be oversynthesized under high light. One of the well-known microalgae for production of β-carotene is *D. salina* [31]. In laboratory conditions, it reached a production of 13.5 mg L−1 d−1 at light intensity in a range of 200–1200 μmol photons m−2 s−1, at 30°C [32].

One of the most important secondary carotenoids produced by microalgae is the red pigment astaxanthin. It is a very powerful antioxidant primarily synthesized by *H. pluvialis*, mainly under high light. However, although its synthesis is not so rapid, as it takes 1 day of sunlight exposure to observe changes in the cells color, from green to red, it can reach a very high content, reaching 5% of the biomass. *H. pluvialis* has been widely studied for its astaxanthin production, due to its high productivity of this carotenoid, and for its robustness. Indeed, most of the studies carried out with *H. pluvialis* have been performed in outdoor cultures, using sunlight to induce astaxanthin production. These studies demonstrated that under environmental conditions, mainly in the summer period, and in very high illuminated areas, this microalga can grow and produce astaxanthin [33, 34].

#### **5.2. Nutrient limitation**

on the kind of light and on the strain, the mechanism of induction may follow different metabolic pathways. For instance, in case of sudden exposure to high light intensity, the cells may react with the induction of the xanthophyll cycle, which is known to occur very quickly, within 15–30 min [24]. This phenomenon has been widely reported in the microalga *C. reinhardtii*, which is considered a model organism for physiological and biochemical study on photosynthesis, because it can be easily manipulated for genetic study, and it can grow very easily both under photoheterotrophic and autotrophic conditions [25]. For this microalga, the induction of zeaxanthin synthesis has been detected within 10 minutes of exposure to 800 μmol photons m−2 s−1, but a partial induction of violaxanthin de-epoxidation to antheraxanthin and then this one to zeaxanthin could be observed already at 300–350 μmol photons

The induction of the xanthophyll cycle may affect also the synthesis of diatoxanthin by the de-epoxidation of diadinoxanthin, which represents an additional xanthophyll cycle in diatoms and dinoflagellates, such as *Phaeodactylum* and *Isochrysis*, respectively, among the main producers of this carotenoid. In *Phaeodactylum tricornutum,* a rapid diadinoxanthin to diatoxanthin conversion has been reported, within 15 min, during exposure to sunlight in outdoor cultures in tubular photobioreactors, with the highest diatoxanthin concentration reached in the central part of the day (highest light intensity) [27]. In addition, these microalgae are well known for the synthesis of fucoxanthin and important antioxidant carotenoid. Fucoxanthin is mainly naturally found in marine microalgae, associated with thylakoid membranes, and it works by transferring excitation energy to chlorophyll *a*, driving electrons to the electrons transport chain [28, 29]. Fucoxanthin is usually found to be 0.22–1.82% in the biomass of these microalgae, but it can reach much higher concentrations in *Isochrysis* cultured at proper light intensity, cell density, and mixing. In particular, it has been observed that in this microalga, the effect of self-shading and low light intensity induced an increase in total carotenoid concentration, probably due to the increase of photosystem number under low light, and conse-

Among the strongest antioxidant carotenoids, the pigment lutein can be overexpressed during high light exposure. It is a very interesting pigment, as it is constitutively present in most of photosynthetic cells, and its synthesis may increase under photooxidative stress. The microalga *Scenedesmus* produced high amounts of lutein (over 5 mg m−2 d−1) in a tubular photobioreactor outdoor, under 1900 μmol photons m−2 s−1 and at 35°C [30]. In this case, the combined effect of high light and high temperature induced the increase of lutein. Indeed, usually, the optimal

Another carotenoid that usually increases during high light exposure is β-carotene. It is a pigment constitutively present in the microalgal cells, which may be oversynthesized under high light. One of the well-known microalgae for production of β-carotene is *D. salina* [31]. In laboratory conditions, it reached a production of 13.5 mg L−1 d−1 at light intensity in a range of

One of the most important secondary carotenoids produced by microalgae is the red pigment astaxanthin. It is a very powerful antioxidant primarily synthesized by *H. pluvialis*, mainly under high light. However, although its synthesis is not so rapid, as it takes 1 day of sunlight

m−2 s−1 [26].

150 Carotenoids

quently of the primary carotenoids.

200–1200 μmol photons m−2 s−1, at 30°C [32].

temperature of growth for microalgal strains is around 25–28°C.

Nutrient limitation is another important stress condition inducing carotenoids synthesis and it is, like high light irradiance, a situation which can occur under environmental conditions. Macronutrient limitation, or starvation, is more incisive on the induction of protective responses than micronutrient limitation, as it directly affects growth, leading, mainly combined with light exposure, to the increase of reducing power, which is well known to activate defensive strategies such as the induction of the synthesis of certain carotenoids.

Nitrogen limitation is among the most studied nutrient-deprivation stress, as it is one of the most important elements in the cell, for its presence in proteins, enzymes, and because it is directly involved in the growth.

As previously reported in *Dunaliella* for β-carotene under high light stress, carotenoid increases in this microalga and this also occurs under nitrogen starvation. In particular, very interestingly, it has been shown that the increase in β-carotene content is concomitant with the synthesis of total fatty acid occurring under high light exposure and in combination with nitrogen starvation [35]. This can be explained by the fact that β-carotene is accumulated in lipid globules, in the cells, and it is supported by the findings that both lipid globules and β-carotene cannot be found when inhibitors of the fatty acid biosynthetic pathway are present [35]. At light intensity of 200 μmol photons m−2 s−1 under nitrogen starvation, a concentration of β-carotene of 2.7% of the biomass can be reached in *D. salina* [36].

A connection between lipid and carotenoid synthesis has been studied in *H. pluvialis*. In particular, the highest carotenoids accumulation has been observed with high light and nitrogen starvation combined, and under these conditions the astaxanthin content resulted more than two times higher than the control [37].

Under nitrogen starvation, astaxanthin synthesis is higher than in the control culture. Transition from the green stage to the red stage occurs during astaxanthin synthesis, due to the cytoplasmatic accumulation of the red pigment, which is observed within 20 h, reaching 1.4% of dry weight in the starved culture.

#### **5.3. Overreduction of PQ-pool: anaerobiosis**

Anaerobiosis is a condition that occurs when microalgal cells are cultivated in closed photobioreactors, in growth conditions that limit the photosynthetic activity; the oxygen evolution rate decreases reaching a value equal or lower than the oxygen respiration rate. Under light exposure, the electrons are driven by light, from water to the electrons transport chain, but if the photosynthetic apparatus is affected, it is not able to use the accumulated electrons, overreducing the cellular environment. Moreover, under anaerobic conditions, the respiration cannot eliminate these reducing electrons, for lack of oxygen that is the final electron acceptor, and therefore, the reduction level of PQ-pool cannot be dissipated.

It has been demonstrated that anaerobiosis has a strongly negative impact on the performance of photosynthetic cells, but on the other hand, it can be a useful means to activate certain metabolic processes sensitive to oxygen, for example, hydrogen production, in some microalgal strains like *Chlamydomonas reinhartdii* [38]. In this microalga, chlorophyll fluorescence and oxygen evolution measurements indicated a strong reduction of photosynthetic activity under sulphur starvation, which leads to the formation of a strongly reductive environment inside the cell compartments. This stress activates an antioxidative response promoting the synthesis of lutein and zeaxanthin [39]. Imposing anaerobic conditions to *C. reinhardtii* in complete medium, it was possible to observe a strong promotion of the xanthophyll cycle; however, under these conditions, the time of induction was not shorter than 5 h, contrary to the short time of induction at high light intensity. After this period, the zeaxanthin content was 12.63 mmol mol−1 Chl*a*. After 24 h it further increased, reaching 29.51 mmol mol−1 Chl*a*. Anaerobiosis induced the overexpression of all the xanthophyll pool, which increased by 15%, indicating a *de novo* synthesis of these xanthophylls, in particular violaxanthin, showing that this type of stress is not able to induce a rapid zeaxanthin synthesis but is strong enough to promote mechanisms of photoprotection on a longer time scale, with accumulation of large amounts of xanthophylls. In addition, increases in lutein content, which more than doubled, and of β-carotene, which increased by 90%, were observed. This strategy was able to preserve cells from photodamage. A very interesting aspect of the microalgal metabolism of carotenoids is that pigment composition may be adjusted by the cells according to the environmental conditions, and that some synthetic pathways can be very fast, in order to optimize the cellular performance and to save energy and storage [40]. In *C. reinahrdtii* cultures where the xanthophyll cycle had been induced, it has been shown that, after 1 h of aerobic dark adaptation, the pigments antheraxanthin and zeaxanthin decreased, as also did lutein and β-carotene, indicating the occurrence of a recovery. These findings underlined the very interesting peculiarity of microalgae, which consists in the strong capability to adapt to strong changes, in a different manner, according to the order of stress.

#### **6. Importance of natural antioxidant compounds from microalgae and application in human health of antioxidants produced by microalgae**

There is an increasing interest in the use of natural compounds in preventing and treating several diseases in humans, animals, and plants. For this reason, the research of a natural source of novel compounds with biological activity, in particular new and safe antioxidants, has gained a lot of importance.

Microalgae and cyanobacteria, under stress conditions, are capable of producing significant amounts of substances with high added value (antioxidant carotenoids, phenolic compounds, and polyunsaturated fatty acids), and for this reason, the study of the physiology of the growth of these microorganisms is of particular interest.

The secondary metabolites produced by photosynthetic organisms find numerous applications in the pharmaceutical, cosmetic, and food industries. In particular, the secondary carotenoids are widely used as antioxidants, acting as targets for highly reactive and toxic oxygen species, counteracting the effect of free radicals, and being effective as antiaging and anticancer agents.

Well known is the implication of carotenoids lutein and zeaxanthin in the pathologies of visual function, and the role of β-carotene in protecting the skin during exposure to the sun, and in the treatment of skin diseases.

It is well known that both lutein and zeaxanthin possess antioxidant properties due to their ability to quench singlet oxygen, reactive oxygen species, and free radicals [26, 41]. In particular, studies reported that an important role is played by lutein and zeaxanthin, constituents of the macular pigment, in the prevention of free radicals formation in the human retina, acting as quenchers [42–44]. This protective role against age-related macular degeneration makes these retinal carotenoids suitable for application as dietary supplements [45].

The antioxidant defense systems are important in maintaining good health, and therefore, an antioxidant-rich diet or antioxidant complements may be necessary as a health-protecting factor.

Interest in the employment of antioxidants from natural sources to increase the shelf life of food is considerably enhanced by the consumers' preference for natural ingredients and concerns about the toxic effects of synthetic antioxidants. Dietary antioxidants include three major groups: vitamins (vitamin C or ascorbic acid and vitamin E or tocopherols), phenols, and carotenoids, which are precursors of some vitamins.

#### **7. Conclusions**

exposure, the electrons are driven by light, from water to the electrons transport chain, but if the photosynthetic apparatus is affected, it is not able to use the accumulated electrons, overreducing the cellular environment. Moreover, under anaerobic conditions, the respiration cannot eliminate these reducing electrons, for lack of oxygen that is the final electron

It has been demonstrated that anaerobiosis has a strongly negative impact on the performance of photosynthetic cells, but on the other hand, it can be a useful means to activate certain metabolic processes sensitive to oxygen, for example, hydrogen production, in some microalgal strains like *Chlamydomonas reinhartdii* [38]. In this microalga, chlorophyll fluorescence and oxygen evolution measurements indicated a strong reduction of photosynthetic activity under sulphur starvation, which leads to the formation of a strongly reductive environment inside the cell compartments. This stress activates an antioxidative response promoting the synthesis of lutein and zeaxanthin [39]. Imposing anaerobic conditions to *C. reinhardtii* in complete medium, it was possible to observe a strong promotion of the xanthophyll cycle; however, under these conditions, the time of induction was not shorter than 5 h, contrary to the short time of induction at high light intensity. After this period, the zeaxanthin content was 12.63 mmol mol−1 Chl*a*. After 24 h it further increased, reaching 29.51 mmol mol−1 Chl*a*. Anaerobiosis induced the overexpression of all the xanthophyll pool, which increased by 15%, indicating a *de novo* synthesis of these xanthophylls, in particular violaxanthin, showing that this type of stress is not able to induce a rapid zeaxanthin synthesis but is strong enough to promote mechanisms of photoprotection on a longer time scale, with accumulation of large amounts of xanthophylls. In addition, increases in lutein content, which more than doubled, and of β-carotene, which increased by 90%, were observed. This strategy was able to preserve cells from photodamage. A very interesting aspect of the microalgal metabolism of carotenoids is that pigment composition may be adjusted by the cells according to the environmental conditions, and that some synthetic pathways can be very fast, in order to optimize the cellular performance and to save energy and storage [40]. In *C. reinahrdtii* cultures where the xanthophyll cycle had been induced, it has been shown that, after 1 h of aerobic dark adaptation, the pigments antheraxanthin and zeaxanthin decreased, as also did lutein and β-carotene, indicating the occurrence of a recovery. These findings underlined the very interesting peculiarity of microalgae, which consists in the strong capability to adapt to strong changes, in a different manner, according

**6. Importance of natural antioxidant compounds from microalgae and application in human health of antioxidants produced by microalgae**

There is an increasing interest in the use of natural compounds in preventing and treating several diseases in humans, animals, and plants. For this reason, the research of a natural source of novel compounds with biological activity, in particular new and safe antioxidants,

acceptor, and therefore, the reduction level of PQ-pool cannot be dissipated.

to the order of stress.

152 Carotenoids

has gained a lot of importance.

Very interestingly, there is an interconversion among carotenoids, as some of them are precursor of others, and their metabolic pathways are often correlated. For example, in one case, the β-carotene can be the precursor of the xanthophyll violaxanthin. Particularly, under a strong oxidative stress, the induction of the xanthophyll cycle, with the deepoxidation of violaxanthin to zeaxanthin, via antheraxanthin, is concomitant to the decrease of β-carotene that contributes to the de novo synthesis of violaxanthin. This phenomenon has been reported in *C. reinhardtii*.

Moreover, zeaxanthin is reconverted to antheraxanthin and violaxanthin by the enzyme epoxidase. The plasticity of the carotenoid metabolism and the strong induction of their synthesis achievable in microalgae make this argument very interesting in terms of biotechnological applications.

#### **Acknowledgements**

This work was supported by Regione Toscana, Italy, in the framework of project PRAF and Officina Profumo Farmaceutica Santa Maria Novella, Florence, Italy, in the framework of a Contract Project.

#### **Author details**

Cecilia Faraloni\* and Giuseppe Torzillo

\*Address all correspondence to: faraloni@ise.cnr.it

Institute of Ecosystem Study, National Research Council, Italy

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156 Carotenoids


**Carotenoid Production by** *Corynebacterium***: The Workhorse of Industrial Amino Acid Production as Host for Production of a Broad Spectrum of C40 and C50 Carotenoids**

Nadja A. Henke, Petra Peters-Wendisch and Volker F. Wendisch

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67631

#### **Abstract**

*Corynebacterium glutamicum* is used as a workhorse of industrial biotechnology for more than 60 years since its discovery as a natural glutamate producer in the 1950s. Nowadays, L-glutamate and L-lysine are being produced with this GRAS organism in the million-ton scale every year for the food and feed markets, respectively. Sequencing of the genome and establishment of a genetic toolbox boosted metabolic engineering of this host for a broad range of industrially relevant compounds ranging from bulk chemicals to high-value products. Carotenoids, the colourful representatives of terpenoids, are high-value compounds whose bio-based production is on the rise. Since *C. glutamicum* is a natural producer of the rare C50 carotenoid decaprenoxanthin, this organism is well suited to establish terpenoidoverproducing platform strains with the help of metabolic engineering strategies. In this work, the carotenogenic background of *C. glutamicum* and the metabolic engineering strategies for the generation of carotenoid-overproducing strains are depicted.

**Keywords:** *Corynebacterium*, C40/C50 carotenoids, biotechnological production, metabolic engineering, decaprenoxanthin, β-carotene, astaxanthin

#### **1. Introduction**

Carotenoids are the dominant pigments for the colouration of food, feed and beverages. The annual demand of the feed additive astaxanthin, for example, is estimated to be 130 tons for

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

aquaculture and poultry breeding [1]. Besides their yellow-to-red colouring properties, this group of terpenoids is drawing attention in the healthcare industry due to their high antioxidant activities. Since the demand of naturally produced carotenoids is rising, and the fact that extraction of these high-value compounds from plant material is rather cost-inefficient, alternative and flexible production systems are favoured [2].

*Corynebacterium glutamicum* is a workhorse for the million-ton-scale production of L-glutamate and L-lysine. As a natural carotenoid producer, this biotechnological established microorganism is in focus as a suitable cell factory for natural carotenoid production. The biotechnological application of this genetically accessible bacterium has been exploited for the production of various natural and non-native products [3], including its potential to produce a range of industrially relevant carotenoids.

#### **2. Carotenoid production with** *Corynebacterium glutamicum*

#### **2.1.** *Corynebacterium glutamicum* **as an established cell factory**

For more than 60 years, *C. glutamicum* is used as a cell factory for the production of amino acids. Because of its GRAS status, this microbe has a long history in the food and feed industry in the million-ton scale [4]. Since its discovery as a natural glutamate producer in the 1950s [4], its production spectrum was broadened. Lysine is the second biggest production volume being generated by this microbe [5, 6]. Sequencing of its genome [7], the development of a well-filled genetic toolbox [8] and establishment of large-scale fermentations [9] boosted investigations of a wide set of industrially relevant compounds.

This cell factory can naturally utilise glucose, fructose, sucrose, mannitol, arabitol, propionate and acetate under aerobic conditions [10, 11]. Moreover, metabolic engineering enabled utilisation of alternative carbon and energy sources such as glycerol [12], amino sugars [13, 14], β-glucans [15], levoglucosan [16], pentoses [17] and starch [18]. Thus, for industrial fermentations usage of carbon sources which are available in high quantities at low prices is possible, while competition with food and feed resources can be avoided.

Although *C. glutamicum* was discovered as a natural glutamate producer, nowadays, several metabolically engineered production strains are available. Besides the proteinogenic amino acids, also non-proteinogenic amino acid like gamma-aminobutyrate (GABA) [19, 20] and citrulline [21], diamines [22, 23], alcohols [24, 25] and organic acids [26, 27] have been produced. Moreover also high-value compounds including the sesquiterpenoid valencene [28] and the C40 carotenoids β-carotene and astaxanthin [29, 30] can be synthesised with this microbe.

The broad product spectrum from bulk compounds, building blocks, food and feed additives and pharmaceutical and bioactive compounds indicates that *C. glutamicum* has developed to a chassis organism of metabolic engineering. Therefore, several efforts for systematic reduction and optimisation of the *C. glutamicum* genome have been made aiming on a reduced metabolic complexity and strength for future purposes of genetic engineering of new routes for new products [31, 32].

#### **2.2. Carotenogenesis in** *Corynebacterium glutamicum*

aquaculture and poultry breeding [1]. Besides their yellow-to-red colouring properties, this group of terpenoids is drawing attention in the healthcare industry due to their high antioxidant activities. Since the demand of naturally produced carotenoids is rising, and the fact that extraction of these high-value compounds from plant material is rather cost-inefficient,

*Corynebacterium glutamicum* is a workhorse for the million-ton-scale production of L-glutamate and L-lysine. As a natural carotenoid producer, this biotechnological established microorganism is in focus as a suitable cell factory for natural carotenoid production. The biotechnological application of this genetically accessible bacterium has been exploited for the production of various natural and non-native products [3], including its potential to produce a range of

For more than 60 years, *C. glutamicum* is used as a cell factory for the production of amino acids. Because of its GRAS status, this microbe has a long history in the food and feed industry in the million-ton scale [4]. Since its discovery as a natural glutamate producer in the 1950s [4], its production spectrum was broadened. Lysine is the second biggest production volume being generated by this microbe [5, 6]. Sequencing of its genome [7], the development of a well-filled genetic toolbox [8] and establishment of large-scale fermentations [9] boosted

This cell factory can naturally utilise glucose, fructose, sucrose, mannitol, arabitol, propionate and acetate under aerobic conditions [10, 11]. Moreover, metabolic engineering enabled utilisation of alternative carbon and energy sources such as glycerol [12], amino sugars [13, 14], β-glucans [15], levoglucosan [16], pentoses [17] and starch [18]. Thus, for industrial fermentations usage of carbon sources which are available in high quantities at low prices is possible,

Although *C. glutamicum* was discovered as a natural glutamate producer, nowadays, several metabolically engineered production strains are available. Besides the proteinogenic amino acids, also non-proteinogenic amino acid like gamma-aminobutyrate (GABA) [19, 20] and citrulline [21], diamines [22, 23], alcohols [24, 25] and organic acids [26, 27] have been produced. Moreover also high-value compounds including the sesquiterpenoid valencene [28] and the C40 carotenoids β-carotene and astaxanthin [29, 30] can be synthesised with this microbe.

The broad product spectrum from bulk compounds, building blocks, food and feed additives and pharmaceutical and bioactive compounds indicates that *C. glutamicum* has developed to a chassis organism of metabolic engineering. Therefore, several efforts for systematic reduction and optimisation of the *C. glutamicum* genome have been made aiming on a reduced metabolic complexity and strength for future purposes of genetic engineering of new routes

alternative and flexible production systems are favoured [2].

**2. Carotenoid production with** *Corynebacterium glutamicum*

**2.1.** *Corynebacterium glutamicum* **as an established cell factory**

investigations of a wide set of industrially relevant compounds.

while competition with food and feed resources can be avoided.

industrially relevant carotenoids.

160 Carotenoids

for new products [31, 32].

*C. glutamicum* is a yellow-pigmented soil bacterium due to the accumulation of a rare C50 carotenoid decaprenoxanthin and its glucosides [33]. Long-chain C50 carotenoids have been mainly isolated from extremely halophilic archaea [34, 35] and Gram-positive bacteria of the order *Actinomycetales* [36]. Among these species few other corynebacteria are known to produce carotenoid pigments such as *C. michiganense* [37], *C. erythrogenes* [38], *C. fascians* [39] and *C. poinsettia* [40]. Nevertheless, the respective pathways are yet poorly understood due to a lack of complete genome information. Carotenogenesis in *C. glutamicum* has been functionally characterized. The genome of *C. glutamicum* possesses two *crt* operons [7], whereas one of them encodes for all the necessary enzymes responsible for the conversion of the C5 precursor molecules dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) to the C50 carotenoid decaprenoxanthin [41].

#### *2.2.1. Organisation of carotenogenic genes within the chromosome*

The precursor molecules derive from the methylerythritol phosphate (MEP) pathway, whose respective genes are partially clustered within the chromosome (**Figure 1**) [30]. The genes *dxs* (cg2083), *ispH* (cg1164) and *idi* (cg2531) are monocistronic, while *dxr* (cg2208), *ispD* (cg2945), *ispE* (cg1039), *ispF* (cg2944) and *ispG* (cg2206) are organised in putative operons. *IspE* is the third gene of the operon cg1037-*ksgA*-*ispE*-cg1040-*pdxK* with genes for a putative resuscitationpromoting factor (cg1037), putative dimethyl adenosine transferase (KsgA) and putative pyridoxamine kinase (PdxK). The genes *ispD* and *ispF* are encoded in the cg2946-*ispDF* operon with cg2946, which codes for a putative CarD-like transcriptional regulator. The genes *dxr* and *ispG* are organised in a transcriptional unit separated by an uncharacterised gene (cg2207) putatively encoding a membrane-embedded Zn-dependent protease. In *C. glutamicum* two prenyltransferases have been characterised [42] which both yield GGPP. Interestingly, the major prenyltransferase IdsA is not encoded within the major *crt* operon [42] but annotated in an operon that is also containing α(1→6) mannopyranosyltransferase (**Figure 1**) [43]. CrtE is the second functional GGPP synthase, which is the first gene of the major *crt* operon (**Figure 1**). Both *crt* operons contain a *crtB* gene; however, the one from the small *crt* operon is weakly expressed [41]. Although both *crt* operons contain gene coding for a phytoene desaturase, only CrtI from the major operon is functional [41]. In addition, *crtEb*, *crtYe* and *crtYf* are part of the major *crt* operon which is necessary for decaprenoxanthin formation (**Figure 4**). Glucosyltransferase gene *crtX* is located in proximity of the major *crt* operon.

Although little is known about carotenogenesis in other corynebacteria, the genomic organisation of corresponding *crt* genes seems to be conserved [41].

#### *2.2.2. Biosynthesis of decaprenoxanthin*

The terpenoid precursor molecules DMAPP and IPP derive from the MEP pathway that uses pyruvate and GAP as substrates from central metabolism in *C. glutamicum*. Eight genes are encoding the enzymes which are necessary to build up the C5 precursors under consumption of NADPH, ATP and CTP (**Figure 2**). It has to be mentioned that DXP, the first intermediate

**Figure 1.** Genomic organisation of carotenogenic genes of *Corynebacterium glutamicum*. The MEP pathway genes involved in the synthesis of the isoprenoid precursors IPP and DMAPP from pyruvate and GAP are depicted in yellow; genes encoding for the decaprenoxanthin biosynthesis pathway from IPP and DMAPP are depicted in orange. Genes that are organised in operons with the respective MEP pathway or decaprenoxanthin pathway genes are shown in grey. Gene names, IDs and respective protein products: *crtEb* (cg0717), lycopene elongase; *crtYf* (cg0718) and *crtYe* (cg0719), hetero-dimeric C50 ε-cyclase; *crtI* (cg0720), phytoene desaturase; *crtB* (cg0721), phytoene synthase; *cmpL3* (cg0722) RND transporter, corynebacterial membrane protein [44]; *crtE* (cg0723), GGPP synthase; *crtR* (cg0725), transcriptional regulator of carotenoid biosynthesis; *crtX* (cg0730), carotenoid glycosyltransferase; *rfp2* (cg1037), resuscitation-promoting factor 2 [45]; *ksgA* (cg1038), putative 16S ribosomal RNA methyltransferase; *ispE* (cg1039), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase; *pdxK* (cg1041), putative pyridoxamine kinase; *xseB* (cg1162), putative exodeoxyribonuclease VII small subunit; *xseA* (cg1163), putative exodeoxyribonuclease VII large subunit; *ispH* (cg1164), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase; *rnd* (cg2081), putative ribonuclease D; *dxs* (cg2083), 1-deoxy-d-xylulose-5-phosphate synthase; *ispG* (cg2206), (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; *rseP* (cg2207), putative membrane-embedded Zn-dependent protease; *dxr* (cg2208), 1-deoxy-d-xylulose-5-phosphate reductoisomerase; *idsA* (cg2384), GGPP synthase; *mptA* (cg2385), α(1→6) mannopyranosyltransferase [43]; *treS* (cg2529), trehalose synthase [46]; *idi* (cg2531), isopentenyl-diphosphate δ-isomerase; *aecD* (cg2536), cystathionine β-lyase [47]; *pknL* (cg2388), serine/threonine protein kinase [48]; *ispF* (cg2944), 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; *ispD* (cg2945), 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase and *carD* (cg2946), putative CarDlike transcriptional regulator.

of the MEP pathway, is also a precursor molecule for thiamine biosynthesis in *C. glutamicum*. Two prenyltransferases IdsA and CrtE use one molecule DMAPP and three molecules IPP to form GGPP (C20) (**Figure 2**) [41]. Two GGPP molecules can be condensed by a phytoene synthase CrtB to phytoene, a colourless C40 structure (**Figure 2**). The red lycopene is formed via the phytoene desaturase CrtI [17].

Lycopene is a central metabolite for all C40 and C50 carotenoids (**Figures 3** and **4**). In *C. glutamicum* this C40 carotenoid is further elongated by the lycopene elongase CrtEb with two DMAPP molecules to form the linear C50 carotenoid flavuxanthin. Finally, a heterodimeric ε-cyclase CrtY<sup>e</sup> Yf introduces ε-cyclic moieties [49] in that linear C50 structure to form decaprenoxanthin (**Figure 4**). Decaprenoxanthin can be glycosylated either at one or at both terminal hydroxy groups by CrtX [50]. The physiological function remains unknown; however, glycosylation of end groups generally yields a more polar molecule structure in comparison to the free decaprenoxanthin and might change integrity into the membrane.

#### *2.2.3. The unusual cell envelope of C. glutamicum and carotenoid accumulation*

The cell envelope of the Gram-positive bacterium *C. glutamicum* possesses a special and complex cell wall structure [51, 52]. Besides the inner plasma membrane, the peptidoglycan and Carotenoid Production by *Corynebacterium*: The Workhorse of Industrial Amino Acid Production... http://dx.doi.org/10.5772/67631 163

of the MEP pathway, is also a precursor molecule for thiamine biosynthesis in *C. glutamicum*. Two prenyltransferases IdsA and CrtE use one molecule DMAPP and three molecules IPP to form GGPP (C20) (**Figure 2**) [41]. Two GGPP molecules can be condensed by a phytoene synthase CrtB to phytoene, a colourless C40 structure (**Figure 2**). The red lycopene is formed

**Figure 1.** Genomic organisation of carotenogenic genes of *Corynebacterium glutamicum*. The MEP pathway genes involved in the synthesis of the isoprenoid precursors IPP and DMAPP from pyruvate and GAP are depicted in yellow; genes encoding for the decaprenoxanthin biosynthesis pathway from IPP and DMAPP are depicted in orange. Genes that are organised in operons with the respective MEP pathway or decaprenoxanthin pathway genes are shown in

(cg0719), hetero-dimeric C50 ε-cyclase; *crtI* (cg0720), phytoene desaturase; *crtB* (cg0721), phytoene synthase; *cmpL3* (cg0722) RND transporter, corynebacterial membrane protein [44]; *crtE* (cg0723), GGPP synthase; *crtR* (cg0725), transcriptional regulator of carotenoid biosynthesis; *crtX* (cg0730), carotenoid glycosyltransferase; *rfp2* (cg1037), resuscitation-promoting factor 2 [45]; *ksgA* (cg1038), putative 16S ribosomal RNA methyltransferase; *ispE* (cg1039), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase; *pdxK* (cg1041), putative pyridoxamine kinase; *xseB* (cg1162), putative exodeoxyribonuclease VII small subunit; *xseA* (cg1163), putative exodeoxyribonuclease VII large subunit; *ispH* (cg1164), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase; *rnd* (cg2081), putative ribonuclease D; *dxs* (cg2083), 1-deoxy-d-xylulose-5-phosphate synthase; *ispG* (cg2206), (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; *rseP* (cg2207), putative membrane-embedded Zn-dependent protease; *dxr* (cg2208), 1-deoxy-d-xylulose-5-phosphate reductoisomerase; *idsA* (cg2384), GGPP synthase; *mptA* (cg2385), α(1→6) mannopyranosyltransferase [43]; *treS* (cg2529), trehalose synthase [46]; *idi* (cg2531), isopentenyl-diphosphate δ-isomerase; *aecD* (cg2536), cystathionine β-lyase [47]; *pknL* (cg2388), serine/threonine protein kinase [48]; *ispF* (cg2944), 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; *ispD* (cg2945), 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase and *carD* (cg2946), putative CarD-

(cg0718) and *crtYe*

grey. Gene names, IDs and respective protein products: *crtEb* (cg0717), lycopene elongase; *crtYf*

Lycopene is a central metabolite for all C40 and C50 carotenoids (**Figures 3** and **4**). In *C. glutamicum* this C40 carotenoid is further elongated by the lycopene elongase CrtEb with two DMAPP molecules to form the linear C50 carotenoid flavuxanthin. Finally, a heterodimeric

noxanthin (**Figure 4**). Decaprenoxanthin can be glycosylated either at one or at both terminal hydroxy groups by CrtX [50]. The physiological function remains unknown; however, glycosylation of end groups generally yields a more polar molecule structure in comparison to the

The cell envelope of the Gram-positive bacterium *C. glutamicum* possesses a special and complex cell wall structure [51, 52]. Besides the inner plasma membrane, the peptidoglycan and

free decaprenoxanthin and might change integrity into the membrane.

*2.2.3. The unusual cell envelope of C. glutamicum and carotenoid accumulation*

introduces ε-cyclic moieties [49] in that linear C50 structure to form decapre-

via the phytoene desaturase CrtI [17].

Yf

like transcriptional regulator.

162 Carotenoids

ε-cyclase CrtY<sup>e</sup>

**Figure 2.** Metabolic pathway of lycopene production in *Corynebacterium glutamicum* starting from GAP and pyruvate. Genes are shown next to the reaction catalysed by the encoded enzyme (see **Figure 1**; *crtE*/*idsA*: prenyltransferase; *crtB*: phytoene synthase and *crtI*: phytoene desaturase).

**Figure 3.** Metabolically engineered platform strains for production of cyclic C40 carotenoids. The biosynthesis of C40 carotenoids from the precursor molecules dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) is depicted. Genes are shown next to the reaction catalysed by the encoded enzyme (*crtE*: GGPP synthase; *crtB*: phytoene synthase; *crtI*: phytoene desaturase; *crtEb*: lycopene elongase; *crtYe/f*: C45/50 carotenoid ε-cyclase; *crtY*: lycopene cyclase; *crtZ*: β-carotene hydroxylase and *crtW*: β-carotene ketolase). Endogenous genes are highlighted in grey boxes and overexpressions indicated by green arrows. Heterologous genes are highlighted in coloured boxes.

**Figure 4.** Biosynthesis of C50 carotenoids starting from the precursor lycopene. The native pathway of decaprenoxanthin biosynthesis (a) is shown with black arrows. Established non-native biosynthesis pathways of sarcinaxanthin (b) and C.p. 450 (c) are indicated by grey arrows. Possible biosynthesis of bacterioruberin (d) from C.p. 496 (BABR) is indicated by dashed grey arrows.

arabinogalactan layer forms a polysaccharide barrier that is esterified to mycolic acids. The presence of the mycolic acid layer is a phylogenetic trait of *Corynebacterianeae* [52]. The plasma membrane consists of polar lipids, mainly phospholipids which form together with proteins the typical lipid bilayer.

Carotenoids are usually attached to or span membranes due to their lipophilic character and rigid structure. Association of carotenoids to membranes often results in a decreased water permeability and increased firmness, thus supporting membrane stability [53, 54]. It is hypothesized that this is closely linked to their function supporting resistances to osmotic stresses, heat or radiation [54–56]. Moreover, it was shown that incorporation of carotenoids into a membrane is more efficient when the carbon backbone length of the carotenoid correlates with the thickness of the phospholipid bilayer [57]. Although decaprenoxanthin is a C50 carotenoid, it is assumed to be integrated into the plasma membrane [58, 59] as it was also shown for most C40 carotenoids of other bacteria [53, 56, 60].

#### **2.3. Metabolic engineering of** *Corynebacterium glutamicum* **for carotenoid production**

Since *C. glutamicum* naturally produces a rare cyclic C50 carotenoid, its potential to produce industrially relevant C40 carotenoids was elucidated over the last years in more detail. First, it was shown that production of both non-native C50 and C40 carotenoids was possible with this production host [50, 61]. Secondly, improvement of the MEP pathway yielded enhanced production [30]. Finally, the production of industrially relevant cyclic C40 carotenoids was shown on the basis of balancing of the enzyme quantities and on the basis of a screen for suitable enzymes for enhanced production [29].

#### *2.3.1. Design of a platform strain for the production of the central intermediate lycopene*

For production of non-native C40 and C50 carotenoids, endogenous decaprenoxanthin production has to be avoided (**Figure 3**). For this reason, a prophage-cured *C. glutamicum* strain MB001 [62] was metabolically engineered by deleting the genes *crtEb* and *crtYe Yf* , resulting in the biosynthesis of the central intermediate lycopene [41, 50]. The supply of the precursor molecule DMAPP and its isomer IPP was successfully engineered through improved expression of *dxs*, encoding for the first committed step in the MEP pathway, on the basis of a chromosomal promoter exchange [30]. Furthermore, it was shown that overproduction of the prenyltransferase (CrtE), phytoene synthase (CrtB) and phytoene desaturase (CrtI) strongly enhanced lycopene production [50]. Chromosomal integration of the artificial operon *crtEBI* under the control of a strong constitutive *tuf* promoter in a Δ*crtEbYe Yf* strain yielded a red phenotype [29].

#### *2.3.2. Metabolic engineering for cyclic C40 carotenoid productions*

**Figure 3.** Metabolically engineered platform strains for production of cyclic C40 carotenoids. The biosynthesis of C40 carotenoids from the precursor molecules dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) is depicted. Genes are shown next to the reaction catalysed by the encoded enzyme (*crtE*: GGPP synthase; *crtB*: phytoene synthase; *crtI*: phytoene desaturase; *crtEb*: lycopene elongase; *crtYe/f*: C45/50 carotenoid ε-cyclase; *crtY*: lycopene cyclase; *crtZ*: β-carotene hydroxylase and *crtW*: β-carotene ketolase). Endogenous genes are highlighted in grey boxes and

**Figure 4.** Biosynthesis of C50 carotenoids starting from the precursor lycopene. The native pathway of decaprenoxanthin biosynthesis (a) is shown with black arrows. Established non-native biosynthesis pathways of sarcinaxanthin (b) and C.p. 450 (c) are indicated by grey arrows. Possible biosynthesis of bacterioruberin (d) from C.p. 496 (BABR) is indicated

by dashed grey arrows.

164 Carotenoids

overexpressions indicated by green arrows. Heterologous genes are highlighted in coloured boxes.

On the basis of lycopene-producing platform strains, a collection of cyclic C40 carotenoids could be generated via different metabolic engineering strategies. First of all, β-cyclisation of lycopene was accomplished via heterologous overexpression of *crtY* from *Pantoea ananatis* [29, 30]. Here, either plasmid-driven or genome-based expressions revealed complete conversion of lycopene to β-carotene (**Figure 3**). This might rely on the fact that β-carotene has a more polar structure due to its β-ionone rings and thus integrates more efficiently in the phospholipid bilayer than lycopene. Heterologous expression of β-carotene hydroxylase from *P. ananatis* in such a β-carotene–accumulating strain yielded production of zeaxanthin [30]. Additional plasmiddriven overproduction of a β-carotene ketolase from *Brevundimonas aurantiaca* showed accumulation of the red C40 carotenoid astaxanthin for the first time in *C. glutamicum* [30]. Analysis of the carotenoid profile showed that conversion of β-carotene to its oxygenated derivatives was not efficient maybe due to an unbalanced expression of involved genes.

Pathway balancing was performed through balancing of the enzyme levels of β-carotene ketolase and hydroxylase (**Figure 3**). In a combinatorial approach, enzyme quantities were varied on the basis of varied translation initiation rates [29]. The corresponding genes were assembled in an expression vector under the strong constitutive *tuf* promoter using different ribosome-binding sites, different spacing lengths and different start codons. The translation initiation rate is depending on the free binding energy of the ribosome-binding site and the 16S rRNA as well as on the free energy of secondary structures of the mRNA [29]. Secondary structures between 5′UTR and coding region are depending on the expressed gene and were calculated with the RBS calculator [63]. Analysis revealed that the higher the TIRs of both genes, the higher the astaxanthin titer [29] in recombinant *C. glutamicum*. With this approach an astaxanthin content of 0.3 mg/g CDW was accomplished indicating that aside pathway balancing also enzyme activities or stabilities could be limiting the production.

The analysis of carotenogenesis identified a transcriptional regulator CrtR [64]. This MarR regulator is binding to the promoter sequence of *crtE* and thus inhibits transcription of the carotenogenic genes. Deletion of the corresponding gene located divergent to the major *crt* operon enhanced both native and non-native carotenoid productions. The production titers of non-native C50 carotenoids sarcinaxanthin and C.p. 450 improved 1.5 and 2-fold, respectively. For the deregulated β-carotene–accumulating strain production, titer doubled to approximately 12 mg/g CDW [29]. This platform strain was used for a second approach to produce astaxanthin with recombinant *C. glutamicum.* In the deregulated β-carotene–producing platform strain, a screening of potential β-carotene ketolases and hydroxylases of rather uncharacterised gene donors for production of canthaxanthin and zeaxanthin was done. Two enzymes from *Fulvimarina pelagi* showed the best conversion of β-carotene to the oxygenated derivatives [29]. Moreover, a combination of those two genes with a two-vector system resulted in a *C. glutamicum* strain producing 1.6 mg/g CDW of astaxanthin [29], which is the highest reported titer obtained with this organism. Since *C. glutamicum* is fast growing even in shake-flask experiments, the volumetric productivity of 0.4 mg L−1 h−1 is competitive to algal and yeast-based productions.

#### *2.3.3. Heterologous gene expression for production of C50 carotenoids*

On the basis of lycopene-accumulating platform strains, production of a range of C50 carotenoids was established (**Figure 4**). The cyclic C50 carotenoids sarcinaxanthin and C.p. 450 can be derived from lycopene via the heterologous expression of corresponding lycopene elongase and linear C50 carotenoid cyclases. For sarcinaxanthin production genes from the *Micrococcus luteus* [36] were cloned and expressed from a plasmid. The lycopene elongase CrtE2 from this organism adds two DMAPP molecules at C2 and C2′ positions which is identical with the catalytic activity of *C. glutamicum* endogenous lycopene elongase. Simultaneously hydroxylation at the C1/C1′ positions is postulated to stabilise the prenylated carbocation. The linear C50 carotenoid flavuxanthin is cyclised to sarcinaxanthin by a heterodimeric γ-cyclase CrtY<sup>g</sup> Yh [36]. This cyclase acts exclusively on a linear C50 backbone and not one lycopene as it holds also true for the ε-cyclase from *C. glutamicum* [36, 49]. Production of C.p. 450 was entailed through plasmid-driven heterologous expression of genes from *Dietzia* sp. CQ4 [65]. Here, elongation of lycopene by LbtBC results in a slightly different linear C50 carotenoid, C.p. 496. Cyclisation by the heterodimeric β-cyclase LbtAB yields C.p. 450. Moreover, starting from C.p. 496, also heterologous synthesis of bacterioruberin could be possible by expression of *cruF*, e.g. from *Haloarcula japonica* [66]; however, this route is not yet established.

than lycopene. Heterologous expression of β-carotene hydroxylase from *P. ananatis* in such a β-carotene–accumulating strain yielded production of zeaxanthin [30]. Additional plasmiddriven overproduction of a β-carotene ketolase from *Brevundimonas aurantiaca* showed accumulation of the red C40 carotenoid astaxanthin for the first time in *C. glutamicum* [30]. Analysis of the carotenoid profile showed that conversion of β-carotene to its oxygenated derivatives was

Pathway balancing was performed through balancing of the enzyme levels of β-carotene ketolase and hydroxylase (**Figure 3**). In a combinatorial approach, enzyme quantities were varied on the basis of varied translation initiation rates [29]. The corresponding genes were assembled in an expression vector under the strong constitutive *tuf* promoter using different ribosome-binding sites, different spacing lengths and different start codons. The translation initiation rate is depending on the free binding energy of the ribosome-binding site and the 16S rRNA as well as on the free energy of secondary structures of the mRNA [29]. Secondary structures between 5′UTR and coding region are depending on the expressed gene and were calculated with the RBS calculator [63]. Analysis revealed that the higher the TIRs of both genes, the higher the astaxanthin titer [29] in recombinant *C. glutamicum*. With this approach an astaxanthin content of 0.3 mg/g CDW was accomplished indicating that aside pathway

The analysis of carotenogenesis identified a transcriptional regulator CrtR [64]. This MarR regulator is binding to the promoter sequence of *crtE* and thus inhibits transcription of the carotenogenic genes. Deletion of the corresponding gene located divergent to the major *crt* operon enhanced both native and non-native carotenoid productions. The production titers of non-native C50 carotenoids sarcinaxanthin and C.p. 450 improved 1.5 and 2-fold, respectively. For the deregulated β-carotene–accumulating strain production, titer doubled to approximately 12 mg/g CDW [29]. This platform strain was used for a second approach to produce astaxanthin with recombinant *C. glutamicum.* In the deregulated β-carotene–producing platform strain, a screening of potential β-carotene ketolases and hydroxylases of rather uncharacterised gene donors for production of canthaxanthin and zeaxanthin was done. Two enzymes from *Fulvimarina pelagi* showed the best conversion of β-carotene to the oxygenated derivatives [29]. Moreover, a combination of those two genes with a two-vector system resulted in a *C. glutamicum* strain producing 1.6 mg/g CDW of astaxanthin [29], which is the highest reported titer obtained with this organism. Since *C. glutamicum* is fast growing even in shake-flask experiments, the volumetric productivity of 0.4 mg L−1 h−1 is competitive to algal

On the basis of lycopene-accumulating platform strains, production of a range of C50 carotenoids was established (**Figure 4**). The cyclic C50 carotenoids sarcinaxanthin and C.p. 450 can be derived from lycopene via the heterologous expression of corresponding lycopene elongase and linear C50 carotenoid cyclases. For sarcinaxanthin production genes from the *Micrococcus luteus* [36] were cloned and expressed from a plasmid. The lycopene elongase CrtE2 from this organism adds two DMAPP molecules at C2 and C2′ positions which is identical with the catalytic activity of *C. glutamicum* endogenous lycopene elongase. Simultaneously hydroxylation

not efficient maybe due to an unbalanced expression of involved genes.

balancing also enzyme activities or stabilities could be limiting the production.

and yeast-based productions.

166 Carotenoids

*2.3.3. Heterologous gene expression for production of C50 carotenoids*

#### **2.4. Future prospects: advancing rational strain engineering for short- and long-chain terpenoid production**

Based on the findings of the research on carotenoid biosynthesis in *C. glutamicum*, new strategies for rational strain engineering on terpenoid productions are now available. First, deletion of endogenous carotenogenic genes will yield accumulation of the terpenoid precursor molecules DMAPP and IPP in a prophage-cured *C. glutamicum* MB001. Secondly, the application of carbon-chain length-specific prenyltransferases will either yield GPP, FPP or GGPP, allowing their conversion to mono-, di-, sesqui- or polyterpenoids. Besides the C40 and C50 carotenoids, it was also shown that *C. glutamicum* is a suitable host for production of short-chain terpenoids like valencene [28]. Valencene is a sesquiterpene which is derived from FPP. It was shown that heterologous expression of *ispA* from *Escherichia coli* yielded the precursor FPP instead of GGPP as synthesised by native prenyltransferases IdsA and CrtE. Therefore, engineering combinations of native, mutated or heterologous prenyltransferases with terpene synthases from other bacteria or plants are the keys to broaden the terpenoid product spectrum of metabolically engineered *C. glutamicum*.

Since many short-chain terpenoids exhibit antimicrobial properties, timed induction of terpene synthase gene expression is a strategy to face this challenge. An optogenetic approach using photolabile caged IPTG as inducer was successfully applied to allow (i) altered expression levels and (ii) non-invasive timed induction of heterologous genes in a valenceneproducing *C. glutamicum* strain [67]. Moreover, recent findings have proven that general transcription machinery engineering (gTME) is an efficient approach to improve carotenoid production in *C. glutamicum* [68]. In this study, carotenoid production was improved in the stationary growth phase either by overexpression of primary sigma factor gene *sigA* or by deletion of alternative sigma factor gene *sigB* [68]. Biosensors have been used in *C. glutamicum* for efficient screening of mutant libraries to find novel targets for metabolic engineering and for positive or negative on-demand control of metabolic pathways or enzymes [64]. An application of a biosensor was already successfully implemented for screening l-lysine-producing *C. glutamicum* strains [69]. Basically, a transcriptional fusion of a regulated promoter and a reporter gene in the presence of the corresponding transcriptional regulator (LysG) enabled intracellular metabolite (lysine) sensing and isolation of lysine-accumulating mutants. On the other hand, intracellular riboswitch-based l-lysine biosensors have been developed to induce the gene of the lysine export system or to repress the citrate synthase gene resulting in increased lysine production [70, 71]. Recently, the CRISPRi/dCas9 system has been established for efficient downregulation of target genes in *C. glutamicum* as exemplified for lysine and glutamate production targets [72]. In summary, a foundation has been laid for employing *C. glutamicum* as a natural terpenoid producer and the recent progress in method and tool development for rational engineering of this biotechnological workhorse foreshadow exciting options for terpenoid production processes using this bacterium.

#### **Author details**

Nadja A. Henke, Petra Peters-Wendisch and Volker F. Wendisch\*

\*Address all correspondence to: volker.wendisch@uni-bielefeld.de

Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Germany

#### **References**


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lished for efficient downregulation of target genes in *C. glutamicum* as exemplified for lysine and glutamate production targets [72]. In summary, a foundation has been laid for employing *C. glutamicum* as a natural terpenoid producer and the recent progress in method and tool development for rational engineering of this biotechnological workhorse foreshadow exciting

Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Germany

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## **Carotenoids in Yellow Sweet Potatoes, Pumpkins and Yellow Sweet Cassava**

Lucia Maria Jaeger de Carvalho, Gisela Maria Dellamora Ortiz, José Luiz Viana de Carvalho, Lara Smirdele and Flavio de Souza Neves Cardoso

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67717

#### **Abstract**

Carotenoids are the most widespread pigments in nature, extremely important for human health, but are highly unstable molecules especially when exposed to light, oxygen and heat. Many authors report the carotenoid's importance, mainly its pro‐vita‐ min A (α‐ and β‐carotene) and, additionally, the antioxidant capacity of some of them. Currently, more than 600 carotenoids are known and characterized by their chemical structures. In vegetables, common pro‐vitamin A carotenoids include β‐carotene and its 9, 13 and 15 isomers, α‐carotene and β‐cryptoxanthin. Other common carotenoids such as lycopene, lutein and zeaxanthin do not have pro‐vitamin A activity but serve as natural antioxidants. They are found in many fruits and vegetables such as carrots, yellow sweet potatoes, yellow sweet cassava and pumpkins. Normally, in these plant materials, the β‐carotene is the most abundant. It is still used as natural food coloring, which is not very expensive, since enough 3–5 g of β‐carotene is used to impart a yellow color characteristic of a ton of margarine. There is also a description of its importance in the formation of compounds responsible for flavors that are of interest fragrance and food industries. The purpose of this chapter is to report the presence of pro‐vitamin A carotenoids, mainly the β‐carotene in pumpkins, yellow sweet potato and yellow sweet and bitter cassava.

**Keywords:** carotenoids, cassava, yellow sweet potato, pumpkin, β‐carotene

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1. Introduction**

Carotenoids are the most widespread pigments in nature, extremely important for human health, but are highly unstable molecules especially when exposed to light, oxygen and heat. Many authors report the carotenoid's importance, mainly its pro‐vitamin A (α‐ and β‐caro‐ tene) and, additionally, the antioxidant capacity of some of them. Currently, more than 600 carotenoids are known and characterized by their chemical structures [1–4]. In vegetables and fruits, common pro‐vitamin A carotenoids include β‐carotene, α‐carotene and β‐cryptoxan‐ thin [2, 5]. Other carotenoids such as lycopene are rich in tomatoes. Lutein and zeaxanthin had no pro‐vitamin A activity but act as natural antioxidants and are the yellow pigments of the human retinal macula [6–9] and are believed to be responsible for the ophthalmological protective effect of carotenoids, acting as both antioxidants and high‐energy blue light filters. In Ref. [10], like spinach, sour cream, corn and egg, lutein and zeaxanthin are the yellow pig‐ ments of the human retinal macula [7–9] and are believed to be responsible for the ophthal‐ mological protective effect of carotenoids, acting as both antioxidants and high‐energy blue light filters [10, 11]. Carotenoids are found in many fruits and vegetables, such as carrots, yellow sweet potatoes, yellow sweet cassava and pumpkins, among other plant materials. The purpose of this chapter is to make an overview on some aspects of the carotenoids such as sources of pro‐vitamin A, functional properties and activities in yellow sweet potatoes, yellow sweet and bitter cassavas and pumpkins.

#### **2. Carotenoids**

Nowadays, there is a new approach on the prevention of some pro‐vtamin A diseases caused by provitamin A deficiencies, at low costs, mainly the β‐carotene. The groups affected by nighblindness, corneal scarring, blindness, measles and, increased mortality in infants (0–12 months), an children (1–6 years) [3] as well as the pregnant and lactating women (blind‐ ness) in the underdeveloped and in developed countries or regions around the world.

Years ago, carotenoids like β‐carotene were and still is used as natural food colorings, since enough 3–5 g of β‐carotene is used to impart a yellow color characteristic in margarines. There is also a description of its importance in the formation of compounds responsible for flavors that are of interest in fragrance and food industries. Besides the food industry, there is the incipient interest of the pharmaceutical industry for its nutritional and functional properties, such as vitamin A precursors and the antioxidant activity [1, 4]. The isomerization is one of the best known chemical properties of carotenoids. Some *cis* (Z) forms can be naturally occur‐ ring, as in the case of the first carotenoid biosynthetic, phytoene and phytofluene route, there are 15 predominantly in the *cis (Z)* configuration. Few *cis* forms were found naturally because the *cis* double bond of the presence creates steric hindrance between neighbor groups, mak‐ ing it less stable molecule. Thus, most carotenoids exist in nature in the *trans* configuration, which is more thermodynamically stable [12]. In case of carotenoids, the *cis (Z)* and *trans (E)* designations are determined by the arrangement of substituents of the C=C double bond. Thus, if the substituents are on the same side of the axis, C=C double bond is called *cis*, and if the substituents are on opposite sides of the axis, C=C double bond is called *trans* [12]. The β‐carotene naturally occurs in all *trans* (*E*) forms, which is thermodynamically more stable and less soluble as said previously. However, the occurrence of *cis* isomer has been reported, frequently, estimated on the theory, that there are 272 possible isomers of β‐carotene, and only 12 were already detected. Among them, easily formed are the *cis* 9, *cis* 13 and *cis*‐15. The carotenoids from plant materials contribute with approximately 68% of vitamin A diet glob‐ ally, and 82% in developed countries. One benefit of pro‐vitamin A carotenoids is that they are only converted into vitamin A when the body needs, therefore avoiding its accumulation. On the other hand, several factors influence on its absorption and utilization, such as the type and physical form in the diet, fat intake, vitamin E, fiber contents and the existence of certain diseases and parasitic infections [13]. There are over 600 known carotenoids, some of these compounds are pro‐vitamin A and other has little or no vitamin A activity [14].

**1. Introduction**

176 Carotenoids

sweet and bitter cassavas and pumpkins.

**2. Carotenoids**

Carotenoids are the most widespread pigments in nature, extremely important for human health, but are highly unstable molecules especially when exposed to light, oxygen and heat. Many authors report the carotenoid's importance, mainly its pro‐vitamin A (α‐ and β‐caro‐ tene) and, additionally, the antioxidant capacity of some of them. Currently, more than 600 carotenoids are known and characterized by their chemical structures [1–4]. In vegetables and fruits, common pro‐vitamin A carotenoids include β‐carotene, α‐carotene and β‐cryptoxan‐ thin [2, 5]. Other carotenoids such as lycopene are rich in tomatoes. Lutein and zeaxanthin had no pro‐vitamin A activity but act as natural antioxidants and are the yellow pigments of the human retinal macula [6–9] and are believed to be responsible for the ophthalmological protective effect of carotenoids, acting as both antioxidants and high‐energy blue light filters. In Ref. [10], like spinach, sour cream, corn and egg, lutein and zeaxanthin are the yellow pig‐ ments of the human retinal macula [7–9] and are believed to be responsible for the ophthal‐ mological protective effect of carotenoids, acting as both antioxidants and high‐energy blue light filters [10, 11]. Carotenoids are found in many fruits and vegetables, such as carrots, yellow sweet potatoes, yellow sweet cassava and pumpkins, among other plant materials. The purpose of this chapter is to make an overview on some aspects of the carotenoids such as sources of pro‐vitamin A, functional properties and activities in yellow sweet potatoes, yellow

Nowadays, there is a new approach on the prevention of some pro‐vtamin A diseases caused by provitamin A deficiencies, at low costs, mainly the β‐carotene. The groups affected by nighblindness, corneal scarring, blindness, measles and, increased mortality in infants (0–12 months), an children (1–6 years) [3] as well as the pregnant and lactating women (blind‐ ness) in the underdeveloped and in developed countries or regions around the world.

Years ago, carotenoids like β‐carotene were and still is used as natural food colorings, since enough 3–5 g of β‐carotene is used to impart a yellow color characteristic in margarines. There is also a description of its importance in the formation of compounds responsible for flavors that are of interest in fragrance and food industries. Besides the food industry, there is the incipient interest of the pharmaceutical industry for its nutritional and functional properties, such as vitamin A precursors and the antioxidant activity [1, 4]. The isomerization is one of the best known chemical properties of carotenoids. Some *cis* (Z) forms can be naturally occur‐ ring, as in the case of the first carotenoid biosynthetic, phytoene and phytofluene route, there are 15 predominantly in the *cis (Z)* configuration. Few *cis* forms were found naturally because the *cis* double bond of the presence creates steric hindrance between neighbor groups, mak‐ ing it less stable molecule. Thus, most carotenoids exist in nature in the *trans* configuration, which is more thermodynamically stable [12]. In case of carotenoids, the *cis (Z)* and *trans (E)* designations are determined by the arrangement of substituents of the C=C double bond. Thus, if the substituents are on the same side of the axis, C=C double bond is called *cis*, and Carotenoids from vegetables account for 80–85% of dietary vitamin A supply, and their role as a source of pro‐vitamin A has attracted great interest due also to the antioxidant potential effect [15].

The biofortification can be an approach to minimize the pro‐vitamin A deficiencies and defined as the enrichment of staple crops with essential micronutrients. At present, it is one of the strategies used to alleviate vitamin A deficiency (VAD) by breeding staple crops with β‐carotene. Staple crops that have been successfully biofortified with β‐carotene under the Harvest*Plus* program are cassava, maize (corn) and sweet potato [16].

Recently, β‐apocarotenoids (cleavage products of β‐carotene formed by chemical and enzy‐ matic oxidations) were identified and quantified in cantaloupe melons and orange‐fleshed honeydew but not in biofortified foods. Biofortified cassava was evaluated; however, there are no detailed analyses of these compounds in biofortified foods and little is known about their bioavailability and intestinal absorption and kinetics of cell uptake and metabolism of β‐apocarotenoids. The β‐apocarotenoids in roots of non‐biofortified cassava varieties were lower than those of biofortified and were hypothesized that these compounds are directly absorbed from the diet similarly to β‐carotene (Caco2 cells) [17]. Two unidentified metabolites (X and Y) of β‐apo‐8′‐carotenal. The cellular uptake of β‐apo‐13‐carotenone was rapid, and this compound was extensively degraded over the time. Understanding the mechanisms of absorption and metabolism of β‐apocarotenoids relative to their quantities in foods is critical in exploring the functions of these metabolites, some of which have been shown to be potent antagonists of vitamin A.

Hess,Thurnham and Hurrel (2005) [18] reported some studies about the influence of provita‐ min A carotenoids on status of iron, zinc and vitamin A, considering the effect of β‐carotene on vitamin A, requirements by consumption of plant foods, link between vitamin A defi‐ ciency and, iron, the possible interactions between vitamin A and, iron metabolism, the link between vitamin A and zinc and, some interventions studies as well the knowledge gap and suggestions for future research. The bioavailability of these micronutrients and their deficien‐ cies in the developed countries in infants, children, and pregnant and lactating women were studied. Another function of some carotenoids like the β‐carotene is the protective ability of these pigments that act as antioxidants, acting in preventing peroxidation. Antioxidants are classified into two categories: chain‐breaking antioxidants that interfere with the propagation step and preventive antioxidants that interfere with the process initiation step [1]. At high concentrations of O<sup>2</sup> , there is a reduction in the antioxidant activity of β‐carotene observed in studies conducted in pulmonary tissues. Since peripheral tissues, the efficiency of carotenoids can be greater because the oxygen pressure is lower [19]. The *Z* isomers of pro‐vitamin A have long been known as fitted with vitamin A activity lower than the *E* isomer (*trans*) match‐ ing [20]. Furthermore, (*all‐E*)‐β‐carotene was absorbed preferentially to (9‐*Z)*‐β‐carotene in humans [21–23]. The analyses for quantification of total carotenoids are very well described in Ref. [2], using UV/vis spectrophotometry at 450 nm, acetone for extraction and the high‐per‐ formance liquid chromatography (HPLC) using petroleum ether for their identification. On the other hand, in pro‐vitamin A carotenoids, the factors that determine a good antioxidant capacity are as follows: the presence of an electron donor substituent or the hydrogen to radi‐ cal, depending on their reduction potential; the radical shift capacity formed in its structure and the ability to chelate transition metals involved in oxidative and access to the site of action process, depending on the hydrophilicity or lipophilicity and its partition coefficient [24]. The chemical characteristics of the antioxidants include solubility, regenerative ability, the struc‐ ture/activity and bioavailability, which are important factors when considering the role of these compounds in human health [25]. In Ref. [26], it was reported that interactions between structurally different compounds and that have variable antioxidant activity promote addi‐ tional protection against oxidative stress. The antioxidants maybe classified as natural or syn‐ thetic. The second antioxidants are widely used in industry, being the most used butylated hydroxyanisole (BHA), butyl hydroxytoluene (BHT), tertiary butyl hydroxyquinone (TBHQ) and propyl gallate (PG). Your choice and concentration vary depending on the food to be used [27–29]. However, due to their potential risks to human health (carcinogenc effects), has increased the interest in research of natural antioxidants [30] present in raw plant materials, processed or not, such as fruit and vegetables, such as tocopherols, ascorbic acid, carotenoids and phenolic compounds [29]. In determination of antioxidant activity of food, in addition to informing its antioxidant potential before ingestion, it is important to assess food's protec‐ tion against oxidation and deterioration reactions that can lead to decreased quality and its nutritional value [31]. Different methods of measurement of capacity / antioxidant activity of substances and foods such as DPPH (α, α‐diphenyl‐β‐picrylhydrazyl (DPPH) free radical scavenging, ORAC (Oxygen Radical Absorbance Capacity) and ABTS (2,2'‐azino‐bis(3‐eth‐ ylbenzothiazoline‐6‐sulphonic acid). These are shown necessary because of the difficulty of measuring each compound separately and the interactions between the different antioxidants in the system [31–33].

#### **2.1. Sweet and bitter yellow cassava**

Cassava (*Manihot esculenta*, Crantz) belongs to the *Euphorbiaceae* family, originated from South America where it was cultivated by the Indians who were responsible for its dissemination in almost all over America. In African, Latin American and Asian continents, it is still one of the main caloric foods to nearly 500 million people, mainly in underdeveloped countries [34, 35]. The variability in total carotenoids and β‐carotene and isomers, in 12 varieties of raw yellow bitter cassava roots as well as the degradation in five varieties after the flour process to observe the heat treatment effect on carotenoids degradation, revealed that total carotenoids varied according to the variety, with the β‐carotene as the most abundant. At the same time, some varieties presented expressive contents of 13 and 9‐*cis*‐β‐carotene isomers. The total degradation of total carotenoids in the flour, after 19th day of storage, showed the necessity of optimizing the drying process to minimize this loss in order to minimize the deficiency in the Brazilian low‐income populations [36]. Studies were conducted in seven raw and cooked roots of yellow sweet cassava to identify total carotenoids, α and β‐carotene and its isomers in new varieties that could contribute in the nutritional quality improvement in the populations with malnutrition problems situated in the tropics and, particularly, in the Brazilian Northeast, where the cassava is almost the one of the main cultivations and sometimes the only nutrient source. The *trans*‐β‐ carotene was predominant; however, isomers 13 and 9‐*cis* were found in significant quantities compared to the total carotenoids content. However, there was no cooking style that stood out regarding carotenoids retention. Total carotenoids varied in raw roots from 14.15 to 2.64 μg g⁻<sup>1</sup> , and total β‐carotene from 10.32 to 1.99 μg g⁻<sup>1</sup> . The highest content was the all‐*E*‐β‐carotene (4.55 μg g⁻<sup>1</sup> ). The highest retention % of total carotenoids was found in two varieties (99.49%) and, in total β‐carotene (94.31%) were both after cooking. Carotenoids' variability presented the individual potential of the varieties, in the retention prevailed the heat effect in each cook‐ ing style applied. However, no cooking style provided a higher retention of total carotenoids or β‐carotene uniformly, in all the varieties, with behavior of each variety of sweet yellow cas‐ sava roots prevailing in the cooking style. This evaluation showed differences in behaviors that can be attributed to the total carotenoids that were initially found. Differences were found in the cooking styles among the cooking styles regarding total carotenoid and β‐carotene in real retention percentage, suggesting and this retention was high for β‐carotene [37]. Another aspect that needs to be point out is the storage of cassava roots after harvest for providing important and fundamental information to plant breeding programs aimed at improving cassava storage root nutritional quality. In Ref. [38], Among the 23 cassava landraces with different types of storage, root color and diverse carotenoid types and profiles, the landrace Cas51 (pink color) had low LYCb transcript abundance, whereas landrace Cas64 (intense yellow storage root) had decreased HYb transcript abundance. Lycopene and total β‐carotene increased in landraces Cas51 and Cas64, respectively [38]. Thirteen cassava accessions from Brazilian Northeast in two crops were evaluated by many characters. There were accessions identified with potential use as parents in plant breeding to increment of β‐carotene BGMC 1221, BGMC 1223 and BGMC 1224 and lycopene BGMC 1222 and BGMC 1223 contents in storage roots [39]. In Ref. [40], the study reported an allelic polymorphism that, in one of the two expressed phytoene synthase (PSY) genes, is capable of enhancing the flux of carbon through carotenogenesis, leading to the accumulation of colored pro‐vitamin A carotenoids in storage roots.

#### **2.2. The yellow‐flesh sweet potato (***Ipomoea batatas* **(L) Lam.)**

classified into two categories: chain‐breaking antioxidants that interfere with the propagation step and preventive antioxidants that interfere with the process initiation step [1]. At high

studies conducted in pulmonary tissues. Since peripheral tissues, the efficiency of carotenoids can be greater because the oxygen pressure is lower [19]. The *Z* isomers of pro‐vitamin A have long been known as fitted with vitamin A activity lower than the *E* isomer (*trans*) match‐ ing [20]. Furthermore, (*all‐E*)‐β‐carotene was absorbed preferentially to (9‐*Z)*‐β‐carotene in humans [21–23]. The analyses for quantification of total carotenoids are very well described in Ref. [2], using UV/vis spectrophotometry at 450 nm, acetone for extraction and the high‐per‐ formance liquid chromatography (HPLC) using petroleum ether for their identification. On the other hand, in pro‐vitamin A carotenoids, the factors that determine a good antioxidant capacity are as follows: the presence of an electron donor substituent or the hydrogen to radi‐ cal, depending on their reduction potential; the radical shift capacity formed in its structure and the ability to chelate transition metals involved in oxidative and access to the site of action process, depending on the hydrophilicity or lipophilicity and its partition coefficient [24]. The chemical characteristics of the antioxidants include solubility, regenerative ability, the struc‐ ture/activity and bioavailability, which are important factors when considering the role of these compounds in human health [25]. In Ref. [26], it was reported that interactions between structurally different compounds and that have variable antioxidant activity promote addi‐ tional protection against oxidative stress. The antioxidants maybe classified as natural or syn‐ thetic. The second antioxidants are widely used in industry, being the most used butylated hydroxyanisole (BHA), butyl hydroxytoluene (BHT), tertiary butyl hydroxyquinone (TBHQ) and propyl gallate (PG). Your choice and concentration vary depending on the food to be used [27–29]. However, due to their potential risks to human health (carcinogenc effects), has increased the interest in research of natural antioxidants [30] present in raw plant materials, processed or not, such as fruit and vegetables, such as tocopherols, ascorbic acid, carotenoids and phenolic compounds [29]. In determination of antioxidant activity of food, in addition to informing its antioxidant potential before ingestion, it is important to assess food's protec‐ tion against oxidation and deterioration reactions that can lead to decreased quality and its nutritional value [31]. Different methods of measurement of capacity / antioxidant activity of substances and foods such as DPPH (α, α‐diphenyl‐β‐picrylhydrazyl (DPPH) free radical scavenging, ORAC (Oxygen Radical Absorbance Capacity) and ABTS (2,2'‐azino‐bis(3‐eth‐ ylbenzothiazoline‐6‐sulphonic acid). These are shown necessary because of the difficulty of measuring each compound separately and the interactions between the different antioxidants

Cassava (*Manihot esculenta*, Crantz) belongs to the *Euphorbiaceae* family, originated from South America where it was cultivated by the Indians who were responsible for its dissemination in almost all over America. In African, Latin American and Asian continents, it is still one of the main caloric foods to nearly 500 million people, mainly in underdeveloped countries [34, 35]. The variability in total carotenoids and β‐carotene and isomers, in 12 varieties of raw yellow bitter cassava roots as well as the degradation in five varieties after the flour process to observe the heat treatment effect on carotenoids degradation, revealed that total carotenoids varied

, there is a reduction in the antioxidant activity of β‐carotene observed in

concentrations of O<sup>2</sup>

178 Carotenoids

in the system [31–33].

**2.1. Sweet and bitter yellow cassava**

*Ipomoea batatas* belongs to the Convolvulaceae family, genus Ipomoea with 50 genera and more than 1000 species. However, it is the most important species and, sometimes, the only staple food crop. The varieties of potatoes with white or pale yellow flesh are less sweet and moist than those with red, pink or orange flesh [41] and are native to the tropical regions in the Americas and Africa [42]. American types with pink yellow flesh contain as high as 5.4–7.2 mg 100 g‐1 of β‐carotene but higher contents. Additionally, more than a dozen African vegetables, this was the richest in folate (1.93–1.96 mg g‐1 ) [43]. Some cultivars are developed by biofortification program as the yellow/orange‐flesh sweet potatoes like the *Beauregard* cul‐ tivar in Brazil, with intense orange pulp, because of its high content of β‐carotene [16] and, among many studies on it, its antioxidant capacity [44]. The β‐carotene is one of the carot‐ enoids with higher pro‐vitamin A activity, which is the largest source of vitamin A and its derivatives in the human diet. It is also the most active carotenoid, comprising 15–30% of all serum carotenoids [45]. Because of its high combined structure, the carotenoids are suscep‐ tible to degradation by light, oxidation by heat, acid or alkaline pH and the presence of metal ion. They can be hydrophobic, lipophilic, insoluble in water and soluble in solvents such as acetone, alcohol and chloroform. Of more than 600 known carotenoids, only about 50 have pro‐vitamin A activity and are antioxidants [46, 47]. On the other hand, studies on the profiles of phenolics, carotenoids and antioxidant capacities of raw and cooked white, yellow, orange, light purple and deep purple sweet potato varieties, grown in Guilin (China), revealed higher anthocyanin contents and antioxidant capacities in purple sweet potato species and higher carotenoid contents in yellow and orange sweet potato. All cooked sweet potatoes exhibited significantly (p < 0.05) lower TPC, MAC, TCC, DPPH and Fluorescence recovery after photo‐ bleaching (FRAP) values compared to the respective raw samples. Steaming samples showed good results in retention of Total Phenolic Compounds, roasting for keeping anthocyanins, and boiling best preserve the carotenoids [48]. Various types of orange sweet potato (*Ipomoea batatas*) are grown in Brazil and in the world having different shapes and sizes and especially differentiated carotenoid contents of pro‐vitamin A. The total carotenoid and β‐carotene as well as its isomers 9:13—cis (*Z*) of β‐carotene from two cultivars of orange sweet potato: an organic cultivar called 'carrot', and the *Beauregard* sweet potato Beauregard showed the high‐ est β‐carotene content among the studied samples being a good source of provitamin A to be cultivated and consumed, mainly, in the areas of low‐income populations and where the deficiency of vitamin A is common among children [16, 44]. *The Beauregard* is a biofortified American cultivar with intense orange pulp because of its high β‐carotene content. The effect of the drying treatment on the β‐carotene and total carotenoid of this cultivar dried at 40°C for 5 h, 50°C for 2 h and at 60°C for 1 h showed total carotenoids, in mg kg‐1 , of 129.85 in raw samples; 124.26 in bleached samples; 760.65 (40°C); 769.76 (50°C) and 832.40 (60°C), respec‐ tively. The results found by Baganha et al. (2016) for total carotenoids, in mg.kg‐1 , were 129.85 ± 2.47 in sweet potatoes raw samples; 124.26 ± 3.40 in bleached samples; 760.65 ± 1.45 (40 °C); 769.76 ± 4.43 (50 °C) and 832.40 ± 6.02 (60 °C), respectively. The mean values for β‐carotene (mg.kg‐1) were 107.93 ± 0.66 (raw); 97.71 ± 4.13 (bleached); 660.08 ± 11.65 (40 °C); 677.03 ± 9.45 (50 °C) and 736.21 ± 3.46 (60 °C), respectively. Drying at 60°C for 1 h showed the highest retention of total carotenoids and β‐carotene, indicating that the shortest time of exposure to heat had a greater influence than the higher temperature [44]. In another study, in India, 15 genotypes of exotic and indigenous orange‐flesh sweet potatoes cooked were evaluated after cooking process. The β‐carotene contents ranged from 28.80 to 97.40 μg g‐1 , and its retention after cooked varied from 76. 90 to 87.76% [49]. Ten sweet potato clones with different orange flesh color were processed in an oven‐drying, boiling, sun‐drying and frying. The carotenoids retention depended on the process applied. The highest retentions of total carotenoids and β‐carotene were observed in oven‐drying (90–91% and 89–96%) followed by boiling (85–90% and 84–90%) and frying (77–85% and 72–86%), and the lowest in both micronutrients were found in the sun‐drying method (63–73%) and β‐carotene (63–73%) [50, 51]. The extraction step is very important in β‐carotene from sweet potato. According to the reference, the best solvent and time of extraction were observed using 91.1% of acetone and 19.6 min of extraction and 278.1 μg g‐1 of β‐carotene in the variety CYY95‐26 and small amounts of the isomers 9 and 13‐cis [52]. Recently, the β‐carotene of four sweet potato varieties from Tanzania (*Jewel*, *Karoti dar*, *Kabode* and *Ejumula*) with different intensities of orange flesh color was evaluated. Sweet potatoes were blanched and boiled. There was a threefold reduction in β‐carotene content when fresh samples were dried. Boiling results in more retention of β‐carotene than blanching in sweet potatoes. The fresh dried had significantly low β‐carotene content and low reten‐ tion on storage compared to boiled and blanched chips, and blanched cowpea leaves retained more β‐carotene after 6 months of storage at room temperature [53].

#### **2.3. Pumpkin (***Cucurbita***)**

by biofortification program as the yellow/orange‐flesh sweet potatoes like the *Beauregard* cul‐ tivar in Brazil, with intense orange pulp, because of its high content of β‐carotene [16] and, among many studies on it, its antioxidant capacity [44]. The β‐carotene is one of the carot‐ enoids with higher pro‐vitamin A activity, which is the largest source of vitamin A and its derivatives in the human diet. It is also the most active carotenoid, comprising 15–30% of all serum carotenoids [45]. Because of its high combined structure, the carotenoids are suscep‐ tible to degradation by light, oxidation by heat, acid or alkaline pH and the presence of metal ion. They can be hydrophobic, lipophilic, insoluble in water and soluble in solvents such as acetone, alcohol and chloroform. Of more than 600 known carotenoids, only about 50 have pro‐vitamin A activity and are antioxidants [46, 47]. On the other hand, studies on the profiles of phenolics, carotenoids and antioxidant capacities of raw and cooked white, yellow, orange, light purple and deep purple sweet potato varieties, grown in Guilin (China), revealed higher anthocyanin contents and antioxidant capacities in purple sweet potato species and higher carotenoid contents in yellow and orange sweet potato. All cooked sweet potatoes exhibited significantly (p < 0.05) lower TPC, MAC, TCC, DPPH and Fluorescence recovery after photo‐ bleaching (FRAP) values compared to the respective raw samples. Steaming samples showed good results in retention of Total Phenolic Compounds, roasting for keeping anthocyanins, and boiling best preserve the carotenoids [48]. Various types of orange sweet potato (*Ipomoea batatas*) are grown in Brazil and in the world having different shapes and sizes and especially differentiated carotenoid contents of pro‐vitamin A. The total carotenoid and β‐carotene as well as its isomers 9:13—cis (*Z*) of β‐carotene from two cultivars of orange sweet potato: an organic cultivar called 'carrot', and the *Beauregard* sweet potato Beauregard showed the high‐ est β‐carotene content among the studied samples being a good source of provitamin A to be cultivated and consumed, mainly, in the areas of low‐income populations and where the deficiency of vitamin A is common among children [16, 44]. *The Beauregard* is a biofortified American cultivar with intense orange pulp because of its high β‐carotene content. The effect of the drying treatment on the β‐carotene and total carotenoid of this cultivar dried at 40°C

180 Carotenoids

for 5 h, 50°C for 2 h and at 60°C for 1 h showed total carotenoids, in mg kg‐1

cooking process. The β‐carotene contents ranged from 28.80 to 97.40 μg g‐1

tively. The results found by Baganha et al. (2016) for total carotenoids, in mg.kg‐1

samples; 124.26 in bleached samples; 760.65 (40°C); 769.76 (50°C) and 832.40 (60°C), respec‐

± 2.47 in sweet potatoes raw samples; 124.26 ± 3.40 in bleached samples; 760.65 ± 1.45 (40 °C); 769.76 ± 4.43 (50 °C) and 832.40 ± 6.02 (60 °C), respectively. The mean values for β‐carotene (mg.kg‐1) were 107.93 ± 0.66 (raw); 97.71 ± 4.13 (bleached); 660.08 ± 11.65 (40 °C); 677.03 ± 9.45 (50 °C) and 736.21 ± 3.46 (60 °C), respectively. Drying at 60°C for 1 h showed the highest retention of total carotenoids and β‐carotene, indicating that the shortest time of exposure to heat had a greater influence than the higher temperature [44]. In another study, in India, 15 genotypes of exotic and indigenous orange‐flesh sweet potatoes cooked were evaluated after

after cooked varied from 76. 90 to 87.76% [49]. Ten sweet potato clones with different orange flesh color were processed in an oven‐drying, boiling, sun‐drying and frying. The carotenoids retention depended on the process applied. The highest retentions of total carotenoids and β‐carotene were observed in oven‐drying (90–91% and 89–96%) followed by boiling (85–90% and 84–90%) and frying (77–85% and 72–86%), and the lowest in both micronutrients were found in the sun‐drying method (63–73%) and β‐carotene (63–73%) [50, 51]. The extraction step is very important in β‐carotene from sweet potato. According to the reference, the best

, of 129.85 in raw

, and its retention

, were 129.85

A large number of pumpkin varieties (Cucurbitaceae), each of which containing different amounts of carotenoids, are cultivated worldwide [54]. In Brazil, *C. moschata* cultivars are known to contain high amount of α‐ and β‐carotene. β‐carotene has almost 100% pro‐vitamin A activity, and α‐carotene has approximately 53% pro‐vitamin A activity [11, 55–57]. Some varieties such as *C. moschata*, *C. maxima* and *C. pepo*, with color ranging from intense yellow to orange, have revealed high levels of carotenoids, particularly, α and β‐carotene, β‐crip‐ toxanthin, lutein and zeaxanthin [11]. The orange‐fleshed pumpkins (*C. moschata*) normally present high levels of carotenoids mainly β‐ and α‐carotene as well the 9, 13 and 15‐β‐carotene isomers. Inspite of the low bioaccessibility and bioavailability of the pumpkin carotenoids, its high contents after the cooking styles can still offer adequate daily dietary. On the other hand, the drying process usually can affect the levels of these micronutrients. In Ref. [6], the carotenoid content within pumpkin and squash measured by HPLC and with colorimeter L\*a\*b\* color space values was correlated, and a range of colors and carotenoid types and con‐ centrations within pumpkins and squash was found as well as strong correlations between colorimetric values and carotenoid content were identified. The authors suggested that the genetic variations should make it possible to increase the nutritional value through cross‐ ing and selection from within and among the different types with high levels of carotenoids. The α‐ and β‐carotene) of pumpkin flours were evaluated using an oven with air circulation and finally milled in temperatures at 45 and 50°C. Pumpkins are cut into slices, blanched at 90°C for 3 min and dried. The drying process at 45°C spent 132 h (5.5 days) was longer com‐ pared with sliced pumpkins dried at 50 (48 hours). In raw pumpkins, total carotenoids were 442.56 μg g‐1 , α‐carotene was 110.87 μg g‐1 , and β‐carotene was 297.37 μg g‐1 . In flours dried at 45 and 50°C, the total carotenoids were 1892.98 and 1668.43 μg g‐1 , respectively. Flours pre‐ sented high contents of carotenoids, as expected, since their moistures were very low (9.17 and 7.83 g 100‐1 ). The flour dried at 45°C preserved 95% of the α‐carotene and 83% of the β‐carotene compared to the flour dried at 50°C. The isomers 9 and 13‐*Z*‐ of the β‐carotene were present in small percentages in both flours. The results showed to be promising by the fact that the use of these flours in meals in scholar‐age children can increase the dietary intake of pro‐vitamin A minimizing the vitamin A deficiencies in underdeveloped countries [58]. As wrote previously, some carotenoids are rich in β‐carotene, but few are converted by the body into retinol, the active form of vitamin A. These carotenoids are susceptible to degradation (e.g., isomerization and oxidation) during cooking. Total carotenoid, α‐ and β‐carotene, and 9 and 13‐*Z*‐β‐carotene isomer contents in *C. moschata* after different cooking styles were evaluated. The raw pumpkin presented 236.10, 172.20, 39.95, 3.64 and 0.8610 μg g‐1 of total carotenoids, β‐carotene, α‐car‐ otene, 13‐*cis*‐β‐carotene and 9‐*Z*‐β‐carotene, respectively. Samples cooked these total carot‐ enoids in boiling water were 258.50, 184.80, 43.97, 6.80 and 0.77 μg g‐1 , respectively. Steamed samples revealed 280.77, 202.00, 47.09, 8.23 and 1.247 μg g‐1 , respectively. Since almost 100% of β‐carotene is converted into vitamin A, these results are promising. All carotenoids increased after the cooking methods, most likely of a higher availability induced by the cooking style [59]. The carotenoids should be more bioavailable after the heat treatments. The total carot‐ enoid and β‐carotene isomers contents, normally, may increase according to the cooking styles applied. Pumpkin consumption in Northeast Brazil could be more, aggressively, promoted to minimize vitamin A deficiency in this geographic area. Landrace pumpkins occur in nature, and their potential as source of pro‐vitamin A, were investigated, in order to be used in con‐ ventional plant breeding or biofortification programs, aiming to increase the total carotenoids and β‐carotene contents. The total carotenoid, α‐carotene, β‐carotene and its isomers in two raw landraces pumpkins (*C. moschata*) (A and B) were evaluated to verify its seed production potential. Total carotenoid content of 404.98 (A) and 234.21 μg g‐1 (B), respectively, were found. The best value for α‐carotene contents 72.99 μg g‐1 . All *E*‐β‐carotene was the most abundant micronutrient varying from 244.22 to 141.95 μg g‐1 in both samples. The 9 and 13‐Z‐carotene isomers were still found in low concentrations. The best β‐carotene content in raw sample (A) revealed to be promising for the production of seeds for cultivation and consumption [37]. Recently, the retention of pro‐vitamin A carotenoids in the pulp from orange‐fleshed pumpkin that was briefly steamed or boiled in either water or water containing 60% sucrose in five genotypes grown in Brazil was investigated and their bioaccessibility in cooked pulp was also determined by *in vitro* digestion and confirmed with Caco‐2. Genotypes varied from 209 to 658 μg g‐1 in pro‐vitamin A carotenoids. The retention after cooking was more than 78%. The bioaccessibility of β‐ and α‐carotene was <4%, which showed high variability, affected by food matrix and cooking. One genotype has the potential to provide more than 40% required for children 4–8 years of age per 100 g serving. Pumpkin (*Cucurbita moschata*) is a food crop targeted for enrichment with pro‐vitamin A carotenoids [60].

Thus, studies on how the pro‐vitamin A carotenoids are assimilated by the human organism, mainly in pumpkins, are relevant and necessary, although, since β‐carotene and α‐carotene in the pumpkin are poorly bioavailable, these levels are high and supply the daily necessities without the amount of daily food being increased.

#### **Author details**

Lucia Maria Jaeger de Carvalho<sup>1</sup> \*, Gisela Maria Dellamora Ortiz<sup>1</sup> , José Luiz Viana de Carvalho<sup>2</sup> , Lara Smirdele<sup>1</sup> and Flavio de Souza Neves Cardoso<sup>1</sup>

\*Address all correspondence to: luciajaeger@gmail.com

1 Natural Products and Food Department, School of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

2 Embrapa Food Technology, Guaratiba, RJ, Brazil

#### **References**

presented 236.10, 172.20, 39.95, 3.64 and 0.8610 μg g‐1

samples revealed 280.77, 202.00, 47.09, 8.23 and 1.247 μg g‐1

potential. Total carotenoid content of 404.98 (A) and 234.21 μg g‐1

targeted for enrichment with pro‐vitamin A carotenoids [60].

and Flavio de Souza Neves Cardoso<sup>1</sup>

\*Address all correspondence to: luciajaeger@gmail.com

2 Embrapa Food Technology, Guaratiba, RJ, Brazil

without the amount of daily food being increased.

The best value for α‐carotene contents 72.99 μg g‐1

micronutrient varying from 244.22 to 141.95 μg g‐1

209 to 658 μg g‐1

182 Carotenoids

**Author details**

Lara Smirdele<sup>1</sup>

Lucia Maria Jaeger de Carvalho<sup>1</sup>

Janeiro, Rio de Janeiro, RJ, Brazil

enoids in boiling water were 258.50, 184.80, 43.97, 6.80 and 0.77 μg g‐1

otene, 13‐*cis*‐β‐carotene and 9‐*Z*‐β‐carotene, respectively. Samples cooked these total carot‐

β‐carotene is converted into vitamin A, these results are promising. All carotenoids increased after the cooking methods, most likely of a higher availability induced by the cooking style [59]. The carotenoids should be more bioavailable after the heat treatments. The total carot‐ enoid and β‐carotene isomers contents, normally, may increase according to the cooking styles applied. Pumpkin consumption in Northeast Brazil could be more, aggressively, promoted to minimize vitamin A deficiency in this geographic area. Landrace pumpkins occur in nature, and their potential as source of pro‐vitamin A, were investigated, in order to be used in con‐ ventional plant breeding or biofortification programs, aiming to increase the total carotenoids and β‐carotene contents. The total carotenoid, α‐carotene, β‐carotene and its isomers in two raw landraces pumpkins (*C. moschata*) (A and B) were evaluated to verify its seed production

isomers were still found in low concentrations. The best β‐carotene content in raw sample (A) revealed to be promising for the production of seeds for cultivation and consumption [37]. Recently, the retention of pro‐vitamin A carotenoids in the pulp from orange‐fleshed pumpkin that was briefly steamed or boiled in either water or water containing 60% sucrose in five genotypes grown in Brazil was investigated and their bioaccessibility in cooked pulp was also determined by *in vitro* digestion and confirmed with Caco‐2. Genotypes varied from

The bioaccessibility of β‐ and α‐carotene was <4%, which showed high variability, affected by food matrix and cooking. One genotype has the potential to provide more than 40% required for children 4–8 years of age per 100 g serving. Pumpkin (*Cucurbita moschata*) is a food crop

Thus, studies on how the pro‐vitamin A carotenoids are assimilated by the human organism, mainly in pumpkins, are relevant and necessary, although, since β‐carotene and α‐carotene in the pumpkin are poorly bioavailable, these levels are high and supply the daily necessities

\*, Gisela Maria Dellamora Ortiz<sup>1</sup>

1 Natural Products and Food Department, School of Pharmacy, Federal University of Rio de

in pro‐vitamin A carotenoids. The retention after cooking was more than 78%.

of total carotenoids, β‐carotene, α‐car‐

, respectively. Since almost 100% of

(B), respectively, were found.

, José Luiz Viana de Carvalho<sup>2</sup>

,

. All *E*‐β‐carotene was the most abundant

in both samples. The 9 and 13‐Z‐carotene

, respectively. Steamed


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http://dx.doi.org/10.5772/intechopen.68279

#### **Abstract**

Vitamin A deficiency (VAD) is a preventable tragedy that affects millions of people, particularly in sub-Saharan Africa. A large proportion of these people rely on diets based on cassava as a source of calories. During the last two decades, significant efforts have been made to identify sources of germplasm with high pro-vitamin A carotenoids (pVAC) and then use them to develop cultivars with a nutritional goal of 15 μg g−1 of β-carotene (fresh weight basis) and good agronomic performance. The protocols for sampling roots and quantifying carotenoids have been improved. Recently, NIR predictions began to be used. Retention of carotenoids after different root processing methods has been measured. Bioavailability studies suggest high conversion rates. Genetic modification has also been achieved with mixed results. Carotenogenesis genes have been characterized and their activity in roots measured.

**Keywords:** carotenogenesis, conventional breeding, dry matter content, physiological deterioration

#### **1. Introduction: cassava as an important food-security and industrial crop**

Cassava (*Manihot esculenta* Crantz) has a Neotropical origin and significant economic relevance. It is an important crop in tropical and subtropical regions of the world, growing from sea level up to 1800 m. Its most common product is the starchy root, but cassava foliage has an excellent nutritional quality for animal and human consumption and offers great potential [1, 2]. Stems are used for commercial multiplication. Therefore, every part of the plant can

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

be used and exploited. Cassava is the fourth most important basic food after rice, wheat, and maize worldwide, but is the second most important food staple (in terms of calories consumed) in sub-Saharan Africa [3–6]. The crop is called Africa's food insurance because it offers reliable yields even in the face of drought, low soil fertility, low intensity management, and because of its resilience to face the effects of climate change [7–9].

Between 2010 and 2014, an average of 22 million ha was annually grown with cassava worldwide (70% in Africa, 18% in Asia, and about 12% in the Americas). The area planted to cassava has grown steadily, and it was 2011 when, for the first time, more than 20 million ha were planted with the crop (FAOSTAT). Markets in cassava are diverse. The crop was initially domesticated for the direct consumption of the roots, which contain plenty of carbohydrates. However, roots contain little protein and few micronutrients [10], when compared to sweet potatoes, potatoes, beans, maize, or wheat. Globally, in the period 1970– 2003, the main uses of cassava roots were for food (54%), followed by feed (30%), and other uses including starch production (4%) [11]. Global use of cassava for feed was affected by the reduction of imports from the European Union in the 1980s. Production of starch, on the other hand, increased considerably in the same period (by 17.5% annually [11]). Today, cassava is the second most important source of starch worldwide [12]. In the 2000s, a considerable amount of cassava roots started to be used for the production of fuel ethanol as well [13, 14].

A comprehensive screening of root quality traits from more than 4000 cassava clones has been published [15]. On average, roots from these genotypes had 33.6% of dry matter content (DMC) of which 84.5% is starch (the main and most valuable product in cassava roots). Cassava starch quality is excellent and has, on average, 20.7% amylose (the remaining 79.3% is amylopectin). Cassava roots spoil quickly (2–3 days after harvest) because of a process called postharvest physiological deterioration (PPD). Therefore, roots need to be processed or consumed soon after harvest.

Vitamin A is an essential micronutrient for the normal functioning of the visual and immune systems, growth and development, maintenance of epithelial cellular integrity, and for reproduction [16]. Vitamin A deficiency (VAD) is the leading cause of preventable blindness in children and increases the risk of disease and death from severe infections. In pregnant women, VAD causes night blindness and may increase the risk of maternal mortality. VAD is a public health problem in more than half of all countries, especially in Africa and South-East Asia, hitting hardest young children (visual impairment and blindness, and significantly increases the risk of severe illness, and even death) and pregnant women (especially during the last trimester when demand by both the unborn child and the mother is highest) in low-income countries. An estimated 250 million preschool children show VAD, and it is likely that, in areas where VAD is prevalent, a substantial proportion of pregnant women is also affected. An estimated 250,000 to 500,000 vitamin A-deficient children become blind every year, half of them dying within 12 months of losing their sight [17].

Vitamin A exists in natural products in many different forms: as preformed retinoids (stored in animal tissues) and as provitamin A carotenoids (pVAC), which are synthesized as pigments by many plants and are found in different plant tissues [16]. The carotenoids are present in both plant and animal food products (in animal products their occurrence results from dietary exposure). Retinoids, on the other hand, are only found in animal products. A comprehensive review on carotenoids has been recently published [18].

Different strategies (dietary diversification, food fortification, and/or supplementation) and considerable efforts have been made to reduce VAD worldwide [19]. These strategies are relatively cost-effective, but have failed to completely eradicate the problem for a diversity of reasons [20]. Moreover, prevalence of VAD has remained unchanged in sub-Saharan Africa and south Asia during the 1991–2013 periods [21]. Recently, different programs such as HarvestPlus (www.harvestplus.org), involving a global alliance of research institutions, initiated the implementation of a fourth strategy (biofortification) to develop micronutrientdense staple crops [19, 22, 23]. A diet rich in pVAC, in addition to reducing the problems related to VAD would also result in other health benefits, including a reduction of cancer incidence [24, 25].

The color of root parenchyma (e.g., pulp) in cassava varies from white to yellow. Pinkish pigmentation has also been reported [26]. Pigmentation of the parenchyma is closely linked to carotenoids content. Most cassava varieties worldwide produce roots with white parenchyma. This is particularly appreciated by the starch industry. However, breeding to develop cassava varieties need to target different end users and it is only recently that the efforts to develop high-carotenoids biofortified germplasm were initiated by HarvestPlus. This is an international, interdisciplinary research initiative that seeks to reduce human malnutrition by increasing micronutrients in staple crops, including cassava. The progress already attained by HarvesPlus and the awareness that has elicited in the plant breeding community resulted in the 2016 World Food Prize Award. Biofortified cassava makes sense considering the importance of this crop in sub-Saharan Africa and the reported prevalence of VAD in that region of the world. The target for these varieties will be not only for human consumption but also for animal feed, particularly poultry [27].

#### **2. Nutritional value of cassava roots**

be used and exploited. Cassava is the fourth most important basic food after rice, wheat, and maize worldwide, but is the second most important food staple (in terms of calories consumed) in sub-Saharan Africa [3–6]. The crop is called Africa's food insurance because it offers reliable yields even in the face of drought, low soil fertility, low intensity management,

Between 2010 and 2014, an average of 22 million ha was annually grown with cassava worldwide (70% in Africa, 18% in Asia, and about 12% in the Americas). The area planted to cassava has grown steadily, and it was 2011 when, for the first time, more than 20 million ha were planted with the crop (FAOSTAT). Markets in cassava are diverse. The crop was initially domesticated for the direct consumption of the roots, which contain plenty of carbohydrates. However, roots contain little protein and few micronutrients [10], when compared to sweet potatoes, potatoes, beans, maize, or wheat. Globally, in the period 1970– 2003, the main uses of cassava roots were for food (54%), followed by feed (30%), and other uses including starch production (4%) [11]. Global use of cassava for feed was affected by the reduction of imports from the European Union in the 1980s. Production of starch, on the other hand, increased considerably in the same period (by 17.5% annually [11]). Today, cassava is the second most important source of starch worldwide [12]. In the 2000s, a considerable amount of cassava roots started to be used for the production of fuel ethanol as

A comprehensive screening of root quality traits from more than 4000 cassava clones has been published [15]. On average, roots from these genotypes had 33.6% of dry matter content (DMC) of which 84.5% is starch (the main and most valuable product in cassava roots). Cassava starch quality is excellent and has, on average, 20.7% amylose (the remaining 79.3% is amylopectin). Cassava roots spoil quickly (2–3 days after harvest) because of a process called postharvest physiological deterioration (PPD). Therefore, roots need to be processed or

Vitamin A is an essential micronutrient for the normal functioning of the visual and immune systems, growth and development, maintenance of epithelial cellular integrity, and for reproduction [16]. Vitamin A deficiency (VAD) is the leading cause of preventable blindness in children and increases the risk of disease and death from severe infections. In pregnant women, VAD causes night blindness and may increase the risk of maternal mortality. VAD is a public health problem in more than half of all countries, especially in Africa and South-East Asia, hitting hardest young children (visual impairment and blindness, and significantly increases the risk of severe illness, and even death) and pregnant women (especially during the last trimester when demand by both the unborn child and the mother is highest) in low-income countries. An estimated 250 million preschool children show VAD, and it is likely that, in areas where VAD is prevalent, a substantial proportion of pregnant women is also affected. An estimated 250,000 to 500,000 vitamin A-deficient children become blind every year, half of

Vitamin A exists in natural products in many different forms: as preformed retinoids (stored in animal tissues) and as provitamin A carotenoids (pVAC), which are synthesized as pigments by many plants and are found in different plant tissues [16]. The carotenoids are present in

and because of its resilience to face the effects of climate change [7–9].

well [13, 14].

190 Carotenoids

consumed soon after harvest.

them dying within 12 months of losing their sight [17].

The nutritional quality of cassava roots in general is low, and contains mainly carbohydrates. Per 100 gram raw weight, white cassava provides 160 kcal mainly as carbohydrates (38 g) and contains further water (60 g), a little protein (1.4 g), fat (0.3 g) and trace elements of iron (0.3 mg), niacin 0.9 mg), thiamin (0.09 mg), riboflavin (0.05 mg), calcium (16 mg), potassium (271 mg), zinc (0.3 mg), and vitamin C (21 g) [28]. People depending on a diet predominantly based on white cassava roots are at greater risk of having iron, zinc and VAD, as was shown in children in Kenya and Nigeria [29].

Cassava also contains variety-dependent concentrations of cyanogenic glucosides (CG), which are toxic for humans and needs to be eliminated during processing before consumption. Therefore, cassava with high concentration in cyanide are better suitable for more processed products like porridge made out of flour and those low in cyanide are more suitable for boiled consumption [30]. CG levels range from low (≤100 ppm) in roots from sweet/cool varieties to very high (≥3000 ppm) in bitter cassava cultivars. CG are eliminated through alternative processing techniques [2]. However, health of people may be affected [31], particularly in years when drought has affected other crops, which increases people's dependency in cassava. In addition to higher consumption of cassava roots, drought generally increases CG levels in them [32].

#### **3. Genetic variation of carotenoids content in cassava roots**

Carotenoids perform essential physiological functions in plants. They are involved in the photosynthesis process, protecting the plant from photo-oxidative damage, and as precursors of regulatory molecules, such as abscisic acid (ABA) and strigolactones [33, 34]. In cassava, carotenoids can be found in leaves and roots. Concentration is much larger in leaves than in roots [35–37]. The earliest reports on pVAC contents in cassava roots (written in Portuguese) were based on cassava samples from the Amazon region of Brazil and the earliest dates back to 1964 [38–40]. Interest in increasing pVAC content in cassava roots began in the 1990s in India [41, 42] and few years later at International Center for Tropical Agriculture (CIAT) in Colombia [43] and at IIITA in Nigeria [37]. HarvestPlus provided financial resources and encouraged systematic work that resulted in the screening of a large sample of cassava germplasm [26, 36].

Quality of data improved over the years. Initially most of the information focused on assessing differences in the intensity of parenchyma pigmentation [44] and often relied on measuring total carotenoids content (TCC) by spectrophotometer. HPLC analysis restricted the number of samples that could be screened. Moorthy reported in 1990 a range of variation for β-carotene from negligible to 7.9 μg g−1 [41]. Unless otherwise specified, all concentration values for carotenoids will be expressed on fresh weight basis. A comprehensive screening of carotenoids content reported in 2005 (1789 genotypes) had an average total carotenoids content (TCC) of 2.46 μg g−1, ranging from 1.02 to 10.40 μg g−1 [26]. **Figure 1** illustrates the strong skewness in frequency distributions for TTC by 2005 with a long tail to the right (low frequency of genotypes with high TCC values).

The basic concept in breeding is to cross outstanding genotypes (e.g., clones) to generate a new generation of segregating progenies that will hopefully have a better average performance. At CIAT those individuals, at the right of the plot in **Figure 1**, were crossed among themselves to obtain botanical seed. Each seed represented a new, genetically unique, genotype. The seed was germinated and the roots from the plants generated were evaluated for their carotenoids content. The best materials (highest pVAC) were then selected and crossed to produce a new cycle of selection. The basic scheme was described by Ceballos and coworkers in 2013 [45]. Very early in this process, it became clear that carotenoids content was not only closely associated with pigmentation of the root parenchyma [41, 43] but also with a reduction or delay of PPD [26, 46, 47]. The beneficial effect of increased TCC in lengthening the shelf life of cassava roots was an important finding that would encourage the adoption

varieties to very high (≥3000 ppm) in bitter cassava cultivars. CG are eliminated through alternative processing techniques [2]. However, health of people may be affected [31], particularly in years when drought has affected other crops, which increases people's dependency in cassava. In addition to higher consumption of cassava roots, drought generally increases

Carotenoids perform essential physiological functions in plants. They are involved in the photosynthesis process, protecting the plant from photo-oxidative damage, and as precursors of regulatory molecules, such as abscisic acid (ABA) and strigolactones [33, 34]. In cassava, carotenoids can be found in leaves and roots. Concentration is much larger in leaves than in roots [35–37]. The earliest reports on pVAC contents in cassava roots (written in Portuguese) were based on cassava samples from the Amazon region of Brazil and the earliest dates back to 1964 [38–40]. Interest in increasing pVAC content in cassava roots began in the 1990s in India [41, 42] and few years later at International Center for Tropical Agriculture (CIAT) in Colombia [43] and at IIITA in Nigeria [37]. HarvestPlus provided financial resources and encouraged systematic work that resulted in the screening of a large sample of cassava germ-

Quality of data improved over the years. Initially most of the information focused on assessing differences in the intensity of parenchyma pigmentation [44] and often relied on measuring total carotenoids content (TCC) by spectrophotometer. HPLC analysis restricted the number of samples that could be screened. Moorthy reported in 1990 a range of variation for β-carotene from negligible to 7.9 μg g−1 [41]. Unless otherwise specified, all concentration values for carotenoids will be expressed on fresh weight basis. A comprehensive screening of carotenoids content reported in 2005 (1789 genotypes) had an average total carotenoids content (TCC) of 2.46 μg g−1, ranging from 1.02 to 10.40 μg g−1 [26]. **Figure 1** illustrates the strong skewness in frequency distributions for TTC by 2005 with a long tail to the right (low

The basic concept in breeding is to cross outstanding genotypes (e.g., clones) to generate a new generation of segregating progenies that will hopefully have a better average performance. At CIAT those individuals, at the right of the plot in **Figure 1**, were crossed among themselves to obtain botanical seed. Each seed represented a new, genetically unique, genotype. The seed was germinated and the roots from the plants generated were evaluated for their carotenoids content. The best materials (highest pVAC) were then selected and crossed to produce a new cycle of selection. The basic scheme was described by Ceballos and coworkers in 2013 [45]. Very early in this process, it became clear that carotenoids content was not only closely associated with pigmentation of the root parenchyma [41, 43] but also with a reduction or delay of PPD [26, 46, 47]. The beneficial effect of increased TCC in lengthening the shelf life of cassava roots was an important finding that would encourage the adoption

**3. Genetic variation of carotenoids content in cassava roots**

CG levels in them [32].

192 Carotenoids

plasm [26, 36].

frequency of genotypes with high TCC values).

**Figure 1.** Frequency distribution for total carotenoids content (μg g−1, fresh weight basis) of cassava germplasm by 2005.

of biofortified varieties. It has been suggested that reduced PPD in high pVAC roots may involve β-ionone-like molecules, derived from β-carotene catabolism, which play a role in the response to biotic stress such as fungal infection [48]. However, PPD is a very variable trait, difficult to measure visually, influenced by the environment, and which depends very much on the storage conditions of the harvested root.

The relationships depicted in **Figure 2** are relevant for the impact of the biofortified varieties released through the HarvestPlus initiative. It is not enough to develop cassava cultivars with high pVAC. The roots from these cultivars should meet consumer preferences. Key parameters defining consumer acceptance are dry matter content (DMC) and cyanogenic potential (particularly in regions of the world where roots are boiled). DMC influences texture after boiling and is also a key parameter in the production of gari, for example (a popular way to consume cassava in Africa). The relationship between carotenoids and DMC is basically nonexistent (**Figure 2**). It is possible, therefore, to identify genotypes with high pVAC and acceptable levels of DMC. The first target of the HarvestPlus initiative was to develop biofortified clones for Africa. The key related trait for the most important ways to consume cassava in Africa would be DMC. Cyanogenic potential is not critical for gari production, but it is critical for table consumption after boiling. This is the most common way to consume cassava in many Latin American and Caribbean (LAC) countries. Only recently CIAT started the development of biofortified cassava with low cyanogenic potential targeting LAC. The relationship between carotenoids and cyanogenic potential in **Figure 2** shows a negative trend, although the coefficient of determination is low (*r*<sup>2</sup> = 0.15). For a variety to be considered "sweet" and apt for table consumption, the maximum HCN levels would be about 150 ppm. At this stage of the breeding process, only a few dozen genotypes have been found to have low HCN, high pVAC and acceptable cooking quality. Efforts are currently made in making crosses among these genotypes to increase the number of segregating materials that can fit the consumer preferences in LAC.

**Figure 2.** Relationship between total carotenoids content (μg/g, fresh weight basis) with dry matter content (TOP); cyanogenic potential (MIDDLE) and all-trans β-carotene (bottom) contents.

About 600 carotenoids have been isolated and characterized in nature, and approximately 10% of these can be metabolized into vitamin A by mammals. The most important carotenoids with vitaminic activity are β- and α-carotenes and cryptoxanthins. Some carotenoids that cannot be converted into vitamin A (e.g., lutein, zeaxanthin, and lycopene) can be found in the parenchyma of cassava root as well. Not all pVAC carotenoids have the same activity. β-carotene has about twice as much vitamin activity as the remaining pVAC carotenoids [49]. Fortunately, as illustrated in **Figure 2**, most of carotenoids in cassava roots are β-carotene. Assessing the nutritional potential of cassava roots, therefore, requires partitioning TCC into its different individual carotenoid components. This is usually done through HPLC chromatograms.

**Table 1** presents information on key root quality traits such as DMC and HCN and a description of the different carotenoids quantified in roots from a large sample of cassava genotypes. The information presented in **Table 1** is clearly unbalanced with large variation in the number of samples used for quantifying different parameters. For example, DMC is available for 4913 samples, whereas HCN was only measured in 981genotypes. Data for all-trans β-carotene is available from 4952 chromatograms, whereas only 49 samples allowed measuring α-carotene. To a large extent, the limited information on α-carotene is due to the low concentration, often below detection level, observed for this carotenoid. It is clear from data presented in **Table 1** that most carotenoids present in cassava roots are β-carotene (TBC), with a prevalence of its all-trans isomer. Similar conclusions were reported by Maziya-Dixon and Dixon in 2015 [50].


**Table 1.** Root quality traits and carotenoid components quantified in a large sample of cassava roots.

About 600 carotenoids have been isolated and characterized in nature, and approximately 10% of these can be metabolized into vitamin A by mammals. The most important carotenoids with vitaminic activity are β- and α-carotenes and cryptoxanthins. Some carotenoids that cannot be converted into vitamin A (e.g., lutein, zeaxanthin, and lycopene) can be found in the parenchyma of cassava root as well. Not all pVAC carotenoids have the same activity. β-carotene has about twice as much vitamin activity as the remaining pVAC carotenoids [49]. Fortunately, as illustrated in **Figure 2**, most of carotenoids in cassava roots are β-carotene. Assessing the nutritional potential of cassava roots, therefore, requires partitioning TCC

**Figure 2.** Relationship between total carotenoids content (μg/g, fresh weight basis) with dry matter content (TOP);

cyanogenic potential (MIDDLE) and all-trans β-carotene (bottom) contents.

194 Carotenoids

#### **4. Inheritance of carotenoids content in cassava roots**

Early attempts to explain the inheritance of carotenoids content in cassava roots were actually based on the visual assessment of intensity of pigmentation in root parenchyma, which was not always linked to the quantification of carotenoids content [44, 51]. Iglesias and coworkers [43] suggested in 1997 that relatively few major genes were involved, based on a hypothesis of two genes with epistatic effects controlling root color. One of these genes would show complete dominance while the second would have partial dominance. These early studies typically describe three classes for the intensity of root pigmentation (white, cream and yellow parenchyma) and agree that the trait shows dominance and is controlled by few genes, thus suggesting high heritability. Akinwale and co-workers [52] also suggested that segregations in TCC can be explained by the genetic control of two genes showing complete dominance.

During the past few years, several studies have been published on the heritability of carotenoids content in cassava roots, but based on their systematic quantification, rather than indirectly assessing the intensity of pigmentation. Parent-offspring regression analysis confirmed high heritability (˃0.60) for the trait [53–55]. High heritability for carotenoids content in cassava is further confirmed in stability studies assessing the relative importance of genotype, environment, and genotype-by-environment interaction [56–58] and estimations of general and specific combining ability effects [59]. An analysis of the segregation for TCC in large full-sib and self-pollinated families was conducted aiming at identifying patterns that could be explained by Mendelian genetics [53, 54]. **Table 2** reproduces the segregating values in 14 self-pollinated (S1 ) families. It is clear that there is an association between the TCC values of the progenitors and the average of their respective S1 progenies (further validating the high heritability of the trait). The range of variation in the resulting S1 progenies is wide, and in most cases, there are individuals with TCC values above those of the respective progenitors (except for one family that only had two individuals). Based on the results of these segregations, it was postulated again that there are at least two genes explaining the high, intermediate, and low TCC values [53, 54].

However, one of these genes (*I-*) would drastically reduce the accumulation of carotenoids and shows complete dominance (one copy of the gene is enough to reduce TCC levels). Breeders may be interested in the homozygous recessive genotype for this gene (*ii*). The other gene (*C-*), would contribute to carotenoids accumulation and shows partial dominance.

The large variation for TCC values in the S1 family AM702 is worth highlighting (**Table 2**). This family had 29 individual genotypes and the range of variation for TCC was from 0.63 to 19.1 μg/g. The performance in family AM697 is also interesting because none of its 38 members had TCC values above 1.0 μg/g. The progenitors of these two families are related to each other since they were siblings from the same full-sib family (GM708). It has been proposed that the progenitor of AM697 was homozygous for the allele reducing TCC accumulation (*II*) and/or was lacking the allele that promoted the accumulation of carotenoids (*cc*) [53, 54].


**4. Inheritance of carotenoids content in cassava roots**

plete dominance.

196 Carotenoids

self-pollinated (S1

(*cc*) [53, 54].

ate, and low TCC values [53, 54].

The large variation for TCC values in the S1

the progenitors and the average of their respective S1

heritability of the trait). The range of variation in the resulting S1

Early attempts to explain the inheritance of carotenoids content in cassava roots were actually based on the visual assessment of intensity of pigmentation in root parenchyma, which was not always linked to the quantification of carotenoids content [44, 51]. Iglesias and coworkers [43] suggested in 1997 that relatively few major genes were involved, based on a hypothesis of two genes with epistatic effects controlling root color. One of these genes would show complete dominance while the second would have partial dominance. These early studies typically describe three classes for the intensity of root pigmentation (white, cream and yellow parenchyma) and agree that the trait shows dominance and is controlled by few genes, thus suggesting high heritability. Akinwale and co-workers [52] also suggested that segregations in TCC can be explained by the genetic control of two genes showing com-

During the past few years, several studies have been published on the heritability of carotenoids content in cassava roots, but based on their systematic quantification, rather than indirectly assessing the intensity of pigmentation. Parent-offspring regression analysis confirmed high heritability (˃0.60) for the trait [53–55]. High heritability for carotenoids content in cassava is further confirmed in stability studies assessing the relative importance of genotype, environment, and genotype-by-environment interaction [56–58] and estimations of general and specific combining ability effects [59]. An analysis of the segregation for TCC in large full-sib and self-pollinated families was conducted aiming at identifying patterns that could be explained by Mendelian genetics [53, 54]. **Table 2** reproduces the segregating values in 14

most cases, there are individuals with TCC values above those of the respective progenitors (except for one family that only had two individuals). Based on the results of these segregations, it was postulated again that there are at least two genes explaining the high, intermedi-

However, one of these genes (*I-*) would drastically reduce the accumulation of carotenoids and shows complete dominance (one copy of the gene is enough to reduce TCC levels). Breeders may be interested in the homozygous recessive genotype for this gene (*ii*). The other gene (*C-*), would contribute to carotenoids accumulation and shows partial dominance.

This family had 29 individual genotypes and the range of variation for TCC was from 0.63 to 19.1 μg/g. The performance in family AM697 is also interesting because none of its 38 members had TCC values above 1.0 μg/g. The progenitors of these two families are related to each other since they were siblings from the same full-sib family (GM708). It has been proposed that the progenitor of AM697 was homozygous for the allele reducing TCC accumulation (*II*) and/or was lacking the allele that promoted the accumulation of carotenoids

) families. It is clear that there is an association between the TCC values of

progenies (further validating the high

family AM702 is worth highlighting (**Table 2**).

progenies is wide, and in

**Table 2.** Descriptive statistics in roots from S1 families derived from progenitors selected because of their high (H), intermediate (I) or low (L) levels of TCC (μg/g fresh weight basis).

It may be convenient to relate the three intensities of pigmentation used in earlier studies to the TCC values of the progenitors in **Table 2**. A root with white parenchyma has TCC values up to 1.5–2.0 μg/g, those with a cream pulp may show TCC values ranging from 1.5 to 3.0 μg/g, whereas a TCC value above 3.0–3.5 μg/g is observed only in yellow roots. It is useful to visualize two levels of variation in carotenoids content in cassava roots. The first level of variation is **qualitative** and relates to the three phenotypes regarding intensity of pigmentation (e.g. white, cream, or yellow roots with the TCC values mentioned above). It is expected that this qualitative variation relates to some of the genes in the carotenoids biosynthesis described below. There is, however, a second level of variation that is **quantitative** in nature. It involves genotypes only with yellow roots and covers a very wide range of variation (from 3.0 to 30.0 μg/g). Understanding the genetic factors controlling the large quantitative variation for TCC among yellow-rooted cassava is critical for breeding cassava with higher levels of pVAC. The distinction between white, cream and yellow roots, on the other hand, is easy to make visually and has already been linked to some of the genes in the carotenogesis pathway.

The pathway in carotenoids biosynthesis has been known for more than three decades. However, it was only after the 1990s that the genes involved in it could be cloned [60]. The diagram in **Figure 3** illustrates the key steps in the process and is helpful for understanding the inheritance of carotenoids synthesis and their accumulation, as crystals, in root chromoplasts. Few conclusions can be drawn from **Figure 3**: (a) The carotenogenesis pathway is relatively simple with few key steps (and therefore few genes); (b) breeding should downregulate the activity of lycopene ε-cyclase (LCYE) to favor the synthesis of β-carotene on the right side of **Figure 3**; and (c) breeding should try to reduce catabolic conversion of β-carotene into zeaxanthin, which eventually leads to the production of abscisic acid (ABA).

Phytoene synthase (PSY) is the enzyme responsible for the synthesis of phytoene, which is the first reaction specifically related to the carotenoid synthesis pathway as illustrated in **Figure 3** and demonstrated in cassava [34], also see later in this chapter. There are three copies of PSY in cassava but transcripts for one of them (PSY3) were negligible [61]. It has been proposed that PSY1 is mostly involved in responses to stress (through ABA), whereas PSY2 would be involved in carotenoids synthesis and accumulation [61]. Genetic transformation works to enhance carotenoids content in cassava roots through the simultaneous over-expression of PSY and CRTI (a bacterial version of phytoene desaturase—PDS) have been successful. Transformed cassava produced yellow roots with considerably higher TCC values compared

**Figure 3.** Diagram of carotenoids biosynthesis in plants (adapted from Stange and co-workers [33]). PSY: phytoene synthase; PDS: phytoene desaturase; ZDS: z-carotene desaturase; CRTISO: carotene isomerase; LCYE*:* Lycopene ε-cyclase; LCYB: Lycopene β cyclase; CεHx: ε-carotene hydroxylase; CβHx: β-carotene hydroxylase; ZEP: zeaxanthin epoxidase.

with the untransformed version of the same genotype that produces roots with white parenchyma [34, 62, 63]. It is clear, therefore, that PSY explains the variation between white and yellow roots (e.g., TCC values below 2.0 μg/g versus those ˃ 3.0 μg/g). It is reasonable to hypothesize that the partially dominant factor (*C-*) in Morillo-Coronado and co-workers [54] is related to PSY.

The pathway in carotenoids biosynthesis has been known for more than three decades. However, it was only after the 1990s that the genes involved in it could be cloned [60]. The diagram in **Figure 3** illustrates the key steps in the process and is helpful for understanding the inheritance of carotenoids synthesis and their accumulation, as crystals, in root chromoplasts. Few conclusions can be drawn from **Figure 3**: (a) The carotenogenesis pathway is relatively simple with few key steps (and therefore few genes); (b) breeding should downregulate the activity of lycopene ε-cyclase (LCYE) to favor the synthesis of β-carotene on the right side of **Figure 3**; and (c) breeding should try to reduce catabolic conversion of β-carotene into zeaxanthin, which eventually leads to the production of abscisic acid

Phytoene synthase (PSY) is the enzyme responsible for the synthesis of phytoene, which is the first reaction specifically related to the carotenoid synthesis pathway as illustrated in **Figure 3** and demonstrated in cassava [34], also see later in this chapter. There are three copies of PSY in cassava but transcripts for one of them (PSY3) were negligible [61]. It has been proposed that PSY1 is mostly involved in responses to stress (through ABA), whereas PSY2 would be involved in carotenoids synthesis and accumulation [61]. Genetic transformation works to enhance carotenoids content in cassava roots through the simultaneous over-expression of PSY and CRTI (a bacterial version of phytoene desaturase—PDS) have been successful. Transformed cassava produced yellow roots with considerably higher TCC values compared

**Figure 3.** Diagram of carotenoids biosynthesis in plants (adapted from Stange and co-workers [33]). PSY: phytoene synthase; PDS: phytoene desaturase; ZDS: z-carotene desaturase; CRTISO: carotene isomerase; LCYE*:* Lycopene ε-cyclase; LCYB: Lycopene β cyclase; CεHx: ε-carotene hydroxylase; CβHx: β-carotene hydroxylase; ZEP: zeaxanthin

(ABA).

198 Carotenoids

epoxidase.

There is no need to deregulate the activity of LCYE in cassava because the pathway naturally favors the accumulation of β-carotene as demonstrated by the values presented in **Table 1**. In fact, only trace values of α-carotene and lutein are found in cassava roots. Downregulation of β-carotene hydroxylases (*CβHx*), the third conclusion above, on the other hand, may be desirable. Reduced activity for this enzyme has been shown to enhance β-carotene concentrations in potato and sweet potato [64, 65]. No information has been published regarding variation for *CβHx* in cassava. However, the putative recessive trait in phenotypic studies (*ii*) may be related to a reduced activity of this enzyme [54].

Molecular markers ranging from microsatellite (SSR) to single nucleotide polymorphisms (SNP), identifying regions in the genome responsible for carotenoids content have been reported as well [34, 66–70]. It would be expected that some of the QTLs identified in these studies are related to the PSY gene. In fact, that was the case reported by Esuma and co-workers in 2016 [67]. Other markers may be linked to other genes in the carotenogenesis pathway (perhaps the putative *I* factor mentioned in Morillo-Coronado et al. article [54]. which may be related to *CβHx*). These genes are likely linked to the qualitative variation mentioned above (e.g., distinguishing white, cream, and yellow roots). From the nutritional point of view, however, much more relevant is the quantitative variation observed among genotypes producing yellow roots (ranging from 3.0 to 30.0 μg/g). Interesting studies in carrot compared roots from three cultivars: one producing white roots with negligible amounts of carotenoids crystals (CC) and two cultivars producing yellow roots with vast differences in CC [71]. In this study, the authors concluded that the difference in CC among the two cultivars producing yellow roots was not due to increased numbers of carotene-containing chromoplasts but rather greater accumulation of carotene per chromoplast. In other words, the carotenogenesis pathways were similar in both cultivars producing yellow roots, but the chromoplasts of the cultivar bred for higher levels of CC show a higher demand or capacity to store them. Perhaps future molecular work in cassava should focus in identifying QTLs related to the *sink strength* of chromoplast in their demand for carotenoids and/or capacity to store them as crystals. These studies should only focus on cassava genotypes producing yellow roots ranging, for example, from 10 to 30 μg/g of TCC.

The relationship between carotenoids and ABA (**Figure 3**) is relevant. ABA has been shown to have important effects as a plant growth regulator [61, 72]. As illustrated in **Figure 2**, there is no relationship between TCC and DMC in the germplasm screened at CIAT. However, many studies in Africa report a clearly negative correlation between the two traits [61, 72]. Perhaps African breeding populations have restricted genetic variability (particularly for *CβHx*) and some of the β-carotene initially produced is further converted into ABA. This hypothesis would be supported by a genetic transformation work in which, parallel to an increase in TCC, there is a reduction in DMC and increase in ABA. The performance of six transformed genotypes with high pVAC has been reported [63]. TCC in the wild type was 0.38 μg/g, whereas the best transgenic line showed 5.73 μg/g. Similarly, TBC values increased from 0.12 μg/g in the wild type to 4.67 μg/g in the best transgenic line. One distinctive feature of the transgenic genotypes is a drastic reduction of DMC. The wild-type roots had 32.6% DMC, whereas the average DMC of the six transgenic lines was 20.2% (ranging from 15.8 to 23.8%). There seems to be a generalized fact that increases of pVAC through genetic transformation result in a (pleiotropic) decrease in DMC. A reasonable explanation for the simultaneous increase in pVAC and reduction of DMC in the transgenic lines would be that the pathway did not stop at the step accumulating β-carotene but continued resulting in a higher production of ABA.

#### **5. Evolution and improvements of sampling protocols for measuring carotenoids in cassava roots**

Only a small tissue section (5 g) is needed for extracting and quantifying carotenoids in cassava roots. At the inception of the HarvestPlus initiative, there was no information regarding the uniformity of pVAC concentration within the root, among roots of the same plant, among roots from different plants of the same genotype, and on the relative importance of environment, age, and genotype-by-environment interaction. For early work, roots were cut longitudinally in four sections: two diagonally opposed sections were used for DMC determination and the remaining two quarters were chopped into small pieces which were thoroughly mixed, and from this bulked tissue, a random sample was used for pVAC quantification.

The first systematic study analyzing sampling variation of pVAC concentration in a cassava clone that produces yellow roots (average TCC 3.90 μg/g) was published in 2008 [73]. Samples along the longitudinal (proximal, central, and distal section) and across the transversal (periphery, mid-parenchyma, and core) axes of the roots were analyzed. Carotenoids and dry matter content were quantified individually in 243 root samples. Average TCC values, on a fresh weight basis, were higher in the proximal sections (4.10 μg/g) and gradually lower in the central (3.86 μg/g) and distal portions (3.73 μg/g). An opposite trend was observed when carotenoids were quantified in dry weight basis. Carotenoids concentrations were higher in the core (4.13 μg/g) and lower toward the mid-parenchyma (4.04 μg/g) and the periphery of the root (3.52 μg/g), both fresh and dry weight basis. Plant-to-plant variation was only significant for dry matter content.

Breeding progress was successful increasing pVAC levels. Along this process, however, laboratory personnel began to observe an increase in the variability of intensity of pigmentation within the root and among roots from the same genotype, even when coming from the same plant. **Figure 4** illustrates some cases with clear differences in intensity of pigmentation particularly across the root. It should be pointed that these differences are not always present and the bottom left photograph shows a case with a more uniform pigmentation. A new and more complete study on sampling variation was published in 2011 [74]. In this new study of many genotypes, most of them with considerably higher levels of pVAC compared with the earlier

would be supported by a genetic transformation work in which, parallel to an increase in TCC, there is a reduction in DMC and increase in ABA. The performance of six transformed genotypes with high pVAC has been reported [63]. TCC in the wild type was 0.38 μg/g, whereas the best transgenic line showed 5.73 μg/g. Similarly, TBC values increased from 0.12 μg/g in the wild type to 4.67 μg/g in the best transgenic line. One distinctive feature of the transgenic genotypes is a drastic reduction of DMC. The wild-type roots had 32.6% DMC, whereas the average DMC of the six transgenic lines was 20.2% (ranging from 15.8 to 23.8%). There seems to be a generalized fact that increases of pVAC through genetic transformation result in a (pleiotropic) decrease in DMC. A reasonable explanation for the simultaneous increase in pVAC and reduction of DMC in the transgenic lines would be that the pathway did not stop at the step

accumulating β-carotene but continued resulting in a higher production of ABA.

**carotenoids in cassava roots**

200 Carotenoids

**5. Evolution and improvements of sampling protocols for measuring** 

Only a small tissue section (5 g) is needed for extracting and quantifying carotenoids in cassava roots. At the inception of the HarvestPlus initiative, there was no information regarding the uniformity of pVAC concentration within the root, among roots of the same plant, among roots from different plants of the same genotype, and on the relative importance of environment, age, and genotype-by-environment interaction. For early work, roots were cut longitudinally in four sections: two diagonally opposed sections were used for DMC determination and the remaining two quarters were chopped into small pieces which were thoroughly mixed, and from this bulked tissue, a random sample was used for pVAC quantification.

The first systematic study analyzing sampling variation of pVAC concentration in a cassava clone that produces yellow roots (average TCC 3.90 μg/g) was published in 2008 [73]. Samples along the longitudinal (proximal, central, and distal section) and across the transversal (periphery, mid-parenchyma, and core) axes of the roots were analyzed. Carotenoids and dry matter content were quantified individually in 243 root samples. Average TCC values, on a fresh weight basis, were higher in the proximal sections (4.10 μg/g) and gradually lower in the central (3.86 μg/g) and distal portions (3.73 μg/g). An opposite trend was observed when carotenoids were quantified in dry weight basis. Carotenoids concentrations were higher in the core (4.13 μg/g) and lower toward the mid-parenchyma (4.04 μg/g) and the periphery of the root (3.52 μg/g), both fresh and dry weight basis. Plant-to-plant variation was only significant for dry matter content. Breeding progress was successful increasing pVAC levels. Along this process, however, laboratory personnel began to observe an increase in the variability of intensity of pigmentation within the root and among roots from the same genotype, even when coming from the same plant. **Figure 4** illustrates some cases with clear differences in intensity of pigmentation particularly across the root. It should be pointed that these differences are not always present and the bottom left photograph shows a case with a more uniform pigmentation. A new and more complete study on sampling variation was published in 2011 [74]. In this new study of many genotypes, most of them with considerably higher levels of pVAC compared with the earlier

**Figure 4.** Illustration of longitudinal (left) and transversal (center) variation in the intensity of parenchyma pigmentation. On right: (**A**): chopped roots; (**B**): homogenized paste after processing roots with a food processor; (**C**): illustration of variation in root color in a full-sib family.

work [73] were analyzed. Variation in aliquot quantifications from the same root was negligible indicating a reliable experimental procedure. A large source of variation for carotenoids was due to differences among the 26 genotypes analyzed (ranging from 2.87 to 12.95 μg/g). In contrast to earlier results, root-to-root variation from the same plant was surprisingly high in some cases and accounted for an average of 25% of the total variation. Plant-to-plant variation was not as high and accounted for 20% of the total variance. Carotenoid content was shown to vary depending on the age of the plant as well, particularly comparing samples harvested at 8 and 10 months after planting. Single-plant evaluations for carotenoid content in cassava, which is a requirement for rapid cycling recurrent selection (described later in this chapter) was acceptable, considering that it reduces in half the time required for evaluation and selection. However, it was suggested that two to three roots per plant are combined together in a sample to better represent each genotype at a standard plant age (10–12 months after planting).

CIAT laboratory began using an industrial-grade food processor that quickly grinds the root samples in to a uniform paste (**Figure 4B**). This approach allowed overcoming the problem of variation in pVAC concentration along and across the root (which resulted in variation in the coarsely chopped pieces, **Figure 4A**), as well as the operational problems of having to use two to three roots per genotype. Early studies made apparent that the variation in DMC along the root influenced the concentrations of pVAC [73]. Variation of DMC across the root, on the other hand, seemed to be less relevant. The influence of variation in DMC in different sections of the root on the quantification of carotenoids was also shown in Ref. [75]. It is important to recognize, therefore, that there is not always a linear relationship between pVAC concentrations reported on a fresh and a dry weight basis. Since DMC is generally around 30–35%, the rule of the thumb dictates that the relationship between TTC and TBC reported on a dry weight basis should be around threefold higher than when reported on a fresh weight basis. However, DMC can vary considerably (from 10 to 50% as shown in **Table 1**) in experimental material. **Figure 5** has been presented to illustrate how a pVAC concentration of 15 μg/g (on a fresh weight basis) will vary when expressed on a dry weight basis, depending on the DMC values of the root. It is important for researchers reporting data on pVAC concentrations to also provide the respective levels of DMC.

**Figure 5.** Illustration of how DMC in the root will affect the concentration of carotenoids expressed on a dry weight basis in a sample that showed 15 μg/g of pVAC on a fresh weigh basis. When DMC is low (22%) pVAC on a dry weight basis tends to be very high (75 μg/g), whereas roots with higher DMC (a desirable trait) show lower pVAC values, if expressed on a dry weight basis (e.g., <45 μg/g).

#### **6. Evolution and improvements of quantifying protocols for carotenoids in cassava roots**

Early work quantifying carotenoids content relied on standard spectrophotometry and HPLC quantifications. Adjustments in the extraction protocol these techniques require, however, had to be made [76, 77].

One of the adjustments was in the separation of the solid and liquid phases that was carried out by centrifugation and not by filtration [26]. Carotenoids are sensitive to ultraviolet (UV) light, pro-oxidants or associated compounds, and high temperature. Thus, steps need to be taken to avoid any adverse changes in this pigment due to such effects protecting them from UV light and avoiding excessively high temperatures. Therefore, special care to avoid direct exposure of ground tissue samples to sunlight is critical. Likewise, the lights in the laboratory need to be protected with UV filters. The typical extraction protocol requires 5 g of root tissue (either fresh or boiled) which is added to a vial with 10 mL acetone. After 10 minutes, 10 ml of petroleum ether are added and mixed using an ultra-turrax for 1 min. Samples are then centrifuged at 3000 RPM, for 10 min, at 10 °C. The organic phase is collected and extraction repeated on the residue with 5 ml of acetone and 5 ml of petroleum ether, followed by centrifugation. Extractions are optimized until it residues turn colorless. Based on preliminary analysis, it was decided that three iterative extractions would be used for fresh root samples and four for boiled samples. The organic phases are combined with 10 mL of 0.1 M NaCl solution and centrifuged (3000 RPM, for 7 min, at 10 °C). This washing process is repeated two additional times. The aqueous phase is extracted with a pipette. Petroleum ether is added to the extracts to adjust volume to 15 ml.

two to three roots per genotype. Early studies made apparent that the variation in DMC along the root influenced the concentrations of pVAC [73]. Variation of DMC across the root, on the other hand, seemed to be less relevant. The influence of variation in DMC in different sections of the root on the quantification of carotenoids was also shown in Ref. [75]. It is important to recognize, therefore, that there is not always a linear relationship between pVAC concentrations reported on a fresh and a dry weight basis. Since DMC is generally around 30–35%, the rule of the thumb dictates that the relationship between TTC and TBC reported on a dry weight basis should be around threefold higher than when reported on a fresh weight basis. However, DMC can vary considerably (from 10 to 50% as shown in **Table 1**) in experimental material. **Figure 5** has been presented to illustrate how a pVAC concentration of 15 μg/g (on a fresh weight basis) will vary when expressed on a dry weight basis, depending on the DMC values of the root. It is important for researchers reporting data on pVAC concentrations to

**6. Evolution and improvements of quantifying protocols for carotenoids** 

**Figure 5.** Illustration of how DMC in the root will affect the concentration of carotenoids expressed on a dry weight basis in a sample that showed 15 μg/g of pVAC on a fresh weigh basis. When DMC is low (22%) pVAC on a dry weight basis tends to be very high (75 μg/g), whereas roots with higher DMC (a desirable trait) show lower pVAC values, if expressed

Early work quantifying carotenoids content relied on standard spectrophotometry and HPLC quantifications. Adjustments in the extraction protocol these techniques require, however,

also provide the respective levels of DMC.

202 Carotenoids

**in cassava roots**

on a dry weight basis (e.g., <45 μg/g).

had to be made [76, 77].

With the extracts obtained, TCC can be determined by visible absorption spectrophotometry, at an absorbance at 450 nm and using the absorption coefficient of β-carotene in petroleum ether (2592) [76, 77]. Partitioning of TCC into concentrations of individual carotenoids is done by HPLC. From the organic phase used for spectrophotometric quantification of TCC, aliquots (15 mL) are taken and partially dried by nitrogen evaporator. Immediately before injection, the dry extract is dissolved in 1 mL of (1:1) methanol and methy tert-butyl ether HPLC-grade and filtered through a 0.22 μm PTFE filter. Separation and quantification of carotenoids are achieved at CIAT using an YMC Carotenoid S-5 C30 reversed-phase column (4.6 mm × 150 mm: particle size, 5 μm), with a YMC carotenoid S-5 guard column (4.0 × 23 mm) in a HPLC, using DAD detector with wavelength set at 450 nm. Peaks are identified by comparing retention time and spectral characteristics against a pure standard and available literature [78].

Breeding projects to increase carotenoids content require screening hundreds of samples in a short period of time. Quantification of carotenoids by spectrophotometry or HPLC limits the number of samples that can be analyzed. Therefore, a large data set linking TCC and TBC data with Near-infrared spectroscopy (NIRS) spectra was gradually developed.

NIRS is based on the absorption of electromagnetic radiation at wavelengths in the range of 780–2500 nm. The interaction between the electromagnetic radiations and the vibrational properties of the chemical bonds results in absorption of a part of the radiation energy. The electromagnetic spectrum is divided into several regions, each of which induces specific molecular or atomic transition and is therefore suited to a specific type of spectroscopy. NIR spectroscopy belongs to the class of methods called vibrational spectroscopy techniques. This class of techniques aims to analyze a product in order to obtain qualitative and/or quantitative information. The principle of absorption can be interpreted as a resonance phenomenon: when the vibrational frequency of a specific chemical bond is equal to the frequency of the infrared radiation, a part of the energy is absorbed by the chemical bond. NIR spectra of foods comprise broad bands arising from overlapping absorptions corresponding mainly to overtones and combinations of vibrational modes involving C-H, O-H, and N-H chemical bonds [79]. Additional information on the theory regarding vibrational NIR spectroscopy is found in more detail in several Refs [80].

The key point is that a NIR spectrum is the resultant of all the elementary absorptions due to the chemical constituents of the product analyzed; the spectrum is as a fingerprint of the sample. This fingerprint contains qualitative and quantitative information about the physical and chemical composition of the sample. Due to this complexity, spectra are treated mathematically in order to extract the relevant information within the spectra linked to the property of interest (carotenoids or other). This step, called calibration, aims to develop mathematical models, which link the reference values to a linear combination of the values of absorbance. The calibration step is based on chemometric methods that applies multivariate analyses such as multiple linear regression (MLR), partial least squares (PLS), or principle components analysis (PCA) to the spectra in order to quantify the property analyzed. Thus, NIRS is a secondary, indirect method, and the calibration step requires a primary method that provides the value of the property for each samples. Once the calibration is developed and validated, it can be applied to new samples and used to directly quantify (actually predict) the property from the spectrum.

In the routine analyses, NIRS method is simple, nondestructive, and rapid, with minimum sample preparation and environmentally friendly. The NIR technique is widely used in the agriculture sector. However, a few studies have been conducted on nutritional properties of fresh tubers or roots using NIR spectroscopy. McGoverin and collaborators inventoried studies on carotenoids in various crops and one related to carotenoids in potato [81]. The efficiency of NIR spectroscopy for predicting TCC, TBC, and DMC in fresh cassava roots has been demonstrated [82]. The models were based on partial least squares (PLS) regression. PLS regression ranged within the linear methods, which assume that the relationship between the independent and dependent variables are linear in nature. However, predictions of a new harvest based on the PLS models were actually "extrapolations" because, year after year, cassava genotypes with higher carotenoids content were obtained by the breeding project. This resulted in a nonlinear response and a tendency to underestimate the highest contents. The use of LOCAL regression algorithm based on large database has circumvented this restriction [83].

The cassava database (6026 samples) has been built over 6 years (2009–2014). For each genotype, two to three commercial-size roots were taken to the lab where they were washed, peeled, and homogenized with a food processor into a homogenous paste. Further analyses were made using aliquots from this homogeneous paste. For NIR analysis, approximately 8 g of ground root tissue was placed in NIR spectroscopy capsules for analysis using a FOSS 6500 monochromator with autocup sampling module. All spectra were recorded from 400 to 2498 nm at 2 nm intervals and saved as the average of 32 scans. Each sample was duplicated. Therefore, spectra from two root subsamples were obtained per genotype. Each of these two samples was measured once. Further analyses were made on the average of the two spectra available per genotype. Spectra were corrected for light scattering using the standard normal variate and de-trend (SNVD) correction. Then, the second derivative of the Log(1/R) spectrum, calculated on five data points and smoothed using Savitzky-Golay polynomial smoothing on five data points, was used in combination with LOCAL regressions to develop prediction models [84].

bands arising from overlapping absorptions corresponding mainly to overtones and combinations of vibrational modes involving C-H, O-H, and N-H chemical bonds [79]. Additional information on the theory regarding vibrational NIR spectroscopy is found in more detail in

The key point is that a NIR spectrum is the resultant of all the elementary absorptions due to the chemical constituents of the product analyzed; the spectrum is as a fingerprint of the sample. This fingerprint contains qualitative and quantitative information about the physical and chemical composition of the sample. Due to this complexity, spectra are treated mathematically in order to extract the relevant information within the spectra linked to the property of interest (carotenoids or other). This step, called calibration, aims to develop mathematical models, which link the reference values to a linear combination of the values of absorbance. The calibration step is based on chemometric methods that applies multivariate analyses such as multiple linear regression (MLR), partial least squares (PLS), or principle components analysis (PCA) to the spectra in order to quantify the property analyzed. Thus, NIRS is a secondary, indirect method, and the calibration step requires a primary method that provides the value of the property for each samples. Once the calibration is developed and validated, it can be applied to new samples and used to directly quantify (actually predict) the property from the spectrum.

In the routine analyses, NIRS method is simple, nondestructive, and rapid, with minimum sample preparation and environmentally friendly. The NIR technique is widely used in the agriculture sector. However, a few studies have been conducted on nutritional properties of fresh tubers or roots using NIR spectroscopy. McGoverin and collaborators inventoried studies on carotenoids in various crops and one related to carotenoids in potato [81]. The efficiency of NIR spectroscopy for predicting TCC, TBC, and DMC in fresh cassava roots has been demonstrated [82]. The models were based on partial least squares (PLS) regression. PLS regression ranged within the linear methods, which assume that the relationship between the independent and dependent variables are linear in nature. However, predictions of a new harvest based on the PLS models were actually "extrapolations" because, year after year, cassava genotypes with higher carotenoids content were obtained by the breeding project. This resulted in a nonlinear response and a tendency to underestimate the highest contents. The use of LOCAL regression algorithm based on large database has circumvented

The cassava database (6026 samples) has been built over 6 years (2009–2014). For each genotype, two to three commercial-size roots were taken to the lab where they were washed, peeled, and homogenized with a food processor into a homogenous paste. Further analyses were made using aliquots from this homogeneous paste. For NIR analysis, approximately 8 g of ground root tissue was placed in NIR spectroscopy capsules for analysis using a FOSS 6500 monochromator with autocup sampling module. All spectra were recorded from 400 to 2498 nm at 2 nm intervals and saved as the average of 32 scans. Each sample was duplicated. Therefore, spectra from two root subsamples were obtained per genotype. Each of these two samples was measured once. Further analyses were made on the average of the two spectra available per genotype. Spectra were corrected for light scattering using the standard normal variate and de-trend (SNVD) correction. Then, the second derivative of the Log(1/R) spectrum, calculated

several Refs [80].

204 Carotenoids

this restriction [83].

The laboratory analyses led to 4277 TCC values that ranged from 0.11 to 29.0 μg g−1 with an average value of 11.6 μg g–1 and 4288 TBC values that ranged from negligible to 20.1 μg g–1 with an average value of 6.9 μg g–1. The standard deviation (SD) was 5.1 μg g–1 for TCC and 3.6 μg g–1 for TBC. All values were measured and expressed on a fresh weight basis. The DMC values (*n* = 5578) ranged between 12.3 and 52.4% with a SD of 5.9%. Between 2009 and 2014, increases in TCC and TBC were 86 and 122%, respectively. The standard error of prediction were 1.38 μg g–1 for TCC and 1.02 μg g–1 for TBC and 1.09% for DMC using LOCAL regression. The scatter plots of NIRS values versus HPLC values for TBC and TCC illustrate the high performances of the models (**Figure 6**). The multiple determination coefficients were higher than 0.9 for both constituents.

After 5 years of harvest and database building, NIR spectroscopy coupled with LOCAL regression led to accurate and robust calibrations for breeding programs aiming at increasing carotenoids content in fresh cassava roots [85]. These results offer immense prospects for many cassava-breeding projects in the world; NIRS overtakes the bottleneck of conventional carotenoids quantification methods. Classically, in a well-equipped laboratory with experienced personnel, 20–30 samples per day can be analyzed; the implementation of NIRS in the analytical chain boosts this number by five to ten. Moreover, the possibility to share data and calibrations between spectrometers make it possible to develop a network for high-throughput phenotyping of fresh cassava roots.

An alternative quantification protocol has been implemented at the International Institute of Tropical Agriculture (IITA) in Nigeria and other cassava research programs in Africa. This method is based on the iCheckTM Carotene technology [86].

**Figure 6.** Scatter plots of TBC (left plot) and TCC (right plot) NIR spectroscopy values versus HPLC values.

#### **7. Evolution and improvements of breeding methods to increase carotenoids in cassava roots**

Cassava breeding relies on a method known as phenotypic recurrent selection. Although it is a simple approach, each cycle of selection requires about 8 years for completion. Elite clones are crossed to produce full- or half-sib families. In the former, the identity of both (male and female) progenitors is known. In the latter, only the female progenitor is known [78]. Large number of botanical seed from the crosses of elite germplasm is generated each year. The seeds are then germinated to produce seedling plants. At this stage, called F1, only one plant per genotype is available. The seedling plants are grown for 11–12 months (the standard age for harvesting commercial cassava) when they are selected based on different traits such as vigor, plant architecture, resistance/tolerance to pest and diseases, and/or starch and root quality traits. Stems of selected plants are harvested and used as a source of planting material for the next stage of selection (single-row trials—SRT). Typically, eight stem cuttings are used to represent each genotype in SRT, which is the first stage where cloned plants are evaluated. The rate of vegetative multiplication (number of cuttings that can be obtained from a given plant) is relatively low in cassava (1:8 to 1:10) and, therefore, it takes several years to have enough planting material from a given genotype to be evaluated in multi-location trials.

Following SRT are the preliminary yield trials (PYT), advanced yield trials (AYT), and uniform yield trials (UYT), which are usually conducted for two consecutive years. Each of these types of trials increases the size of the plots, the number of replications, and/ or the number of locations used in the evaluation process. Usually, 4000–5000 seeds are germinated, and about 4000 seedlings transplanted for the F1 stage. The number of genotypes in SRT, PYT, ADYT, and UYT is gradually reduced from about 2500, 200, 60, and 20, respectively. The entire process takes about 8 years for completion. This lengthy process is necessary because yield data is prone to large experimental errors and affected by genotype-by-environment interaction.

Breeding for increased levels of pVAC, however, is much easier because of the high heritability of the trait. Breeders do not need data from replicated trials using large plots in many locations to identify a genotype with higher levels of pVAC. In fact, a single plant is enough as results from studies on sampling variation demonstrated. Therefore, a special breeding approach—rapid cycling recurrent selection RCRS—was implemented [45]. In this scheme, seedling plants were evaluated for carotenoids content and the best genotypes preselected based on single plant evaluations. Preselection was based on a visual assessment for the intensity of pigmentation that discarded genotypes producing roots with white, cream, or pale yellow roots. Yellow roots from genotypes preselected in the field were then sent to the laboratory for carotenoids quantification. Selection was based primarily on TCC/TBC levels but other traits such as DMC and root yield potential were also taken into account. Selected genotypes were immediately incorporated into the crossing blocks to be used as progenitors.

Each recurrent selection cycle, therefore, lasted 2–3 years (depending on how quickly the selected materials flowered). Selected genotypes were also incorporated into the normal selection process described above (SRT, PYT, AYT, and UYT) to identify genotypes that not only had excellent levels of pVAC but also acceptable to outstanding agronomic performance. Number of genotypes involved was different compared with ordinary cassava breeding. The F1 seedling stage was considerably larger with 8000 to 10,000 seeds germinated and about 5000 to 8000 plants grown through the season. About 1000 to 1500 plants were selected in the field and about 500–800 of them eventually screened for pVAC levels in the laboratory. This breeding approach was very successful and resulted in three to fourfold increases in TCC and TBC in the maximum values observed at the F1 trials in a decade [45]. This kind of genetic progress was in fact unprecedented in cassava and is largely due to the high heritability of the trait.

are crossed to produce full- or half-sib families. In the former, the identity of both (male and female) progenitors is known. In the latter, only the female progenitor is known [78]. Large number of botanical seed from the crosses of elite germplasm is generated each year. The seeds are then germinated to produce seedling plants. At this stage, called F1, only one plant per genotype is available. The seedling plants are grown for 11–12 months (the standard age for harvesting commercial cassava) when they are selected based on different traits such as vigor, plant architecture, resistance/tolerance to pest and diseases, and/or starch and root quality traits. Stems of selected plants are harvested and used as a source of planting material for the next stage of selection (single-row trials—SRT). Typically, eight stem cuttings are used to represent each genotype in SRT, which is the first stage where cloned plants are evaluated. The rate of vegetative multiplication (number of cuttings that can be obtained from a given plant) is relatively low in cassava (1:8 to 1:10) and, therefore, it takes several years to have enough planting material from a given genotype to be evaluated in multi-location trials.

Following SRT are the preliminary yield trials (PYT), advanced yield trials (AYT), and uniform yield trials (UYT), which are usually conducted for two consecutive years. Each of these types of trials increases the size of the plots, the number of replications, and/ or the number of locations used in the evaluation process. Usually, 4000–5000 seeds are germinated, and about 4000 seedlings transplanted for the F1 stage. The number of genotypes in SRT, PYT, ADYT, and UYT is gradually reduced from about 2500, 200, 60, and 20, respectively. The entire process takes about 8 years for completion. This lengthy process is necessary because yield data is prone to large experimental errors and affected by

Breeding for increased levels of pVAC, however, is much easier because of the high heritability of the trait. Breeders do not need data from replicated trials using large plots in many locations to identify a genotype with higher levels of pVAC. In fact, a single plant is enough as results from studies on sampling variation demonstrated. Therefore, a special breeding approach—rapid cycling recurrent selection RCRS—was implemented [45]. In this scheme, seedling plants were evaluated for carotenoids content and the best genotypes preselected based on single plant evaluations. Preselection was based on a visual assessment for the intensity of pigmentation that discarded genotypes producing roots with white, cream, or pale yellow roots. Yellow roots from genotypes preselected in the field were then sent to the laboratory for carotenoids quantification. Selection was based primarily on TCC/TBC levels but other traits such as DMC and root yield potential were also taken into account. Selected genotypes were immediately incorporated into the crossing blocks to be used as progenitors.

Each recurrent selection cycle, therefore, lasted 2–3 years (depending on how quickly the selected materials flowered). Selected genotypes were also incorporated into the normal selection process described above (SRT, PYT, AYT, and UYT) to identify genotypes that not only had excellent levels of pVAC but also acceptable to outstanding agronomic performance. Number of genotypes involved was different compared with ordinary cassava breeding. The F1 seedling stage was considerably larger with 8000 to 10,000 seeds germinated and about 5000 to 8000 plants grown through the season. About 1000 to 1500 plants were selected in the field and about 500–800 of them eventually screened for pVAC levels in the laboratory. This breeding

genotype-by-environment interaction.

206 Carotenoids

RCRS, however, faced some limiting bottleneck in its initial scheme. Selection for TCC/TBC of seedling plants required extraction and quantification of pVAC that was time-consuming. Only six to eight samples per day could be analyzed through HPLC. This resulted in many logistic problems that were gradually identified. The harvesting season lasted up to 4 months rather and 2–3 weeks. This was necessary to screen at least 500–800 samples in the laboratory. Extending the harvesting season for such a long period implied that some genotypes were harvested during the dry season and others after the arrival of the rains. This, in turn had some impact on DMC of the roots because this parameter is highest at the end of the dry season but is considerably reduced after the rains began. The availability of irrigation at CIAT reduced the difference in dry and wet season, but only partially. As the problem of a lengthy harvesting season became evident, CIAT began the development of the protocol for predicting pVAC and DMC based on NIRS, as described above [82, 84]. The possibility of selecting for high pVAC based on reliable NIRS predictions was a major breakthrough. It allowed increasing the number of samples analyzed (from 500–800 to 2000–2500), while reducing the harvesting season (from 4 months down to 3–4 weeks).

An improved RCRS scheme has been recently described [85]. In this scheme, seedling plants (F1 stage) are grown only for 6 months. Since plants are young, only three vegetative cuttings can be taken from selected genotypes. These cuttings are planted to grow a new stage (F1C1) that was not used previously. In the F1C1, each genotype has been cloned and is represented by three plants. Selection is conducted in two steps. The visual assessment done in the field in the old system is still done at the F1 stage, with the difference that it is done when harvested plants are only 6-months old. Only genotypes producing yellow roots are selected, but other traits such as resistance to thrips, adequate vigor, and acceptable yield potential are also taken into consideration. A key feature is that seedlings are transplanted off-season, and the harvest of the plants takes place during the normal harvesting/planting season. Therefore, planting of the F1C1 is done in the usual season. The F1C1 is then grown for a full season and plants harvested at 10–12 months of age. However, in the new system, three plants per genotype are available.

One of these plants is harvested at the end of the dry season for NIRS quantification of pVAC and DMC. Stems from selected genotypes can be used for planting a new crossing block. The remaining two plants of selected genotypes are left in the field and are used as a source of planting material for further phenotypic selection for good agronomic performance in two separate SRT planted in two locations. In the old system, the seedling plant was used for two purposes: as a source of roots for pVAC quantification and the stems were used as a source of planting material. In the new system, these functions are performed by different plants. Quantification of pVAC is done only during the dry season, thus avoiding the variation due to changes in DMC that is somewhat related to the timing of harvest. Harvesting of the stems to be used as a source of planting material takes place only when the rains have arrived in the target environment. The planting material does not need to be stored for (sometimes) a long period of time, waiting for the rains to arrive.

#### **8. The transgenic approach**

As already demonstrated in this chapter, conventional breeding has been very effective in increasing carotenoids content in the storage roots of cassava. However, breeding cassava is time-consuming and cumbersome due to its heterozygous nature. Adoption of new, improved varieties where cassava plays a key food security role is often low. Farmers tend to be reluctant to shift away from the varieties they have grown for decades [6, 87]. The alternative of turning farmers' preferred varieties into vehicles for delivering pVAC through genetic transformation is very appealing. The technology could deliver exactly the same variety that farmers have grown for years but with increased nutritional value. This is a product that conventional breeding could not offer. Therefore, biotechnology tools have also been included among the strategies that have been considered to deliver biofortified cassava to farmers.

The accumulation of carotenoids in the root involves several genes that, as described above, may have anabolic or catabolic function. This further complicates the conventional breeding strategy, since putting all the desirable alleles of the relevant genes into a single variety are complex and takes time. This would be particularly true if the objective is to silence a gene whose function is to catabolize carotenoids, for example, into ABA (**Figure 3**). Not only enzymes that are directly involved in the making or degrading of carotenoids should be the target of breeding. It has been recently shown that the ORANGE (OR) protein is a posttranscriptional regulator of phytoene synthases (PSYs) in plants [88], which adds another level of intervention (conventional or transgenic) for enhancing carotenoids in cassava roots.

Genetic transformation is not only a viable approach to produce cassava clones with increased pVAC in the roots, but it is also a powerful tool for understanding the individual impact of different genes and alleles in carotenogenesis. Examples of the importance of polymorphisms of single nucleotides—SNPs—from relevant genes such as phytoene synthase have been published [34, 63, 89]. The different studies have demonstrated that the substrates necessary for the activity of relevant enzymes were present in roots of most genotypes. Without this precedent, it would be more difficult to design a genetic modification strategy to increase pVAC contents by inserting new gene combinations in commercial varieties, well established in the markets and accepted by consumers.

As stated above, genetic transformation would add nutritional value to farmers' preferred varieties. In addition, this approach would reduce the time required for developing new varieties since it implies handling few genes of the carotenoid synthetic pathway alone, not an entire genome. Finally, it could ensure that the transgenic variety produces a minimum pVAC as has been the case for genetically modified potato or the Golden Rice [90, 91].

There are already at least five examples of crops in which pVAC contents have been substantially increased using transgenes from the pathway of carotene synthesis: rice, maize, potato, tomato, and canola. They have been guided by promoters that express these genes in specific organs, or constitutively. Genetic transformation of rice, with genes from the carotene pathway [92, 93] has shown that it was possible to increase the TCC in the grain up to 27 times (maximum 37 μg/g, DW), of which more than 80% (>30 μg/g, DW) corresponded to β-carotene. In canola, the β-carotene increase was 50-fold [94]. It has been shown that β-carotene in the potato tuber can be increased >3600 times, reaching 47 μg/g DW, with which 250 g of potato satisfy half the RDA [90].

**8. The transgenic approach**

208 Carotenoids

markets and accepted by consumers.

As already demonstrated in this chapter, conventional breeding has been very effective in increasing carotenoids content in the storage roots of cassava. However, breeding cassava is time-consuming and cumbersome due to its heterozygous nature. Adoption of new, improved varieties where cassava plays a key food security role is often low. Farmers tend to be reluctant to shift away from the varieties they have grown for decades [6, 87]. The alternative of turning farmers' preferred varieties into vehicles for delivering pVAC through genetic transformation is very appealing. The technology could deliver exactly the same variety that farmers have grown for years but with increased nutritional value. This is a product that conventional breeding could not offer. Therefore, biotechnology tools have also been included among the strategies that have been considered to deliver biofortified cassava to farmers.

The accumulation of carotenoids in the root involves several genes that, as described above, may have anabolic or catabolic function. This further complicates the conventional breeding strategy, since putting all the desirable alleles of the relevant genes into a single variety are complex and takes time. This would be particularly true if the objective is to silence a gene whose function is to catabolize carotenoids, for example, into ABA (**Figure 3**). Not only enzymes that are directly involved in the making or degrading of carotenoids should be the target of breeding. It has been recently shown that the ORANGE (OR) protein is a posttranscriptional regulator of phytoene synthases (PSYs) in plants [88], which adds another level of

intervention (conventional or transgenic) for enhancing carotenoids in cassava roots.

Genetic transformation is not only a viable approach to produce cassava clones with increased pVAC in the roots, but it is also a powerful tool for understanding the individual impact of different genes and alleles in carotenogenesis. Examples of the importance of polymorphisms of single nucleotides—SNPs—from relevant genes such as phytoene synthase have been published [34, 63, 89]. The different studies have demonstrated that the substrates necessary for the activity of relevant enzymes were present in roots of most genotypes. Without this precedent, it would be more difficult to design a genetic modification strategy to increase pVAC contents by inserting new gene combinations in commercial varieties, well established in the

As stated above, genetic transformation would add nutritional value to farmers' preferred varieties. In addition, this approach would reduce the time required for developing new varieties since it implies handling few genes of the carotenoid synthetic pathway alone, not an entire genome. Finally, it could ensure that the transgenic variety produces a minimum pVAC

There are already at least five examples of crops in which pVAC contents have been substantially increased using transgenes from the pathway of carotene synthesis: rice, maize, potato, tomato, and canola. They have been guided by promoters that express these genes in specific organs, or constitutively. Genetic transformation of rice, with genes from the carotene pathway [92, 93] has shown that it was possible to increase the TCC in the grain up to 27 times (maximum 37 μg/g, DW), of which more than 80% (>30 μg/g, DW) corresponded

as has been the case for genetically modified potato or the Golden Rice [90, 91].

Both the rice grain and the potato tuber have complemented the carotene synthesis route, which in the case of rice was not very active, with encouraging results suggesting that a similar strategy could be attempted for cassava. In the case of maize, genes of the carotenoids synthesis pathway have been introduced in different combinations producing increases β-carotene and other carotenoids, including complex mixtures of hydroxycarotenoids and ketocarotenoids [95, 96].

The synthesis and accumulation of carotenes in plant storage roots such as cassava and sweet potato are just beginning to be understood at the molecular level. It is not yet very clear how the promoters of carotene synthesis genes are regulated in roots. On the other hand, the perception of foods derived from transgenic crops is not yet totally favorable, with exceptions, although they are safe for human consumption [97]. This forces us to think of strategies to reduce opposition to acceptance, such as replacing bacterial genes, which are used today to genetically modify crops, by plant genes. Genome edition using CRISPR/Cas9 or similar molecular scissors is an alternative for modifying alleles in cassava [62]. However, there are at least three factors that must be taken into account when using genetic modification to increase carotene content in cassava roots: the genes themselves (their coding sequences and their origins), controlling sequences for transcriptional control, and interacting proteins such as PSY and OR for posttranslational control of carotenoid production in plants.

As an example, in the case of cassava, having genetically transformed a wild-type genotype that produces white roots with combinations of the bacterial versions of key carotenoid biosynthetic genes (crtB, crtY, and crtI) significantly increased pVAC levels. However, transgenesis could not exceed the maximum pVAC levels attained by conventional breeding (about 90 μg/g DW in the 2016 harvest, unpublished data). These experiments, however, demonstrated that there were bottlenecks for the synthesis of carotenoids in roots with white parenchyma, such as the absence of a PSY capable of effectively synthesizing phytoene, to keep the route operating so that enough carotenoids were produced and accumulated in the root until the harvest time (usually 11–12 MAP). This bottleneck was solved with the introduction of the CRTB (the bacterial version of PSY in plants) enzyme alone. This modification was responsible for increasing TCC from 0.4 to 22 μg/g (DW) and turning roots from white to yellow [34]. In addition, in the same work, it was confirmed that the PSY enzyme was a limiting step of the pathway, demonstrating that a single SNP in the coding region of the PSY gene could partially explain the difference between white and yellow roots. Thus, deficiency in the carotenoid synthesis pathway in the white cassava root was complemented by providing enzymes more effective than the endogenous ones, possibly not regulated by the plant [34, 62], and by showing allele diversity correlated with better efficiency among PSY endogenous enzymes.

Compared with the increases in TCC obtained in potato by transgenesis (maximum 114.4 μg/g DW; [90]), the 13–31 μg/g (DW) obtained in cassava [34, 63, 98, 99], can be considered only moderate. However, these comparisons are made on a dry weight basis that would favor potato (**Figure 5**) because of its considerable lower DMC and starch contents compared with cassava. The same three genes (crtB, crtY, and crtI) were used in cassava and potato, under the control of the same patatin promoters (in fact it was the same construct). However, the TCC baseline of the nontransgenic control in potato was 5.8 μg/g DW, while for cassava, it was only 1.1 μg/g DW, which improved the chances of increasing TCC in the former. If we accept that the genetic modification of cassava still has a potential to raise the carotenoid content in the root, at levels similar to or higher than those reached in potato, the results with the tuber would indicate the way forward with cassava: raise TCC would be more effective by transgenesis if yellow-rooted (nonwhite) plants were modified.

#### **9. Retention and bioavailability studies**

The impact of biofortified cassava roots in the reduction of VAD depends on two factors: (a) how much of the carotenoids present in the raw root at harvest time reach the individual consuming the cassava product; and (b) how much retinol can the individual make with the consumed cassava product. This section describes information regarding these two key steps.

As stated in the introduction, cassava roots have a short shelf life due to PPD. Roots therefore need to be consumed or processed 1–3 days after harvest. A diversity of processing methods has been developed by different cultures resulting in many ethnic products. The easiest and most direct way to consume cassava roots is boiling or steaming them. However, this approach does not allow storing the roots and other processing methods that allow storage for long periods have been developed. Roots can be dried and ground into flour. Drying can be done in an oven, in open air under shaded conditions or by sun drying. Two popular processing methods in West Africa are fufu and gari [100].

A comprehensive review of true retention of carotenoids in cassava roots after alternative processing methods and different storage period has been published [101]. Different authors have highlighted that retention of carotenoids is not only affected by the processing method and length of storage period but also by the cassava genotype [102–104]. Boiling the root is a very simple and popular processing method in many regions of the world, which results in relatively high retentions. There is large variation reported depending on genotypes. Average retention after boiling ranged from 68 [105], around 70 [104], and 74% [100]. DMC in the roots influences true retention of carotenoids after boiling [75, 106]. When the effect of DMC prior to boiling is taken into consideration, retention of carotenoids after this processing method was relatively high (87%) and uniform (retention ranged from 76 to 97%) [75].

Drying the roots soon after harvest is an important method to prevent PPD. Significant differences were observed regarding the drying method employed. The highest β-carotene retention was obtained by oven drying (72%), followed by shade drying (59%), and sun drying (38%) [102]. Similar conclusions were obtained by other authors but with some variation in the average retentions that may be due to the genotype effect [43, 101, 104, 107].

Retention of carotenoids after gari preparation varies widely. Final carotenoids content in gari is a function of the intensity and duration of roasting as well as the duration of the fermentation prior to roasting [101]. Retention reported by Failla and collaborators in 2012 was about 66, 62, and 29%, respectively, for boiling, fufu, and gari [63]. On the other hand, a different study reported average retentions of 74% after boiling, 41 and 22% in raw and cooked fufu, and about 45% in gari [100].

Carotenoids are also lost during storage of the processed product. Chávez and co-workers measured the β-carotene retention during a 4-week storage period with flour and chips that had been either oven-dried or sun-dried [102]. Oven-dried cassava initially retained 72% of β-carotene, which decreased to 40 and 32% after 2 and 4 weeks of storage at room temperature, respectively. Sun-dried cassava had a lower initial β-carotene retention (38%), which was further decreased to 24 and 18% after 2 and 4 weeks of storage, respectively. Retention values for cassava chips were very similar to those of cassava flour. Lower retention levels after storage (at room temperature and in the absence of light) have been reported [108]. Carotenoid content of gari products decreased markedly with time and temperature [109].

The absorption, bioavailability, and conversion of β-carotene, the most prominent carotenoid in yellow cassava, into retinol in the human body depend on several factors. These factors are food- and host-related and depended on, for example, host genotype, availability of fat in the diet, and the food matrix [110]. Beta-carotene in cassava is stored in parenchyma cells, which are more easily destructed in the gastrointestinal tract than, for example, chloroplasts membranes, the primary location of β-carotene in green leafy vegetables. Little is known on the histology of carotenoids stored in cassava roots because of the density of amyloplast. An animal study with gerbils showed a bioconversion factor of 3.7 μg β-carotene to 1 μg of retinol [111]. In a study with 10 healthy American adults, this conversion factor was estimated to be 4.2 μg, but the individual variation between the humans was high (range 0.3–10.6) [112]. A randomized-controlled efficacy study with Kenyan primary school children showed a significant increase of both retinol and β-carotene in the blood after 4 months of feeding with boiled yellow cassava as compared to boiled white cassava [113]. In conclusion, we can say that the β-carotene from cassava is absorbable, bioavailable and converted into retinol in the human body. More research is currently being conducted to provide evidence on the effect of processing in different recipes, as well as for different age groups countries.

#### **10. Conclusions and perspectives**

cassava. The same three genes (crtB, crtY, and crtI) were used in cassava and potato, under the control of the same patatin promoters (in fact it was the same construct). However, the TCC baseline of the nontransgenic control in potato was 5.8 μg/g DW, while for cassava, it was only 1.1 μg/g DW, which improved the chances of increasing TCC in the former. If we accept that the genetic modification of cassava still has a potential to raise the carotenoid content in the root, at levels similar to or higher than those reached in potato, the results with the tuber would indicate the way forward with cassava: raise TCC would be more effective by trans-

The impact of biofortified cassava roots in the reduction of VAD depends on two factors: (a) how much of the carotenoids present in the raw root at harvest time reach the individual consuming the cassava product; and (b) how much retinol can the individual make with the consumed cassava product. This section describes information regarding these two key steps.

As stated in the introduction, cassava roots have a short shelf life due to PPD. Roots therefore need to be consumed or processed 1–3 days after harvest. A diversity of processing methods has been developed by different cultures resulting in many ethnic products. The easiest and most direct way to consume cassava roots is boiling or steaming them. However, this approach does not allow storing the roots and other processing methods that allow storage for long periods have been developed. Roots can be dried and ground into flour. Drying can be done in an oven, in open air under shaded conditions or by sun drying. Two popular pro-

A comprehensive review of true retention of carotenoids in cassava roots after alternative processing methods and different storage period has been published [101]. Different authors have highlighted that retention of carotenoids is not only affected by the processing method and length of storage period but also by the cassava genotype [102–104]. Boiling the root is a very simple and popular processing method in many regions of the world, which results in relatively high retentions. There is large variation reported depending on genotypes. Average retention after boiling ranged from 68 [105], around 70 [104], and 74% [100]. DMC in the roots influences true retention of carotenoids after boiling [75, 106]. When the effect of DMC prior to boiling is taken into consideration, retention of carotenoids after this processing method

Drying the roots soon after harvest is an important method to prevent PPD. Significant differences were observed regarding the drying method employed. The highest β-carotene retention was obtained by oven drying (72%), followed by shade drying (59%), and sun drying (38%) [102]. Similar conclusions were obtained by other authors but with some variation in

Retention of carotenoids after gari preparation varies widely. Final carotenoids content in gari is a function of the intensity and duration of roasting as well as the duration of the

was relatively high (87%) and uniform (retention ranged from 76 to 97%) [75].

the average retentions that may be due to the genotype effect [43, 101, 104, 107].

genesis if yellow-rooted (nonwhite) plants were modified.

**9. Retention and bioavailability studies**

210 Carotenoids

cessing methods in West Africa are fufu and gari [100].

Most of the information described in this chapter relates to the work coordinated and financed by the HarvestPlus initiative. It all began about two decades ago with the simple, yet relevant, idea that the nutritional value of crops could be improved. Since then, the significant progress achieved in different crops was highlighted with the 2016 Food Prize Award to a group of researchers lead by Dr. Howard Bouis.

The significant gains in carotenoids content in cassava roots could only be obtained with the concerted effort of many researchers working in a wide range of disciplines. The first group of cassava varieties with increased levels of carotenoids has been already released in Brazil and in Africa. A second generation of new varieties with higher levels of pVAC and better agronomic performance will soon follow. In addition to the strategic relevance of the germplasm generated, valuable information has been generated ranging from the relationship between carotenoids and DMC in the roots to retention and bioavailability information. Enhanced levels of carotenoids resulted in an unexpected reduction of PPD. The new protocol to screen for carotenoids content based on NIR was developed and prompted further changes in the breeding approach. The different constructs for genetic transformation not only resulted in varying degrees of success but also exposed unexpected responses from the plant, such as the reduction in DMC.

The release of varieties, rich in pVAC, requires strategies for the efficient production of planting material to be distributed to farmers as well as participatory approaches to promote their adoption. Deploying these varieties will provide excellent opportunities for nutritional studies and development of new food products and alternative processing methods. The concerted effort of many researchers and institutions and the valuable financial support of key donors and investors have been motivated by the magnitude of VAD. Hopefully, the scientific community will soon be able to document the impact of these efforts in the livelihood of millions of people affected by VAD.

#### **Author details**

Hernán Ceballos1,2\*, Fabrice Davrieux<sup>3</sup> , Elise F. Talsma<sup>2</sup> , John Belalcazar1 , Paul Chavarriaga1 and Meike S. Andersson<sup>2</sup>


3 Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement (CIRAD), UMR Qualisud, St Pierre, Reunion Island, France

#### **References**


[4] Nweke F. New Challenges in the Cassava Transformation in Nigeria and Ghana. EPT Discussion Paper No. 118. Washington DC, USA: International Food Policy Research Institute (IFPRI); 2004

better agronomic performance will soon follow. In addition to the strategic relevance of the germplasm generated, valuable information has been generated ranging from the relationship between carotenoids and DMC in the roots to retention and bioavailability information. Enhanced levels of carotenoids resulted in an unexpected reduction of PPD. The new protocol to screen for carotenoids content based on NIR was developed and prompted further changes in the breeding approach. The different constructs for genetic transformation not only resulted in varying degrees of success but also exposed unexpected responses from the

The release of varieties, rich in pVAC, requires strategies for the efficient production of planting material to be distributed to farmers as well as participatory approaches to promote their adoption. Deploying these varieties will provide excellent opportunities for nutritional studies and development of new food products and alternative processing methods. The concerted effort of many researchers and institutions and the valuable financial support of key donors and investors have been motivated by the magnitude of VAD. Hopefully, the scientific community will soon be able to document the impact of these efforts in the livelihood of mil-

, Elise F. Talsma<sup>2</sup>

3 Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement

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Hernán Ceballos1,2\*, Fabrice Davrieux<sup>3</sup>

2 HarvestPlus Organization, Cali, Colombia

\*Address all correspondence to: h.ceballos@cgiar.org

(CIRAD), UMR Qualisud, St Pierre, Reunion Island, France

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*Edited by Dragan J. Cvetkovic and Goran S. Nikolic*

Carotenoids are one of the most widespread pigment groups distributed in nature; more than 700 natural carotenoids have been described so far. These pigments are known for versatile roles they play in living organisms; however, their most pivotal function is involvement in scavenging of reactive oxygen species and photoprotection. In the same time, carotenoids as natural pigments with important biological activities, such as antioxidant and provitamin A activity, have a great potential in the food, feed and pharmaceutical industries. They can be either extracted from plants and algae or synthesized by various microorganisms, including bacteria, yeasts, filamentous fungi and microalgae.

Carotenoids

Carotenoids

*Edited by Dragan J. Cvetkovic* 

*and Goran S. Nikolic*

Photo by LIgorko / iStock