Demystifying Emulsifiers as Additives through Molecular Gastronomy: A Contribution to Rise Consumer's Sovereignty

*Lígia Rebelo Gomes, Cláudia Silva and Catarina D. Simões*

## **Abstract**

According to the Food and Agriculture Organisation of the United Nations, one third of food produced annually for human consumption results in food losses or wastage, which is environmentally degrading, economically unviable, ethically incorrect, and does not contribute to sustainable development. The use of additives can help prevent the waste of food that is still fit for consumption in a world where about 16% of the world's population goes hungry. Food additives may overcome the problem of limited supply of natural ingredients, increase the shelf life of foods and simplify the complex cooking procedures. To raise the consumer's knowledge about food additives, this work presents and explain in a simple manner some physical/ chemical properties of emulsifiers, namely fatty acids esters and sucrose esters of fatty acids. Moreover, this work reviews and illustrates, recurring to recipes of molecular gastronomy, how these additives are used in food preparation to achieve and maintain certain desirable characteristics, how they contribute to obtain a better result in final preparation, and how they can be used in modern cuisine. Preparations coming from the discipline of molecular gastronomy have been chosen since they are based on laboratory related procedures and only use few ingredients, including an additive.

**Keywords:** food additives, emulsifiers, sucrose esters, mono- and diglycerides of fatty acids, molecular gastronomy

## **1. Introduction**

Despite all the literature available concerning the food additives, the availability of easy reading yet scientifically sounding information about the nature, chemical composition, role, and safety of the additives is scarce. It appears that, in a world where fake information is quickly spread in social media and food additives start to be perceived as harmful, the development of tools that permit the demystification of their use and therefore their acceptance, is essential as they, at least, contribute to the expansion of food's the shelf life and fight waste.

Following the work that our group has undertaken, to raise the consumer's knowledge about additives, we desire to present and explain in a simple manner some physical/chemical properties of emulsifiers, EU codes, in the 400–499 range, namely fatty acids esters and sucrose esters of fatty acids. How those additives are used in food preparation in order to achieve and maintain certain desirable characteristics, how they contribute to obtain a better result in final preparation and how they can be used in modern cuisine will be illustrated and reviewed recurring to recipes of molecular gastronomy. Preparations coming from the discipline of molecular gastronomy have been chosen since they are based on laboratory related procedures and they use only a few ingredients, including an additive.

This review chapter is a means to attain a final objective which is to develop tools for the demystification of food additives targeted to wider target people audience than researchers as, for example, undergraduate students of chemical, biological and nutrition sciences, students of gastronomy, chefs and high school teachers, in the hope of encouraging them to spread genuine scientific information based, as far as possible, on the current state-of-art. This will contribute to the raising of the level of knowledge of those additives thus allowing the consumer to make informed decisions.

Emulsifying agents are discussed first, the chapter begins with a simple review of emulsification and interfacial tension. Then the E471 and E473 class of additives will be presented (chemical constitution and relevant physical properties). For each class the most common uses in food processing will be discussed as well as their role in a particular recipe. Finally, a brief summary concerning their safety will be made.

### **2. Interfacial tension and emulsions**

A mixture is formed when it is thermodynamically more stable than the states given by its separate components. That occurs when the interaction energies between molecules of the mixture are higher than the sum of the interaction energies of the primary constituents [1]. This condition is usually satisfied when the molecular constituents that are to be mixed have cohesive forces of similar magnitude. Otherwise, they do not tend to mix, even when shaken and whipped, and phase separation occurs spontaneously [2]. Anyhow, components must contact in the interfaces and interfaces are particular: surface phenomena such as surface tension (or interfacial tension) applies. The term interfacial tension usually relates to the liquid/liquid and liquid/solid phase boundaries; the term surface tension applies to the liquid/gaseous interface and the term surface free energy applies for the solid/ gaseous. The surface free energy affects wettability [3].

Interfacial tension and surface tension are defined as the work that must be done to maintain the unit of interface or free surface, respectively [1]. By way of an example, consider the interface of two immiscible liquids (oil and water), see **Figure 1**. The molecules placed in the boundary surface are interacting with molecules of both liquids, as such, the sum of interactive forces that act in the boundary molecule is unbalanced. That means that there is a net force that tends to push those molecules into their respective bulk. Thus, in order to keep the surface or the interface, a network of molecules is formed against the pull, as so, work must be done in order to maintain or increase the size of the interface. As consequence, when a water solution is vigorously stirred with an oil solution, the droplets formed after stirring tend to aggregate and phase separation occur. This happens because the system will minimise its energy when it reaches the smaller surface of contact [3].

The additives that will be here presented can act as surfactants that is, they have the capability of modify the surface tension/interfacial tension/surface free energy of the heterogeneous systems. As so, they can act as emulsifiers and/or humectants. This ability comes from their chemical constitution: they are amphipathic molecules, *e.g*., they are unbalanced in their charge distribution, which means that one

*Demystifying Emulsifiers as Additives through Molecular Gastronomy: A Contribution to Rise… DOI: http://dx.doi.org/10.5772/intechopen.96743*

#### **Figure 1.**

*Schematic representation of the differences on the net forces in two liquid that are not mixable, e.g., polar water molecules (in red circles) and non-polar oil molecules (blue curved lines): in the bulk of each liquid the net forces between molecules are balanced while on the interface the net forces acting on the molecules are not, as consequence of the differences on the mutual interaction energies. Such molecules tend to undergo a displacement to the respective bulk solution and new molecules are displaced to the inner solution to replace them. This dynamic is kept in order to maintain the interface and the energy required to do it, per surface unit, is called the interfacial tension.*

can recognise a part of the molecule that is polar (hydrophilic) and another that is non-polar (hydrophobic), see **Figure 2(a)** for an example (a sucrose ester). Those kinds of molecules are named as surfactants agents due to their ability to modify the interface/surface tension value. If this value is lowered by action of the surfactant agent, then they stabilise the interface. The mechanism by which this is possible is related to the amphiphilic nature of the molecule: the polar part of the molecule will interact preferentially with the polar liquid (water) and the non-polar preferentially with the oil, see **Figure 2(c)** and **(d)**.

Two common two-phase systems in food products are emulsions and foams. Emulsions are composed of two immiscible liquids that may be stabilised by a type of surfactant named emulsifier [3]. Foams are dispersions of gas bubbles in a liquid continuous phase containing foaming agents, i.e., surface-active substances that ensure the stability of the system [2].

An emulsion is characterised by the existence of small droplets that constitute the dispersed phase imbibed in an immiscible liquid, which forms the continuous phase, see **Figure 2(d)**. Foams are a state where air (gas) is confined as bubbles into a continuous phase that could be a water solution, fat, or a hydrocolloid. When the emulsion is called oil–water (O/W), the oil is the dispersed phase and water is the continuous phase and the reverse happens for a water–oil emulsion (W/O). An emulsifier agent is a tension-active molecule (or mixture of molecules) that has the ability to be adsorbed at the O/W/W/O interface, lowering the tension and preventing the dispersed-phase droplets from aggregating [4]. The type of emulsion formed when an aqueous solution is mixed with an oil and with an emulsifying agent depends on the solubility of that agent: "The phase where the emulsifier is most soluble will be the continuous phase", hence, even if a preparation has 60% of constituents of oily origin an O/W emulsion will be obtained if the emulsifier chosen is soluble in water [1].

The mechanisms by which the emulsifiers stabilise emulsions and dispersions are based in electrostatic or/and steric phenomena. Non-ionic emulsifiers, as sucrose esters of fatty acids and mono and di-glycerides of fatty acids are good

#### **Figure 2.**

*(a) Structure of a sucrose di-ester (derived from stearic acid). The polar part of the molecule is identified as hydrophilic and the non-polar as hydrophobic (or lipophilic), (b) usual scheme representation of the molecule, (c) the surface-active agent has the ability to stabilise the interface: The polar part of the molecule interacts preferentially with the polar liquid (water) and the non-polar preferentially with the oil; (d) when the mixture is shaken, droplets of oil disperse into the water phase (continuous phase).*

steric stabilisers. In most cases, they cope with presence of any ions in solution and do not react with them, allowing for a wide application in various systems, especially in water of unknown hardness [1, 5].

Thus, in order to stabilise an emulsion, the appropriate emulsifier must be selected. One of the challenges is to select an emulsifier with an adequate hydrophilic/lipophilic balance (HLB) suitable for the system. The HLB is a measure of the hydrophilicity of an emulsifier: higher HLB values indicate a higher hydrophilicity. Emulsifiers with HLB values of 3.5–6 tend to be the most suitable for W/O emulsions, and those with HLB values from 8–18 are best suited for O/W emulsions. Surfactants with values ranging from 7–9 are good wetting agents [1, 5].

Most commercial emulsifiers, especially glycerides of fatty acids and sucrose esters, are mixtures of mono-, di- and tri-esters. Emulsifier HLB values are largely dependent on the monoester content; a greater monoester content leads to a higher HLB value [5]. The HLB value also depends on the chain length(s) of the fatty acids attached to the emulsifier; short fatty acid chains lead to higher HLB values [6].

Another factor to take into account is the molecular weight of the surfactant: emulsifiers with low molecular weight are usually more rapidly adsorbed onto the phase interface and prevent droplet coalescence during homogenisation, they are a preferable choice for making micro-emulsions, as those used in food processing [7, 8].

*Demystifying Emulsifiers as Additives through Molecular Gastronomy: A Contribution to Rise… DOI: http://dx.doi.org/10.5772/intechopen.96743*

**Figure 3.**

*A graphical representation of the surface tension (*γ*) of water solutions of a fatty acid ester of a glucopyranoside at various concentrations. The arrow indicates the value of the critical micelle concentration (CMC): before the CMC there is a strong change of the surface tension; after the CMC that value is minimum and stays constant and the air micelle in water is stabilised. Adapted from [9, 10].*

#### **2.1 The critical micelle concentration**

A micelle is considered a supramolecular aggregate shape of molecules with spherical that is formed by surfactant molecules. Micelles in water are aggregates with the hydrophilic part of the molecule contacting with surrounding solvent and the hydrophobic tail regions in the micelle centre. The micelle content can be air (see **Figure 3**) or a fat. In colloidal and surface chemistry, the critical micelle concentration (CMC) is defined as "the concentration of surfactants above which micelles form and all additional surfactants added to the system will form micelles".

**Figure 3** depicts a graphical representation of the surface tension (γ) of water solutions of a fatty acid di-ester of a glucopyranoside at various concentrations. When the concentration of the surfactant is low, the whipping of the solution will not give "stable bubbles", but this will occur after the surfactant concentration reaches the CMC (value indicated by the arrow). As seen, before the CMC there is a strong change of the surface tension and after the CMC that value reaches a minimum and stays independent of the concentration, giving stability to the air droplets. For the mono and di-glycerides of fatty acids and as well for sucrose esters the stability of the micelles is given by the strong hydrogen bonds that the sugar moiety can make with water [9, 10].

The CMC depends on the temperature, the pressure and it also changes with the composition of the water solution, namely with the presence of other surface-active compounds.

#### **3. E473 and E471 classes of additives**

This revision will concern about two classes of food additives, the sucrose esters of fatty acids (E473) and the mono- and diglycerides of fatty acids (E471). Both are widely used nowadays in food industry and thus present in the preparations available in supermarket shelves. The sucrose esters of fatty acids are used to obtain mostly O/W emulsion (but not only) and mono- and diglycerides of fatty acids are used to obtain W/O emulsions. Both are good agents for form stable foams.

#### **3.1 Sucrose esters of fatty acids (sucrose esters), E473**

Sucrose esters of fatty acids class of compounds (E473) are tensioactive agents that have fatty acids esterified in sucrose. They synthesised by the esterification of the available hydroxyl groups of sucrose (O-α-D-glucopyranosyl-(1 → 2)-β-Dfructofuranoside) and fatty acids. In **Figure 4** the structure of sucrose is depicted: this is a disaccharide formed from condensation of glucose and fructose to produce α-D-glucopyranosyl-(1 → 2)-β-D-fructofuranoside. The free hydroxyl groups are numbered as red labels; the primary hydroxyl groups are more reactive since they are not so steric hindered, as such, their reaction with fatty acids is most probable giving the sucrose mono-, di-, or tri-esters. They can be obtained with a very wide range of variation in the molecular structure due to the different combinations between the length of the fatty acid chain, the degree of saturation of the fatty acid and the degree of esterification. The length of the fatty acid chain generally varies between 4 and 18 carbon atoms, but the most used fatty acids for esterification are i) the saturated lauric, myristic, palmitic and stearic acids, see **Figure 5(a)**, and ii) the unsaturated oleic and erucic acids, **Figure 5(b)**.

Sucrose esters can be synthesised either chemically or enzymatically. In industrial scale they are obtained by trans-esterification (see **Figure 6**): the reactants are sucrose, a triglyceride (TG), potassium carbonate as catalyst in *N*,*N*dimethyformamide (DMF, **CAS:** 68–12-2) or dimethylsulfoxide (DMSO, **CAS:** 67–68-5) as solvents. This reaction gives a product that is a mixture of the sucrose monoester (> 50%), sucrose di-esters (~10%) higher esters, sucrose and TAG [11]. This processing method has issues connected with the removal of the solvents that have high boiling points [426.2 ± 0.7 K (~153°C) for DMF and 463 ± 1 K (~189°C) for DMSO] and the separation of the monoester from the other by-products. Sucrose monoesters may be also produced in large scale by the reversible transesterification process, see **Figure 6**, using sucrose and a fatty acid methyl ester as reagents and sucrose esters and methanol will be obtained as products. This this a reversible process, so, the methanol should be removed during the reaction. Under the optimal conditions this protocol can provide material containing upwards of 70% of the monoester [11].

Chemical synthesis uses solvents such as DMSO and/or DMF that display toxicity [12–14]. In alternative, enzymatic based reaction procedures have been developed more recently [15, 16], and present some advantages over the chemical reactions: i) they can take place under lower temperatures ii) they use less toxic solvents and iii) they allow better control over the reaction final products. However, the main obstacle to commercial sucrose ester production by the enzymatic means is the fact that the reaction has to be run in batch rather than flow mode [17]. The most promising enzymes are a group of esterases that catalyse the hydrolysis of lipids. In the present context, important lipases are the

#### **Figure 4.**

*The structure of produce* α*-D-glucopyranosyl-(1* → *2)-*β*-D-fructofuranoside (sucrose). The labelled atoms are carbons atoms containing hydroxyl groups available for sterifications. The primary C1', C6*′ *and C6 are the most reactive.*

*Demystifying Emulsifiers as Additives through Molecular Gastronomy: A Contribution to Rise… DOI: http://dx.doi.org/10.5772/intechopen.96743*

**Figure 5.**

*The most used fatty acids for esterification into sucrose (a) saturated fatty acids (b) unsaturated fatty acids.*

**Figure 6.** *Reaction pathways to obtain the sucrose esters.*

triacylglycerol lipases that selectively hydrolyse insoluble triacylglycerol's at the substrate-water interface [18, 19].

Most of the physical–chemical characteristics of a determined sucrose ester are related to the kind of fatty acids used for esterification. In **Figure 7** the chemical formula of a di-ester of sucrose is show, where the esterification occurred in the 1 and 6' position and the fatty acid used was the stearic acid.

Sugar esters are considered as typical low molecular weight surfactants, they decrease the surface tension on interfaces and form micelle sub-structures of oil and air in water after reaching the CMC. The CMC depends on the structure of the sugar ester. Having low molecular weight, sucrose esters are soluble in water and they will make the stabilisation of O/W emulsions. The HLB value depends on the type of fatty acids used in esterification [20]. The relationship between HLB watersolubility and composition in the sucrose esters is dependent on three factors: (a) the degree of substitution; (b) the alkyl chain length in the ester group; and (c) the presence of dienoic or trienoic acyl groups. Providing the right blend of mono- and di- sucrose esters linear values of HLB range from 10–16. If mixtures are made with glycerides and sucrose esters the HLB range from 4–16 can be covered [21]. The HLB scale of sucrose esters is usually defined by suppliers and it should be merely considered as an index of ranking: from the most hydrophilic (high HLB) to the most lipophilic (low HLB) within the sucrose ester family.

#### **Figure 7.**

*The chemical formula of (a) a mono-acyl-sucrose ester and of a (b) di-acyl sucrose ester.*

Some authors [22] worked on the clarification of the real HLB for the sucrose esters. They concluded that the experimental HLB of sucrose monoesters, would be around 11–12 for short fatty chains (6 to 12 carbons) and around 10–11 for long fatty chains (14 to 18 carbons). Those values are slightly different than the values presented by the producers that range from 2–16. Sucrose esters powders may come with residual amounts of fatty acids and those, by their turn, may appear in the neutral form or in the protonated form. Fatty acids in neutral state are also able to act as surfactants and they are good to stabilise the O/W emulsions.

The easiest parameter to evaluate the thermal stability of sucrose esters is the melting point, it can vary within the 40–60°C range depending on the type of fatty acids that have been esterified. Nevertheless, the emulsions can be heated to about 180°C (they decompose above 220°C) without compromise their emulsification properties of the surfactants (however a colour change may be observed in high temperatures if sugars are present due to caramelisation). The thermal properties of sucrose esters depend on the various HLB values: high or medium HLB values tend to be vitrified by melting. Since sucrose esters are usually a mixture, apart from crystallisation, they also present amorphous structures which slowly crystallise in time [23].

Sucrose esters are stable in the pH range of 4 to 8, so they can be used as an additive in most foods. At pH higher than 8, saponification (hydrolysis of the ester bond to release the original sucrose and the salt of fatty acids) might occur. Hydrolysis could also occur at pH lower than 4.

#### *3.1.1 Common uses for sucrose esters*

Monoesters of sucrose esters are most commonly studied, although applications of di-esters or higher esters have also a wide range of applications. The sugar esters can be used in white sauces, dairy products that are alternative to milk, aerated dairy desserts, like ice cream, mousses, bakery, confectionery, preparation of cereal based products [18].

Thus, no wonder that the use of sucrose esters as surfactants in the food industry has been increasing exponentially. Apart from the emulsification power that will be discussed next, they also present advantages concerning about their biodegradability: after ingestion, the sucrose esters are hydrolysed by digestive enzymes into sucrose and their component fatty acids that are further metabolised in the usual way [5].

In a general approach the use of sucrose esters as additive in food preparations will allow the formation of O/W emulsions. The presence of the sucrose ester will replace the oil (fat) continuous phase by smaller globules/oil droplets imbibed into a water

*Demystifying Emulsifiers as Additives through Molecular Gastronomy: A Contribution to Rise… DOI: http://dx.doi.org/10.5772/intechopen.96743*

phase. The encapsulation of the oil will protect it against oxidation, which occurs in the presence of oxygen and light. This will improve the stability of the prepared food and will increase the shelf life. They can also act as humectants as they can make the stabilisation of the water/air interface, as so, they are popular in preparations of foams. In addition of acting as emulsifiers and texturizers, they present several other advantages such as the protection of proteins from flocculation, the prevention of sugar crystallisation and they form stable structures resulting from interaction with starch.

#### *3.1.1.1 Preparations based in oil/water emulsions*

Typical O/W emulsions are mayonnaise and dressings. Sucrose esters are used in their preparation as an alternative to egg yolk. Sucrose esters with a high HLB value are soluble in cold water so the preparation can be made at room temperature. They also do permit the formation of stable emulsions with very small droplets; this is only possible due to their ability of significantly lowering the tension in the oil–water interface. Another important feature is that emulsions can be made with homogeneous droplet radius; this will avoid coalescence of the small droplets into to bigger, ones once the former have higher Laplace pressure.

Visually the emulsions made with sucrose esters appear like a white sauce and they impart a creamy sensation. A large number of small oil droplets give the impression of a higher fat content, thus, the creamy mouth feel and flavour perception of the preparations and will mimic the sensation of taste provided by fats. In addition, the small droplets will scatter the light into various directions increasing the brightness of the preparation and giving a whiter appearance. In the preparation of mayonnaise, the emulsion must be made before adding the acidic components in order to prevent the hydrolysis of the ester that can occur at values below pH 4.

Sucrose esters can replace milk proteins in the preparation of white sauces and improve the stability of the emulsion. There are sucrose esters (or mixture of sucrose esters) in the market that have higher O/W emulsification power than proteins. The small size of the oil droplets obtained and their homogeneity, provide high stability against flocculation and the emulsions may not require a high viscous continuous phase. The viscosity of the continuous phase is, generally, obtained by adding starch (in this case it will work as a stabiliser). The advantage in gastronomic terms is to have sauces that are less sticky, more satiny and with good power to release flavours. Furthermore, the preparation does not decompose by Maillard reaction1 during pasteurisation or sterilisation. Drinks based on soy, almond, rice and coconut are popular alternative products to milk. The addition of sucrose esters in their processing allows the stabilisation of the oil emulsion in a continuous hydrophilic phase that usually displays low viscosity. Again, "the milk alternative product" will appear whiter. Usually the mono-esterified esters are chosen since they interact better with proteins that are pH sensitive due to their isoelectric point. The tension-active agent will interact with these proteins, stabilising preparations against flocculation [28].

<sup>1</sup> Reaction between amino acids and reducing sugars that initiate a complex cascade of reactions during heating, resulting in the final formation of substances brown called melanoidins. It begins with the nucleophilic attack of the α-carbonyl group of a reducing sugar, for example, to the amino group of proteins. The occurrence of the reaction in food depends on several factors: high temperatures (above 40°C), water activity in the range of 0.4 to 0.7, pH in the range of 6 to 8 (preferably alkaline), relative humidity of 30% at 70%, the presence of transition metal ions such as Cu2 + and Fe2 +, which can catalyse the reaction [24–27].

#### *Natural Food Additives*

Pre-preparations of fat-soluble substances for further water dilution: fat soluble substances such as omega-3 and some natural dyes may be previously mixed with sucrose esters and kept as a concentrated emulsion for further incorporation into water-dispersed form in beverages, dairy products, and confectionery.

### *3.1.1.2 Preparations based in oil/water emulsions with significant sugar content*

In preparations with sugar like chewy soft candy and ices, sucrose esters are also widely used nowadays. The reason for that is related to the capacity of sugar esters to control sugar crystallisation. Sucrose esters promote the formation of secondary grains during crystal growth process and prevent the agglomeration by making a homogenisation of the crystal size. The delay on sucrose crystallisation may be due to the decrease of the molecular mobility [29] or to the disrupting the crystal lattice due to molecular interactions between sucrose and the additive [30]. The polar group of the sucrose ester interferes with crystallisation after cooling precluding the crystal growth: during cooling the non-polar fatty acid of the sucrose ester will move from the polar sugar solution to the less polar sugar crystal. As a result, the sugar crystals will be surrounded by a layer of fatty acids that will prevent further crystal growth see **Figure 8**. The re-solubilisation and re-crystallisation of the sugar will be inhibited. The sugar ester layer around the micro-crystalline nuclei stabilises the outer shell of water and prevents water evaporation from the sugar mixture (in fondants). This will result in a gritty mouth feel.

Bakery products with sucrose esters as additives are very usual. The esters form a flexible network with gluten making the preparations more resistant to mechanical stress during processing. In addition, they contribute to maximise gas retention due to their properties of stabilisation of the air/solid interface (see 3.2.1.3). Sucrose esters are also able to interact with starch: they have the ability to complex with the linear and helical amylose and the branched amylopectin. In consequence the temperature and enthalpy of gelatinization will change [31]. The capacity of interaction with starch varies with the length of the monoester and the degree of saturation. The sucrose fatty acids interact preferentially with amylose and form a helical complex during gelatinization. This reaction prevents retrogradation of the starch and increases the long-life term [32].

#### *3.1.1.3 Aerated oil/water emulsions preparations*

Sucrose esters are good humectant agents since they significantly decrease the surface tension value of water solutions or hydrocolloids. They help into aeration and promote foam stabilisation.

Foams correspond to the macroscopic manifestation of an air/liquid or air/ solid interface, air appearing as bubbles. The dispersive phase can be a water

#### **Figure 8.**

*Effect of sucrose ester into the crystal growth of sugar (a) without the surfactant; (b) with surfactant: the crystals are surrounded by the fatty acid chain that protect the micro-crystals from further growth.*

*Demystifying Emulsifiers as Additives through Molecular Gastronomy: A Contribution to Rise… DOI: http://dx.doi.org/10.5772/intechopen.96743*


**Table 1.**

*Values for hydrophilic/lipophilic balance (HLB) and surface tension (Du Noüy method) for several sucrose esters surfactants and for water at T = 20°C [33]. Concentration above the critical micelle concentration.*

solution, fat, protein or hydrocolloids. Water has a high surface tension thus, without a surfactant, it is difficult to retain air inside. The addition of a sugar ester to the preparation reduces the surface tension in the interphase and makes the foam stable. **Table 1** gives the values for surface tension of several sucrose surfactants [33, 34].

The lower the surface tension the easier to prepare the foam and to keep it over time. The value for the surface tension of air/water interfaces that is obtained at concentrations higher than the CMC tends to be in the range of 24–44 mN/m. One of the most effective surfactants is the sucrose laurate with HLB of 15 and surface tension of 28.5 mN/m.

The presence of three phases (water/oil/air) puts additional challenges to the stabilisation of the preparation. First, the presence of fatty materials destabilises the aqueous foams because the water film around the air bubble will be squeezed by the presence of the fat: this will decrease its thickness leading to the collapse of the air bubble. If an emulsion is made previously to aeration the surfactant must be used to stabilise the fat droplets first in order to preclude the interference of the fat with the water layer around the bubble. For this purpose, the use of a surfactant with medium HLB value (6–11) is the better choice since they can make stable oil-in-water emulsions. However, to ensure a longer shelf life without syneresis and retraction, preparations must contain stabilising and gelling agents.

The second challenge deals with the presence of proteins. Proteins are surface active agents and compete with the sucrose esters in the surface of air/water and in the interface of oil/water droplets and, as seen, generally the formation of the oil/water emulsion precedes the aeration process. In preparations of creams those proteins are egg and milk proteins. It seems that egg proteins are more effectively absorbed in the oil/water interface than milk proteins [35], but low weight surfactants are able to displace the proteins from the fat layer drops, lowering the steric stabilisation made by the macromolecules, especially if they are daily proteins [36]. This will increase the probability of the coalescence of the emulsion during whipping. Those effects have been studied at least one decade ago and it seems that the content of sucrose ester is a key to obtain a good stability for the foam: in concentrations lower than 0.1% the amount of sucrose ester that is absorbed into the surface oil films is insignificant while in concentrations higher than 0.1125% most of the proteins are displaced by the surfactant in the oil/water interface [37], that is, little protein is left around the oil droplets of the emulsion due to the preferential adsorption of sucrose ester over milk proteins (**Figure 9**).

Additionally, the shear stress given by whipping promotes a higher desorption of milk proteins on the oil/water interface, that is, the egg proteins are better for

#### **Figure 9.**

*There is a competitive absorption in the oil–water interface between milk proteins: in consequence a mixed surfactant protein film is formed around oil droplets. This last film is weaker than that obtained when only protein is present and will contribute to the destabilisation of the emulsion. The higher the content of sucrose ester in the preparation, the higher the degree of substitution.*

preparation of stable foams, but in order to get a stable foam, partially coalescence of fat droplets should occur [38, 39].

*3.1.2 Examples for the use of sucrose esters in molecular gastronomy*

## *3.1.2.1 Preparation of a butter cream*

The ingredients for the preparation of a butter cream are: egg whites (4 units); sugar (267 g) and water (60 g); unsalted butter (454 g); salt (1.5 g) and sucrose esters (5.4 g) (**Figure 10**).

Preparation consists in:



This is a typical O/W emulsion even if it looks like the opposite; although the amount of fat is higher than that of the water, the nature of the surface-active agent (its HLB) dictates the type of the obtained emulsion. On first step, protein egg whites (including globulins as albumins and mucoproteins) that can act also as tensioactive agents are mixed with the sucrose esters. After the preparation of the sugar syrup the tensioactive agents are to be mix with the aqueous phase. Sucrose esters interact in stabilisation of sugar crystallisation process but here the sugar syrup is kept always warm, as so, sugar esters will play a role into the solid/liquid interface only after aeration. Careful must be taken with temperature: not too low to avoid sugar crystallisation; not too high to avoid proteins denaturation (sugar esters protect protein against from flocculation). The oil phase is added slowly at room temperature in order to form the emulsion. In step 5 the whipping allows the air to be retain in the pre-obtained emulsion, that will be stabilised by the sucrose esters in the air/(O/W) emulsion.

## *3.1.2.2 Cream bubbles*

Ingredients: Cream (250 g); milk (125 g); water (87.5 g); water for sugar syrup (37.5 g); sugar (37.5 g); sucrose ester (5 g) and spices (seeds of 1 piece cardamom, seeds of 1 piece of vanilla, ginger flower).

Preparation consists in:


Foams and whipped emulsions are stabilised by dairy proteins. The illustrated preparation has fats, proteins, hydrocolloids, and colloidal components. The presence of the sucrose ester contributes, together with the proteins, to stabilising the interacting components of the preparation as an emulsion when they are first boiled and mix (homogenisation). Then, at last minute, the aeration process to make the foam should be implemented (aeration).

Proteins (namely dairy proteins) and the sucrose ester present in the bulk preparation compete at interfaces air/water and oil/water. At high concentrations, the water-soluble sucrose ester is more effectively adsorbed at those interfaces and displace the proteins. This mechanism occurs depending on the ratio protein/surfactant. As so, proteins they are to be apportioned between the bulk phase (water) and interfaces (oil/water).

The mix of the sucrose ester with proteins in the interfacial layers makes the surface film to be weaker when compared only with proteins, contributing to the destabilisation of the emulsion. This is important to the aeration process: when a cream is whipped, shear forces are acting into an aqueous dispersive system with a high interface area that encloses, oil and gaseous phases brought together. The destabilisation of the emulsion will make the incorporating of air easier during the foam manufacture and at same time the aqueous phase behaves like a reservoir of active material surface, say, protein and surfactant. This is beneficial for building a network of partially coalesced fat globules around the air bubbles and for stabilising new formed air-water interfaces.

#### *3.1.3 Safety and maximum daily intake*

Sucrose esters were approved and registered by European Food Safety Authority or EFSA under the E number of 473. The authorization for the use of sucrose esters of fatty acids (E 473) was made in 1995 in various preparations but not for colour stabilisation and sweetening preparations but, having a wide range of applications, sucrose esters are used in many industrial preparations and the European Food Safety Authority (EFSA) requested to the food industries extra data about the use of the sucrose esters in baked goods and flavoured drinks (chocolate milk, cocoa, eggnog, drinking yogurt and whey based drinks) in 2004 [40].

The and the panel responsible for the evaluation concluded that the daily the exposure to the additive exceeded the acceptable daily intake of 40 mg/ kg/bw/day.

Meanwhile health concerns on high consumption of the sugar esters of fatty acids raised since it is used in several categories of food of preparations where data was not provided from producers. As so, the current exposes could be higher than that advised by the panel.

In 2010, the panel recommend the collection of reported used form industry for fine bakery and flavoured drinks and in the light of new data, ANS Panel indeed concluded that the use of sucrose esters of fatty acids (E 473) may lead to an exposure higher than the 40 mg/kg/bw/day [41].

The uncertainty regarding the amount of E473 that could be ingested by the population, due to the wide use of these additives, led the commission to launch of a public call for data aiming at collecting reported use levels from industry or analytical data and a motorization of the presence of the surfactant on the label of foods in 2014. A refined analysis was made based on the preference of the population for consuming certain food categories and brands (flavoured drinks, fine bakery, only dairy-based and almond drinks). The panel concluded that the refined exposure in the brand-loyal scenario could be estimated as 54 mg/kg/bw/per day for babies (12–35 months) and as 124 mg/kg/bw/per day for children (3–9 years), but as not all categories were included this value may be overestimated [42].

The panel in 2018 recommended a higher detail in data collection in order to provide the scientific experts with data for a more realistic estimation. Thus, the actual studies point that there is an over cumulative intake of this additive by the European population due to quantity of preparations available in marked that use sucrose esters of fatty acids [42].

#### **3.2 Mono- and diglycerides of fatty acids (E471)**

Monoglycerides and diglycerides are lipid molecules composed of a single fatty acid or two fatty acids, respectively, esterified with hydroxyl groups of the glycerol [43, 44]. The food additive itself, consists of a mixture of glycerol mono-, di- and

*Demystifying Emulsifiers as Additives through Molecular Gastronomy: A Contribution to Rise… DOI: http://dx.doi.org/10.5772/intechopen.96743*

tri-esters of fatty acids derived from edible oils and fats [45, 46]. The amount of mono- and di-esters should be at least 70% according to the European Regulation (2012), and may also contain a small quantity of free glycerol (maximum 7%) and fatty acids [45]. Therefore, it is a mixture of substances which may contain different mono- and diglycerides of fatty acids, depending on the fatty acid sources. The fatty acids in mono- and diglycerides may be saturated or unsaturated, being the lauric, linoleic, myristic, oleic, palmitic, and stearic fatty acids the most prevalent. [46].

A revision concerning the synthesis of monoglycerides was published some years ago [46]. Their synthesis can be made according to schematic reactions presented in **Figure 11**. As seen, mono- and diglycerides of fatty acids may be produced by chemical methods, such as inter-esterification (glycerolysis) of fatty acid esters with glycerol and direct esterification of glycerol with fatty acids [47–49]. Some methyl esters of the fatty acids used are depicted in **Figure 12**. Both reactions occur under alkaline conditions at a high temperature (200–250°C), and yield a mixture of mono-, di- and triglycerides in addition to a reduced fraction of free glycerol [46, 50]. Alternatively, mono- and diglycerides of fatty acids may be produced by enzymatic hydrolysis of triglycerides [51, 52]. The advantages of enzymatic synthesis are higher yields and mild reaction conditions, resulting in products of higher quality and lower energy consumption [53].

Regardless of the chosen method, it is usually necessary to separate and to concentrate these glycerides [48]. Monoglycerides may be separated from the

**Figure 11.**

*Synthesis of monoglycerides, diglycerides and triglycerides via trans-esterification (glycerolysis) using methylfatty acids and glycerol in a basic reaction medium (adapted from [46]).*

**Figure 12.**

*Examples of methyl esters fatty acids used in the esterification of glycerol.*

diglycerides, triglycerides, and glycerol by molecular distillation. This a method to separate and purify thermally unstable compounds and substances with low vapour pressure and higher molecular weight, without significant thermal decomposition [48]. Molecular distilled monoglycerides contain an equilibrium of 1-monoglycerides and 2-monoglycerides dependent on the temperature used during the process. The monoester content is usually 90–95% in commercially distilled monoglycerides [50]. Distilled monoglycerides have a better dispersibility in water than the monoand diglycerides of fatty acids because they present high purity and well-defined molecular structure, forming a liquid crystalline mesomorphic phase with ordered bilayers of the fatty acid chains, separated by water layers associated with the polar groups [50]. This feature is useful in food products where interactions with watersoluble ingredients (e.g., starch components) or the aerating properties of fat-free products are of importance [54]. When distilled monoglycerides are heated to their melting point with water, a gel is formed in which the water layers alternate with lipid bilayers [55]. The melting point depends on the chain length of the fatty acid and on the purity of the monoglyceride [50].

Mono- and diglycerides of fatty acids are amphipathic molecules, which can be represented as depicted in synthetic pathways of **Figure 11**. They can be absorbed in the water/oil surfaces once the hydrophilic head has a high affinity for water, and a lipophilic tail to the oil [49]. The structure of mono- and di-glycerides of fatty acids classifies them as surfactants, i.e., surface-active agents or molecules that migrate to the interface between two phases (solid, liquid, or gas).

The functional properties of mono- and diglycerides of fatty acids are determined by the HLB of the mixture that will depend on i) the type of fat used as the base ingredient, i.e., if the original fat is saturated or unsaturated, ii) the length of the hydrocarbon chain fatty acids and iii) the percentage of monoglycerides. The mono- and di-glycerides of fatty acids are non-ionic oil soluble surfactants, predominantly lipophilic and only sparingly soluble in water, having a low HLB. Therefore, they stabilise W/O emulsions such as margarines and spreads, by forming reversed micelles in oil [50, 56]. They are also used to inhibit fat crystallisation in some O/W emulsions because this improves the stability of the food product to refrigeration conditions, for example, in dressings. Moreover, oil-soluble emulsifiers can be used in combination with water-soluble emulsifiers to facilitate protein displacement from fat droplets during the manufacturing of ice creams, whipped creams, and toppings.

#### *3.2.1 Common uses for mono- and diglycerides of fatty acids*

The food additive mono- and diglycerides of fatty acids offers the food industry many solutions for optimal product formulation and processing. When used as additive in food products such as bread and baked products and ice cream, monoand diglycerides of fatty acids require a high concentration of monoglycerides [57, 58]. The high interest of the food industry in the utilisation of mono- and diglycerides of fatty acid additives is due to the fact they are safe for consumption and have useful properties that improve the production of food products [59].

Food-grade mono- and diglycerides of fatty acids have been studied as surfaceactive substances or foaming agents. Diglycerol monomyristate influences the foamability and stability of olive oil foams, depending on the surfactant concentration, and the size of its solid particles [60]. Moreover, the addition of a mixture of distilled mono- and diglycerides of edible saturated fatty acids from vegetable origin with a total content in monoglycerides larger than 90% result in high stable foams of rapeseed oil, in which a dense layer of surfactant crystals at the oil-air interface protects the air bubbles, thus preventing the dissolution and coalescence

*Demystifying Emulsifiers as Additives through Molecular Gastronomy: A Contribution to Rise… DOI: http://dx.doi.org/10.5772/intechopen.96743*

of the system [61]. In addition, there is reported evidence of Pickering stabilisation of gas bubbles by monoglyceride crystals at the air–oil interface. Gunes *et al*. observed that the air bubbles produced in sunflower oil with a monoglyceride (98% saturated) were covered by a layer of monoglyceride crystals preventing rapid dissolution and coalescence of the oil foam [62]. In addition, whipping is an important mechanical process for the effective coverage of the bubble surface with the stabilising layer of fat crystals as compared to foams obtained by depressurization [62].

## *3.2.2 Examples for the use of mono- and diglycerides of fatty acids in molecular gastronomy*

*3.2.2.1 Mono- and diglycerides of fatty acids to prepare "olive oil foam"*

To prepare "olive oil foam", two ingredients are necessary: extra virgin olive oil (200 g) and mono- and diglycerides flakes (200 g) **Figure 13**.

Preparation consists in:


## *3.2.3 Safety and maximum daily intake*

According to the European Regulation the presentation of the food additive E-471 varies from a pale yellow to pale brown oily liquid to a white or slightly offwhite hard waxy solid. The solids may be in the form of flakes, powders, or small beads [63].

Mono- and diglycerides of fatty acids are the most widely used food emulsifiers, accounting for around 70% of the world production of food emulsifiers [3, 56]. Commercial mono- and diglycerides of fatty acids usually contain 45–55% monoglycerides, 38–45% diglycerides, 8–12% triglyceride and 1–7% free glycerol [50]. Mono- and diglycerides of fatty acids are also produced in the gastrointestinal tract during the hydrolysis of the food derived triglycerides by gastric and pancreatic lipases [64]. Mono- and diglycerides of fatty acids have been evaluated by the Joint

**Figure 13.** *A olive oil foam prepared with diglycerides flakes.*

FAO/WHO Expert Committee on Food Additives in 1974. The commission recommended a specific risk assessment for its use in food products for infants under the age of 12 weeks [65]. In 2017, the safety of mono- and diglycerides of fatty acids when used as a food additive was re-evaluated by the EFSA Panel on Food Additives and Nutrient Sources added to Food for ages above 12 weeks of age [66]. Since the hydrolysis of mono- and diglycerides of fatty acids is likely to occur in the gastrointestinal tract with production of free glycerol and free fatty acids. For this reason, the Panel also evaluated the safety of the food additives glycerol (E 422) and fatty acids (E 570). No evidence for adverse effects was reported in toxicity studies that assessed these additives. Neither genotoxic nor carcinogenic effects have been observed [67]. On the other hand, exposure to mono- and diglycerides of fatty acids may be compared with the fat consumed, due to the similarity in their metabolic breakdown. US dietary guidelines recommend an overall fat consumption of 30% of total energy intake and saturated fat consumption of 10% of total energy intake [68]. High saturated fat intake has been associated with higher risk of coronary heart disease and mortality [69]. Although not all saturated fats have the same biological activity, the saturated fatty acids palmitic acid and myristic acid, often used in E-471 additives, may have the most negative impact on serum lipidic levels [70]. However, EFSA Panel reports a small contribution of mono- and diglycerides of fatty acids to the daily fat intake, representing around 0.8–3.5% of the recommendation [67]. EFSA Panel concluded that there was no safety concern regarding the use of these food additives for the general population and that there was no need for a numerical acceptable daily intake [66]. In addition, since the dietary exposure to emulsifiers has not increased over the 10-year period there is no reason to suspect that the dietary exposure may cause a safety concern [47].

As mentioned before, the additives here discussed act as surfactant food additives. Independently of the assessment and evaluation, it is worthy to be mention that there are studies concerning their possible effects on intestinal barriers and microbiota that probably will keep the experts watchful. Recent studies suggest that high doses of emulsifier consumption may contribute to the rising incidence of several diseases involving the gastro-intestinal tract [71–73]. The rise of scientific reports and population studies will help risk assessors make adequate re-evaluations concerning the authorised food emulsifiers.

## **4. Conclusions**

The utilisation of sucrose esters of fatty acids and mono- and diacylglycerides of fatty acids as food additives has been increasing exponentially. Those molecules act as surfactants and, depending of their mixtures and nature of the esterified fatty acids, they can present a wide range of HLB values. As so, they are useful for promoting the stabilisation of oil–water; water–oil and water/oil-gas emulsions. They are a common ingredient in industrial bakery, white sauces, alternative dairy to milk, aerated dairy desserts, ice creams, mousses, confectionery and preparation of cereals based products. They are obtained by chemical or enzymatic synthesises from natural reagents as fatty acids, sucrose and glycerol by esterification present advantages concerning about their biodegradability: after ingestion, they are hydrolysed by digestive enzymes into sucrose/glycerol and their component fatty acids that are further metabolised in the usual way. Despite this, concerns about their safety are rising since the daily intake doses may become higher than the maximum safe daily intake doses. Evaluation panels made studies about refined exposure in the brand-loyal scenario concluding that the daily intake doses could be overestimated specially in babies and children.

*Demystifying Emulsifiers as Additives through Molecular Gastronomy: A Contribution to Rise… DOI: http://dx.doi.org/10.5772/intechopen.96743*

## **Author details**

Lígia Rebelo Gomes1,2\*, Cláudia Silva2,3 and Catarina D. Simões3,4

1 LAQV-REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences of the University of Porto, Porto, Portugal

2 FP-ENAS - UFP Energy, Environment and Health Research Unit, University Fernando Pessoa, Porto, Portugal

3 Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal

4 CIBIO-InBIO Research Center in Biodiversity and Genetic Resources, University of Porto, Vairão, Portugal

\*Address all correspondence to: lrgomes@ufp.edu.pt

© 2021 The Author(s). Licensee IntechOpen. 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.

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## **Chapter 10**

## Molecular Breeding of Sweetpotato Carotenoids

*Muhammad Zubair Khan, Miho Takemura, Takahashi Maoka, Jun-ichiro Hattan, Motoyasu Otani and Norihiko Misawa*

## **Abstract**

Sweetpotato [sweet potato; *Ipomoea batatas* (L.) Lam.] is the seventh most valued food crop of the world. It has an inherent ability to grow under diverse agroecological and microclimatic zones ranging from tropical and subtropical zones to temperate areas with its tuberous roots enriched with the secondary metabolites of immense nutritional value. Among these, carotenoids are the most conspicuous one for having their use in nutritional, pharmaceutical, food, feed, aquaculture, and cosmetic industries. In food industries, carotenoids are used as food additives being antioxidants with attractive colors. Despite the immense economic importance, sweetpotato has received lesser attention in terms of its breeding with improved varieties. The conventional method of breeding by crossing has not been much successful due to the complexity of genome sterility and cross-incompatibility. Hence, the modern molecular breeding approaches, e.g. genetic, genomic, and metabolic (pathway) engineering, have been applied to this crop by some of researchers in Japan, Korea, and China to generate various cultivars with improved quantities and qualities of carotenoids. This has also opened a new gate for molecular breeders to engineer new sweetpotato cultivars enriched with carotenoids under current global scenario of dramatically rising climatic changes where novel food resources are bitterly needed, especially under alarmingly growing world population, the majority of which suffers from malnutrition.

**Keywords:** sweetpotato, carotenoids, molecular breeding, metabolic engineering, pathway engineering

#### **1. Introduction**

Sweetpotato [*Ipomoea batatas* (L.) Lam.], also described as "sweet potato," belongs to the family Convolvulaceae and occupies the seventh position among the food crops of the world after wheat, rice, maize, potato, barley, and cassava [1, 2]. The largest genus in the family Convolvulaceae is *Ipomoea,* consisting of 600–700 species, among which, only *I. batatas* is cultivated widely as a food crop around the world [3, 4]. In comparison with other tuber crops, sweetpotato comprises higher contents of carbohydrates, many minerals, and more protein estimates than other vegetables [5, 6]. It also contains much higher levels of provitamin A, vitamin C, and minerals than those of rice or wheat [7]. Hundred grams of raw sweetpotato contain 1.57 g of protein, 20.12 g of carbohydrates, 3 g of total dietary fiber, 41.8 g

of total sugars, 30 mg of calcium, 0.61 mg of iron, 25 mg of magnesium, 47 mg of phosphorous, 337 mg of potassium, 55 mg of sodium, 0.3 mg of zinc, 2.4 mg of vitamin C, 0.5 mg of niacin, 0.2 mg of vitamin B6, 14,187 IU of vitamin A (VA), 0.2 mg of vitamin E, 11 μg of vitamin B-9, and 8509 μg of β-carotene (β,β-carotene) [8]. The starch in sweetpotato is easy to digest. Therefore, it is a valuable constituent in the preparation of excellent weaning meals [9]. It is a source of food supply to combat malnutrition in the developing nations, since the tuberous roots (tubers) are enriched with starch and dietary fiber, along with carotenoids, anthocyanin, ascorbic acid, potassium, calcium, iron, and other bioactive ingredients [10–13]. For people of South East Asia and Africa, this crop is the main source of β-carotene [10, 14]. The tubers of the Japanese cultivars are diverse concerning carotenoids accumulation [15]. Sweetpotato may exert diverse health positive effects, since it contains high amounts of numerous phytochemicals in roots or leaves [6, 16]. *I. batatas* cultivars with color-fleshed tubers have been reported for their excellent bioactivities, such as antimutagenic [17], free radical scavenging [18], hepatoprotactive, reduction of liver injury [19, 20], anticancer [21–23], antioxidative activities [23–25], antimicrobial activity, antihypertension, anti-inflammatory, antidiabetic, anticaries effect, ultraviolet protection [23], and chemopreventive activities [26]. Previous reports also suggest that its tubers may be useful for treating peptic ulcers [27]. The genome of *I. batatas* is structurally complex and has a size of 4.8–5.3 pg/2C nucleus [28]. Due to the existence of polyploidy, sweetpotato is a hexaploid species (2n = 6x = 90) that has a basic chromosome number of 15, [3] with a huge genome size of 2200–3000 Mbp [29]. The genetic studies on this species are exhausting, since it is difficult to generate seeds and to evaluate the effects of polyploidy on the genome [30, 31]. Complex structure of its genome also manifests self and cross-incompatibility, causing barrier for genetic studies on important agronomical characters [3, 32]. Its tubers exhibit various colors, such as white, yellow, orange and purple orange, and yellow and orange-fleshed lines, were shown to contain β-carotene as the predominant carotenoid [11–13, 33, 34]. Annual yield of sweetpotato is currently exceeding the value of 105 million metric tons, 95% of which is shared by the developing countries. China is the world's leader among all in sweetpotato consumption that counts about 66% of the total global consumption. China is followed by Nigeria and Tanzania, though, each of these last two countries shares only 4% of the total global consumption [35]. By applying the conventional breeding, biofortification of sweetpotato involved the selection of orange-fleshed varieties, to combat vitamin A deficiency among the developing nations [36]. In Japan, high β-caroteneaccumulating varieties, such as "Benihayato,*"* "J-Red,*"* and "Sunny-Red," were initially developed by Japanese breeders at the Kyushu-Okinawa Agricultural Research Center (formerly the Kyushu National Agricultural Experiment Station), Miyakonojo, Miyazaki, Japan [37–39]. The enhancement of sweetpotato with provitamin A carotenoids (PVACs) has also been the area of research focus for the HarvestPlus (a company headquartered in Washington, DC, USA, involved in the development of nutritious food crops through biofortification and promotion of such crops) since the launch of its projects on biofortification [40]. Genetic modification of sweetpotato by using the transgenic tools and in order to improve the nutritional quality offers huge scope, and numerous research reports have already been published on genetic modification of sweetpotato using molecular gene engineering technologies [41]; however, it is the immense need to overcome hidden hunger, specially the one related with the insufficiency of provitamin A carotenoids among the poorly fed but rapidly growing populations in the developing countries by molecular breeding of sweetpotato varieties on sustainable basis.

## **2. Carotenoids and their distribution**

Carotenoids, the visible colors of life, are the 40-carbon isoprenoids synthesized naturally by fungi, bacteria, algae, and cyanobacteria [42–44] and conspicuously by green plants including bryophytes [45] and higher plants [44, 46, 47]. Being intracellular, carotenoids are commonly located in the membranes of chloroplasts, mitochondria, or endoplasmic reticulum [48]. Approximately 750 carotenoids have been reported so far [49, 50].

Maoka et al. (unpublished) analyzed carotenoids that were extracted from the orange tubers of the cultivar W71. It was consequently found that there were β-carotene-5,8,5′,8′-diepoxide (13.8% of the total carotenoids), β-carotene-5,6,5′,8′ diepoxide (9.2%), β-carotene-5,8-epoxide (4.6%), β-cryptoxanthin (3.2%), β-cryptoxanthin-5′,6′-epoxide (2.2%), lutein (2%), and zeaxanthin (trace amounts), in addition to β-carotene (59.3%). The biosynthetic pathway of these carotenoids is proposed in **Figure 1**. The carotenoids with 5,6-epoxy-β-ring or with 5,8-epoxyβ-ring are unique to sweetpotato. The tubers of the Japanese cultivar "Benimasari" were also found to accumulate not only the unique carotenoids, such as β-carotene-5,8,5′,8′-diepoxide (40.5% of the total carotenoids), β-carotene-5,8-epoxide (6.5%), β-cryptoxanthin-5′,8′-epoxide (10.5%), and β-carotene (10.5%), but also typical carotenoids that included 5,6-dihydroxy-β-ring (named ipomoeaxanthins) [15].

### **2.1 Role of carotenoids in animals**

Animals, with very few exceptions [51, 52] are unable to synthesize them [53, 54]; however, carotenoids are accumulated by crustaceans, crabs, fish, crayfish, prawns, mammals, and in insects such as butterflies. The animal and human diet must include carotenoids as essential nutrients [44].

In marine animals, astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) has been reported as the most commonly stored carotenoid pigment [55]. It is

#### **Figure 1.**

*Proposed carotenoid biosynthetic pathway in sweetpotato orange tubers [10].*

responsible for the red/pink coloration of crustaceans [56, 57] and the flesh of salmonoids [58]. Astaxanthin has received much attraction for its likely role in preventing cardiovascular diseases and aging caused by UV light in human body [59]. Chemical structures of some major dietary carotenoids are shown in **Figure 2.**

Both á -carotene and β-carotene have provitamin A activity and are converted to retinol in the human body [60–64]. Carotenes such as the lycopene and β-carotene play a potential role in human nutrition and act as protectants against diseases, such as lycopene protects against cardiovascular [65], aging-related diseases, macular degradation of eye [66, 67], and certain types of cancers including gastrointestinal, cervix, breast, and prostate cancer [47, 61, 68–73].

Beneficial effects of dietary carotenes, α-carotene, and β-carotene on human health related to enhancement of immune system and minimizing the risk of cancer are due to their antioxidant potential [69, 74]. β-Carotene, α-carotene, and β-cryptoxanthin are provitamin A carotenoids (PVACs) and hence they are the main precursors of vitamin A (VA) in the human body [75].

**Figure 2.** *Structures of some major dietary carotenoids.*

## **2.2 Carotenoids use in disease prevention**

Carotenoids, especially astaxanthin, have been reported to enhance both the nonspecific and specific immune system and protect cell membranes and cellular DNA from mutation [44, 76]. Intake of fruits and vegetables rich in carotenoids mainly lycopene, α-carotene, β-carotene, β-cryptoxanthin, zeaxanthin, and lutein lowers the risk of morbidity and mortality by cardiovascular diseases and atherosclerosis [69]. Epidemiological studies have reported that lycopene can lower the risk of prostate cancer [77] and in its ability to quench singlet oxygen; it is 2- to 10-fold stronger than β-carotene and α-tocopherol, respectively [78]. Clinical studies have reported that the lycopene-enriched foods are protective against oxidative DNA damage in leukocytes in vitro [79] and prostate tissue in vivo [80]. β-Carotene is useful in reducing the risk of ischemic heart disease and myocardial infarction [81]. In the macular region of human eye including eye lens, two xanthophylls, lutein and zeaxanthin, exist in high concentrations and are regarded very important carotenoids for eye health. Reports suggest that these two carotenoids protect eye from high energy UV light and are excellent reactive oxygen species scavengers [82]. The role of lutein and zeaxanthin as macular pigments and their function in eye health has been reported in previous studies [83]. It has been anticipated that phytoene and phytofluene which are colorless precursors of other carotenoids possess light absorption in UV-A and UV-B range and protect skin by their photo-protective characteristics [84, 85]. Astaxanthin is also known as the super antioxidant. Since, it contains particular molecular configuration, making it extremely powerful antioxidant consequently, protecting cells against oxidation by quenching singlet oxygen and dissipating the energy as heat. It has the strong potential for scavenging free radicals and effectively breaks peroxide chain reactions [86, 87]. Studies have showed that the low-density lipoprotein (LDL) high cholesterol levels in mice decreased when supplemented with astaxanthin. Neither β-carotene nor canthaxanthin produced the same effect. Astaxanthin or other carotenoids can decrease the oxidation of the lipid carriers and thereby reduce the risk of atherosclerosis [88]. It also has positive effects in case of antitumor activity [89].

### **2.3 Industrial uses of carotenoids**

All carotenoids show antioxidants activities appearing in a variety of colors in red, yellow, and orange; therefore, carotenoids are used as natural pigments in food, food supplements, nutraceuticals, pharmaceuticals, and cosmetic industry and various biotechnological purposes [90, 91]. Global carotenoids market touched \$1.5 billion (\$1500 million) in 2017 with a projection of \$2.0 billion by 2022 [92]. In a previous report, the global market for carotenoids was \$766 million in 2007. The expected projection for the year 2015 was \$919 million with a compound annual growth rate (CAGR) of 2.3%. In 2007, β-carotene alone shared the market value at \$247 million; this segment was predicted to be worth \$285 million by 2015 with CAGR of 1.8% [91, 93]. In horticultural crops, they appear as a trait of attractiveness, adding value to the marketing potential of fruits and vegetables [94, 95]. Green algae Haematococcus pluvialis*,* which is the natural source of astaxanthin, has been reported for huge amounts ranging from 10,000 to 40,000 ppm (mg/ kg) of astaxanthin in addition to other important carotenoids such as β -carotene, lutein, and canthaxanthin [58, 96]. Industrially, astaxanthin has been utilized as a feed supplement for cultured fish and shellfish [97, 98]. Other diverse biological functions of astaxanthin include an involvement in cancer prevention [99], enhancer of immune responses [100], and a free radical quencher [58, 101]. It is evident, therefore, that astaxanthin is a biomolecule with huge biofunction potential both to the pharmaceutical and food industries [58].

## **3. Sweetpotato carotenoids**

The carotenoids present in the sweetpotato leaves can scavenge free radical agents as singlet-oxygen quenchers [102–105]. In a recent analytical report [105], the total phenol, carotenoid, anthocyanin, and flavonoids contents of the sweetpotato leaves ranged from 2.0 to 22.5 (g/100 g DW), 0.9 to 23.4 (β-carotene equivalents/100 g; BET/100 g), 2.2 to 24.5 (color value/g DW), and 62.8–272.2 (catechin equivalents; μg/g), respectively [105]. Consumption of sweetpotato in Asia ranges from its use as additional food of minute status to a very vital supplementary food to rice and/ or other root and tuber crops [106]. It is cooked or used to make cakes, chapatis, mandazia, bread, buns, and cookies [107]. In the United States and some other developed countries, sweetpotato is strictly used as a luxury food. In Japan, it is used in novel plant products and/or nutraceuticals [108]. By using absorption spectroscopy, Ishiguro et al. [109] analyzed carotenoids from eight cultivars of yellow-fleshed sweetpotato and compared them in terms of their carotenoids. By HPLC analyses, they revealed some 17 different carotenoids from yellow- and orange-fleshed sweetpotato. In yellow-fleshed sweetpotato, the major carotenoids included β-carotene-5,8, 5′,8′-diepoxide (32–51%) and β-cryptoxanthin 5,8-epxide (11–30%), whereas β-carotene with amounts ranging from 80 to 92% were dominant in the orange-fleshed cultivars. For other orange cultivars, e.g. W71 and "Benimasari," carotenoid composition in the tubers has already been described along with a comprehensive metabolic pathway [10, 15]. Kammona et al. [110] analyzed and compared the carotenoid composition in some Malaysian orange, yellow, purple, and white sweetpotato tubers. They reported the highest total carotenoid contents from orange sweetpotato followed by yellow, purple, and white sweetpotato. Among the individual carotenoids analyzed, β-carotene existed in all types ranging from 91.95 ± 2.05 μg/g DW in white sweetpotato to 376.03 ± 11.05 μg/g DW in orange sweetpotato tubers. Traces of zeaxanthin were reported with values 5.44 ± 3.23 μg/g DW and 20.47 ± 2.03 μg/g DW in yellow and white sweetpotato, respectively. Lutein was available only in orange sweetpotato at trace amount of 0.91 ± 1.03 μg/g DW. Purple sweetpotato contained only β-carotene (113.86 ± 14.17 μg/g DW) with absence of other carotenoids [110].

Islam et al. [111] performed HPLC analyzes of *trans*- and *cis*-β-carotene from raw and boiled sweetpotato which included three orange-fleshed, three yellowishcream-fleshed, and one white-fleshed varieties of sweetpotato. The deep-orangefleshed variety Kamalasundari (BARI SP-2) showed the highest amounts of β-carotene among all the varieties followed by yellow varieties. On the other hand, from one of the two white-fleshed varieties, only trace amounts of β-carotene were obtained with no amounts at all from the other one. Their results proposed that the orange-fleshed varieties of sweetpotato contain the highest amounts of β-carotene in raw as compared to those which were boiled.

Despite huge economic value, sweetpotato has not received due importance as compared with common staple crops such as wheat, maize, and rice. World increasing hidden hunger, especially in developing countries, needs new foods and nutrition sources on sustainable bases. In this regard, sweetpotato not only offers immense nutritional, medicinal, industrial, and potential benefits but is also a new horizon in modern industrial biotechnological uses for biofunction development through the latest molecular tools and technologies of molecular plant breeding.

#### **3.1 Isolation and functional identification of carotenoids biosynthesis genes**

The heterologous complementation expression system in *Escherichia coli* offers unique tool for functional analysis of isolated new carotenoids biosynthesis genes

#### *Molecular Breeding of Sweetpotato Carotenoids DOI: http://dx.doi.org/10.5772/intechopen.101849*

from different organisms [112]. Carotenoid biosynthetic pathway in microorganisms, such as *Erwinia uredovora* and *Erwinia herbicola* (reclassified as *Pantoea ananatis* and *Pantoea agglomerans*, respectively), is specified by a gene cluster, encoding biosynthetic enzymes that function in a pathway starting with the synthesis of geranylgeranyl pyrophosphate (*GGPP*) and ending in the synthesis of zeaxanthin glucosides [113, 114]. Complete carotenoid gene clusters or part of it from *E. uredovora* or *E. herbicola* have been introduced into *E. coli,* which is otherwise a nonpigmented bacterium, and such transformed *E. coli* engineered in a way that they accumulate a range of colorful carotenoids [114, 115]. Since carotenoids are derived from isoprenoid precursors, *E. coli* can accumulate carotenoids by coupling an endogenous isoprenoid biosynthetic pathway with enzymes encoded by transformed genes of carotenogenic organisms such as *E. uredovora*. Hence, the biosynthetic pathway can be reconstructed in vivo even if the enzymes are of such diverse origin as those encoded by bacteria and plants [116–118]. The expression of carotenoid genes in *E. coli* has been useful for identifying function of gene products [118–120], the manipulation of the pathway [121, 122], investigating transcriptional regulators of carotenoids biosynthesis genes [123], and the isolation of new genes encoding enzymes of the carotenoid biosynthetic pathway [124] or enzymes catalyzing the synthesis of carotenoid precursors [125].

Misawa et al. [55] isolated and functionally identified the carotenoids biosynthesis genes cluster that included *crtB* (phytoene synthase), *crtI* (phytoene desaturase), *crtW* (β-carotene ketolase), and *crtZ* (β-carotene hydroxylase) from *Agrobacterium aurantiacum* (reclassified as *Paracoccus* sp. strain N81106). The functional identification of the isolated gene cluster led them to propose astaxanthin biosynthetic pathway for the first time.

Misawa et al. [114] isolated and functionally identified the carotenoid biosynthesis genes, such as *crtE* (GGPP synthase), *crtX* (Zeaxanthin glucosyltransferase), *crtY* (lycopene β-cyclase), *crtI* (phytoene desaturase), *crtB* (phytoene synthase), and *crtZ* (β-carotene hydroxylase), form *E. uredovora* by analyzing carotenoids accumulated in *E. coli* transformants in which these genes were expressed. By analysis of accumulated carotenoids in the transformed *E. coli* by these individual genes, they found that carotenoids in this pathway appeared to be close to those in higher plants rather than to those in bacteria. Although HPLC is a routine analytical tool to analyze various metabolic products from plants, highly developed and comprehensive metabolome analytical techniques with respect to particular tissues now offer precise analytical approaches such as nuclear magnetic resonance (NMR; COSY and NOESY) and accurate mass spectrometry (MS) techniques [47, 126]. A foreign *crtW* gene was expressed in the W71 cultivar of sweetpotato, and carotenoids generated there have been successfully analyzed by UV-vis, ESI-MS, <sup>1</sup> H-NMR, and CD spectral data [127]. As a result, novel carotenoids, shown in **Figure 3**, i.e. echinenone 5′,8′-epoxide, echinenone 5′,6′-epoxide, and 3′-hydroxyechinenone 5′,6′-epoxide, were identified besides ketocarotenoids including astaxanthin.

## **3.2 Sweetpotato carotenoids biosynthesis genes, cloning, and genetic engineering**

Although, sweetpotato is highly important as a valuable source of carotenoids especially β-carotene, very little research has been done on molecular biological aspects of its carotenoid biosynthesis [10, 14, 31, 128]. The development of an efficient and reproducible transformation system is needed for genetic manipulation of sweet potato to either improve the crop or establish it as a novel "transgenic plant bioreactor" [129]. Otani et al. [130] developed and reported the first successful transformation protocol for the production of transformed (transgenic) sweetpotato

#### **Figure 3.**

*Novel carotenoids produced in the tuber of the transgenic sweetpotato engineered with the crtW gene of bacterial origin.*

plants that was based on the formation of hairy roots using leaf disks as explants for *Agrobacterium rhizogenes*. However, the regenerated transgenic plants showed some morphological abnormalities such as short storage root and internodes. Later on, to overcome such anomalies, a modified and successful *Agrobacterium tumefaciens-*mediated transformation protocol was developed via somatic cell embryogenesis [131–133]. Liao et al. [14] isolated and functionally characterized an isopentenyl diphosphate isomerase (*idi*) gene from sweetpotato cultivar YUSU 303 from Southeast China. They isolated a full-length cDNA of *idi* gene by SMART™ RACE cDNA Amplification Kit (Clontech, USA). Isolated *idi* was 1155 bp with an open reading frame of 892 bp encoding a polypeptide of 296 amino acids (GenBank accession No. DQ150100). Isolated *idi* gene was cloned in pTrc expression vector and was fed to *E. coli* which contained pAC-BETA plasmid for β-carotene accumulation. *E. coli* were cultured and carotenoids were analyzed by color complementation. Cultures of *E. coli* which were transformed with *idi* gene turned orange indicative for β-carotene and suggested its potential activity in promoting β-carotene biosynthesis. Kim et al. [134] isolated a partial sequence of phytoene synthase (*PSY*) which contained 354 bp from a cultivar Shinhwangmi (accession No. HQ828092). It showed 94% sequence identity with a *PSY* isolated from Ipomoea species Kenyan (GenBank accession No. AB499050.1). However, no gene function of isolated *PSY* from sweetpotato could be reported. Ling et al. [135] isolated a lycopene ε-cyclase (*LCYe*) gene from sweetpotato cultivar Nongdafu 14 from China. However, they did not functionally characterize it. They isolated a full-length cDNA of *idi* gene by GeneRacer TM Kit (Invitrogen Carlsbad, CA, USA). Isolated *LCYe* was 1805 bp with an open reading frame of 1236 bp encoding a polypeptide of 411 amino acids. Quantitative real-time PCR analysis showed that *IbLCYe* expression levels were desirably higher in roots as compared to those in leaves. Isolated *LCYe* gene was expressed in tobacco cultivar Winconsin 38. Carotenoids from transgenic tobacco plants were extracted and analyzed by HPLC which revealed transgenes accumulating more β-carotene as compared to control plants. Kim et al. [128] isolated a partial lycopene β-cyclase (*IbLCYb*) from a cultivar Yulmi of sweetpotato. They synthesized primers by using a partial sequence of *IbLCYb* from database with accession number JX393306 and amplified a partial cDNA of *IbLCYb* by

#### *Molecular Breeding of Sweetpotato Carotenoids DOI: http://dx.doi.org/10.5772/intechopen.101849*

RT-PCR. By using isolated *IbLCYb*, an IbLCYb-RNAi vector was constructed and then used to transform white-fleshed sweetpotato. Transformed sweetpotatoes were cultured and analyzed for accumulated carotenoids. Their results showed a total increase in the carotenoids contents along with increase in resistance against salt stress in transgenic sweetpotato as compared to the control. Significant levels of carotenoids genes expression were observed in all plant parts with highest expression in leaves to lowest in the fibrous roots. But in case of transgenic calli, expressions of *IbLCYb* were dramatically reduced and found high in non-transgenic calli. Lycopene was not produced both by transgenic and non-transgenic sweetpotato. In an another experiment, Kim et al. [134] cloned a partial cDNA encoding β-carotene hydroxylase (*BHY*) from storage roots of sweetpotato cultivar Shinhwangmi and constructed an RNA-i-IbCHY-β vector for transformation of white-fleshed cultivar Yulmi and evaluation of inhibition effects of β-carotene hydroxylase (*BHY*) in transgenic lines. Downregulation of *IbBHY* gene expression altered the content and degree of carotenoids between transgenic and non-transgenic cells with an increase in the β-carotene and total carotenoids in transgenic sweetpotato cells along with an increase in their antioxidation potential.

## **3.3 Metabolic engineering of the carotenoid biosynthetic pathway to enhance carotenoid contents in higher plants**

The pathway engineering approach using a variety of carotenoid biosynthesis genes is becoming a potential approach as one of the most effective methods to generate large quantities of structurally diverse carotenoids [59, 136, 137]. Astaxanthin (3,30-dihydroxy-4,40-diketo-β-carotene) is a high-value ketocarotenoid that is biosynthesized only by a few organisms typically at low levels. This red pigment (produced through chemical synthesis) has been used in large amounts in aquaculture. Currently, natural astaxanthin is employed as a health boosting food and is investigated for the treatment of a number of human diseases including cancers [138]. The limited renewable sources and growing demand for natural astaxanthin have attracted tremendous interest in its engineering into heterologous hosts, especially plants with the ability of sequestering 10- to 50-fold higher carotenoids than microorganisms, to produce the high-value pigment, during the past decade [139, 140]. The most promising approach reaching high astaxanthin yields was by chloroplast transformation using a bacterial ketolase gene [141]. Plastid genome transformation of lettuce (*Lactuca sativa*) has similarly been site-specifically modified with the addition of three transgenes, which encoded β,β-carotenoid 3,3′-hydroxylase (*crtZ*) and β,β-carotenoid 4,4′-ketolase (4,4′-oxygenase; *crtW*) from a marine bacterium *Brevundimonas* sp. strain SD212, and isopentenyl-diphosphate-isomerase (*idi*) from a marine bacterium *Paracoccus* sp. strain N81106. The resultant transplastomic lettuce leaves generated 49.2% astaxanthin fatty acid diester, 18.2% astaxanthin monoester, and 10.0% astaxanthin in its free forms along with the 17.5% of other ketocarotenoids. The ketocarotenoids produced in transplastomic lettuce were 94.9% of total carotenoids. The wild-type native carotenoids analyzed were 3.8% lactucaxanthin and 1.3% lutein in the transplastomic lettuce [142]. Likewise, by the introduction and heterologous expression of *crtW* gene, astaxanthin and other intermediates have been produced and reported in carrot (*Daucus carota*) roots [143], canola (*Brassica napus*) seeds [144], and maize (*Zea mays*) endosperms [145]. A comprehensive carotenoid biosynthetic pathway in these higher plants is shown in **Figure 4** with a summarized illustration for the metabolic pathway engineering with heterologous *crtW* and *crtZ* genes expression.

Through pathway engineering that utilizes the marine bacterial carotenoid 4,4′-ketolase (4,4′-oxygenase) gene named *crtW,* unique keto-carotenoids such as

#### **Figure 4.**

*Carotenoid biosynthetic pathway in higher plants. A summarized illustration for the introduction and function of heterologous crtW and crtZ genes expressed in tobacco [141] and lettuce [142] leaves. The carotenoids shown in black represent native carotenoids accumulated by both tobacco and lettuce, and black and underlined are those reported from both of the transgenic tobacco and lettuce. Carotenoids underlined red are reported from lettuce only, where GGPS, is geranylgeranyl pyrophosphate synthase, PSY is phytoene synthase, PDS is phytoene-desaturase, ZDS is ζ-carotene desaturase, CRTISO is carotenoid isomerase, LCYb is lycopene β-cyclase, LCYe is lycopene ε-cyclase, LsLCYe is lettuce LCYe, LsCYP97C is lettuce heme-containing cytochrome P450-type carotene ε-ring hydroxylase, BHY is non-heme di-iron-type carotene β-ring hydroxylase, EHY is carotene ε-ring hydroxylase, ZEP is zeaxanthin-epoxidase, VDE is violaxanthin de-epoxidase, and NSY is neoxanthin synthase.*

astaxanthindiglucoside, 2,2′-dihydroxyastaxanthin, and 2,2′-dihydroxycanthaxanthin have been produced in *Escherichia coli* [146, 147] and 4-ketoantheraxanthin in tobacco (*Nicotiana tabacum*) plants [148]. Breitenbach et al. [149] also synthesized α-echinenone (4-keto-α-carotene) in rice callus using *crtW*. Recently, 4-ketozeinoxanthin was produced in *E. coli* cells by introducing the bacterial *crtW* gene and carotenogenic genes from liverwort [150].

#### **3.4 Metabolic engineering of the carotenoids biosynthetic pathway in sweetpotato**

Metabolic engineering of carotenoid biosynthetic pathway using a combinatorial approach has led to the efficient production of interesting carotenoids of high commercial value and pharmaceutical potential [44, 59, 151].

Starting with transgenic approach, prerequisite is to have a sound knowledge on the metabolic pathways regulating the carotenoid biosynthesis and their accumulation. Due to efforts of many scientists, the carotenoid metabolic pathway and the function of the biosynthetic enzymes involved in carotenoids biosynthesis have been elaborated well [152]. It was reported that sweetpotato contained not only β-carotene but also several epoxy carotenoids unique to the sweetpotato tubers, e.g*.* β-carotene-5, 8-epoxside and β-carotene-5, 8, 5′8′-diepoxside [15]. Therefore, it

#### *Molecular Breeding of Sweetpotato Carotenoids DOI: http://dx.doi.org/10.5772/intechopen.101849*

was assumed that the new structural carotenoids with epoxy and keto groups can be produced by expressing the ketolase *crtW* gene in sweetpotato tubers. Recently, marine bacterial genes that include the *crtW* gene encoding carotenoid 4,4′-ketolase [148] was introduced into sweetpotato cultivar W71 under the control of the CaMV promoter. Consequently, novel carotenoids with epoxy and keto groups 1, 2, and 3 were obtained along with a series of ketocarotenoids. The structural elucidation of these novel epoxy-keto carotenoids along with biosynthetic pathway in sweetpotato was also proposed [127]. A tabulated summary of recent developments in molecular breeding of sweet potato by genetic, metabolic, and pathway engineering approaches is presented in **Table 1**.

"White Star" (WS) and W71, which produce white- and orange-fleshed tubers, respectively, are important sweetpotato cultivars, since they are amenable to *Agrobacterium*-mediated transformation [10, 127, 158, 159]. Chemical analysis of the carotenoids and isolation and functional characterization of the carotenoids biosynthesis genes of these two cultivars was reported in more details by Khan et al. [10] for the first time. One of the initial works that led to the sweetpotato genetic improvement for enhancing provitamin A amounts was done by Kim et al. [160] which involved isolation and functional analysis of the orange (*Or*) gene, from orange-fleshed sweetpotato controlling the carotenoids accumulation in the transgenic calli of sweetpotato*.* White-fleshed sweetpotatoes were transformed by the orange (*Or*) gene, which resulted in the 10-fold increased accumulation of β-carotene and total carotenoids. Later, identical results were presented by Park et al. [161] who observed that the overexpression of *IbOr* gene boosted the carotenoid composition in purple-flesh sweetpotato cultivar. In higher plants, the biosynthesis of carotenoids from lycopene involves the enzymatic activity


#### **Table 1.**

*Role of metabolic engineering in carotenoids enhancement in sweetpotato\* .* of lycopene ϵ-cyclase (*LCYe*) gene, via β-branch-specific biosynthetic pathway yielding β-carotene. By downregulating the expression of *IbLCYe* through RNA interference (RNAi) technology, higher amounts of β-carotene were recorded [162]. To increase the β-carotene contents in sweetpotato, researchers have also made use of the molecular markers along with the evaluation and screening of available germplasm.

This combined approach is thought very useful in selecting the desirable parents for breeding new sweetpotato varieties with the higher levels of β-carotenes [163]. To analyze the gene diversity and evolutionary relationships among various cultivars of sweetpotato, Hwang et al. [164] have applied the use of Simple Sequence Repeats (SSRs*)*. Their results showed that polycross-derived cultivars have higher levels of genetic diversity suggesting the application of polycross breeding that overcomes the challenges of cross-incompatibility. For breeding high β-carotene sweetpotato varieties, Quantitative Trait Loci (QTLs) for β-carotene content in a cross sweetpotato were reported for the first time by Cervantes-Flores et al. [165] which led to the understanding of the inheritance pattern and is considered the foundation of the development of marker-assisted breeding techniques for breeding high β-carotene (provitamin A) accumulating sweetpotato cultivars.

Orange-fleshed sweetpotato, which is a genetically modified crop, is now well accepted by consumers [166, 167] and has appeared as a sound supply of provitamin A. To achieve the daily provitamin A needs, mere 125 g of fresh orange-fleshed sweetpotato roots from most varieties are enough [35].

## **4. Conclusion**

The rapidly increasing world population demands sustainable supply of ample quantities of quality food, especially under the changing climatic conditions. Food insecurity accompanied with already existing malnutrition among the developing countries is a grand challenge of the day. Foods rich in phytonutrients not only contribute toward enhancing the health but also reduce the risk of many diseases including early aging. In this regard, genetic improvement of the major staple crops such as sweetpotato needs additional strategies of the molecular plant breeding to overcome the genetic complexity. The application of metabolic engineering supplemented with the omics and the recently developed gene editing tools and technologies are the potential strategies to be adopted which promise scope for improving the quantity of phytonutrients, especially the carotenoids in sweet potato. It will contribute to prevent the malnutrition and the diseases linked with foods deficit in quality nutrients. There is a dire need to apply multiple gene engineering approaches for multi-phytonutrients improvement to meet the need.

#### **5. Recent development and future scope**

Recent developments in carotenoids gene manipulations have helped to make insight that engineering sweetpotato with IbOr gene manipulations would be a potential strategy to improve the total carotenoids and specially the β-carotene through enhancing sink strength in storage roots of sweet potato. Moreover, site-directed mutagenesis supplanted with the genome editing tools such as CRISPR-Cas9 such as CRISPR-Cas9 and its different modifications will further lead to a fruitful biofortification of sweet potato for nutritional enhancement through carotenoids improvement.

## **Acknowledgements**

Principal and the corresponding author Dr. Muhammad Zubair Khan is highly thankful to the MEXT, Japan, for fully funding the research and studies conducted for the completion of doctoral degree. The author is also very much thankful to the laboratory technical staff members, Ms. Chisako Fuchimoto, Miyuki Murakami, Ayaka Atsumi, and Megumi Hashida for technical supports in the laboratory experiments. Author is highly thankful to the president of Ishikawa Prefectural University, Director, and office staff of the Research Institute for Bioresources and Biotechnology and the administrative staff of the Ishikawa Prefectural University, Ishikawa, Japan.

## **Conflict of interest**

The authors declare that they have no conflict of interest.

## **Notes/thanks/other declarations**

Corresponding author is highly thankful to the Prefectural government offices of Kanazawa city hall, Nonoichi city hall and Nagoya Regional Immigration Bureau Kanazawa Branch Office for facilitating stay pertaining to the whole term of the doctoral research and studies conducted.

## **Author details**

Muhammad Zubair Khan1 \*, Miho Takemura2 , Takahashi Maoka3 , Jun-ichiro Hattan<sup>2</sup> , Motoyasu Otani2 and Norihiko Misawa<sup>2</sup>

1 Faculty of Agriculture, Department of Plant Breeding and Molecular Genetics, University of Poonch Rawalakot, Rawalakot, Azad Jammu and Kashmir, Pakistan

2 Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa, Japan

3 Research Institute for Production Development, Kyoto, Japan

\*Address all correspondence to: zubairkhan@upr.edu.pk

© 2022 The Author(s). Licensee IntechOpen. 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.

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## **Chapter 11**
