Food Research International 76 (2015) 735–750
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Food Research International journal homepage: www.elsevier.com/locate/foodres
Review
Carotenoids from fruits and vegetables: Chemistry, analysis, occurrence, bioavailability and biological activities Ramesh Kumar Saini ⁎, Shivraj Hariram Nile, Se Won Park ⁎ Department of Bio-Resources and Food Sciences, College of Life and Environmental Sciences, Konkuk University, Seoul 143-701, South Korea
a r t i c l e
i n f o
Article history: Received 27 May 2015 Received in revised form 23 July 2015 Accepted 31 July 2015 Available online 3 August 2015 Keywords: Carotenoids Lutein β-Carotene Lycopene Biosynthesis Physiology Processing
a b s t r a c t Fruits and vegetables are generally considered as important contributors to a healthy diet and their intake is extremely helpful to reduce the risk of specific diseases like cancers, cardiovascular diseases, neural tube defects, and cataracts. Bioactive constituents from fruits and vegetables, such as carotenoids, folic acid and dietary fiber appear to play important roles in the prevention of these diseases. Carotenoids and their derivatives are versatile isoprenoids and play a vital role in plants and animals, starting from cellular antioxidant to gene regulation and so their importance at cellular and molecular level is well established. The most significant aspect of carotenoids in our diet is the antioxidant and provitamin A activity, and also the color that they impart to our food. The composition and bioavailability of carotenoids in food are significantly influenced by processing and other post-harvest technologies. This review discusses the theoretical aspects and recent developments in structural properties, biosynthesis and enhancement, processing, methods of analysis, composition in fruits and vegetables, and bioaccessibility and bioavailability of carotenoids. Additionally, future research challenges in this context are identified. © 2015 Published by Elsevier Ltd.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Chemistry of carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Carotenoids in fruits and vegetables . . . . . . . . . . . . . . . . . . . . . . . 4. Post-harvest physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Biosynthesis of carotenoids in plants . . . . . . . . . . . . . . . . . . . . . . . 6. Bioavailability and bioaccessibility of carotenoids . . . . . . . . . . . . . . . . . 7. Functions, biological activity and recommended daily allowance (RDA) of carotenoids 8. Genetic engineering of carotenoid pathway . . . . . . . . . . . . . . . . . . . . 9. Enhancement of carotenoids with non-GM based approaches . . . . . . . . . . . 10. Methods of analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Carotenoids are natural pigments which are metabolized by plants, algae, and photosynthetic bacteria; which are responsible for the ⁎ Corresponding authors. E-mail addresses:
[email protected] (R.K. Saini),
[email protected] (S.W. Park).
http://dx.doi.org/10.1016/j.foodres.2015.07.047 0963-9969/© 2015 Published by Elsevier Ltd.
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yellow, orange, and red colors in various fruits and vegetables (Namitha & Negi, 2010). Carotenoids can be classified into two groups on the basis of functional groups; xanthophylls, containing oxygen as functional group, including lutein and zeaxanthin, and carotenes, which contain only parent hydrocarbon chain without any functional group, such as α-carotene, β-carotene and lycopene. Addition of polar groups (epoxy, hydroxyl and keto) alters the polarity of carotenoids and affects biological functions (Britton, 2008). Fruit and vegetables
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are the principal source of carotenoids and play an important role in diet due to vitamin A activity (Haskell, 2013). Apart from this, carotenoids are also important for antioxidant activity, intercellular communication and immune system activity (Skibsted, 2012; Stephensen, 2013). Epidemiological studies demonstrated that the consumption of diets rich in carotenoids is associated with a lower incidence of cancer, cardiovascular diseases, age related macular degeneration and cataract formation (Meyers et al., 2014; Sharoni et al., 2012). Deficiency of carotenoids results in clinical signs of conjunctiva and corneal aberrations including, xerophthalmia, night blindness, keratomalacia, corneal ulceration, scarring, and resultant irreversible blindness (Sommer, 2008). Other than the above, deficiency of provitamin A carotenoids leads to visiondisability in human and increased mortality due to a weakened innate immunity and adaptive immunity (Stephensen, 2001). Lycopene is the powerful antioxidant carotenoids; however it lacks vitamin A activity. This potent antioxidant activity of lycopene is generally responsible for the protection of cellular system from a variety of reactive oxygen (ROS) and reactive nitrogen species (RNS), and also helps in preventing cardiovascular diseases risk (CVD) in human (Müller, Caris-veyrat, Lowe & Böhm, 2015). The versatile use of carotenoids in feed, food, cosmetic and pharmaceutical industries, makes them potential candidates for enhancement and manipulation. Over the past decades advances in molecular genetics and biotechnological approaches have led to the understanding of carotenoid biosynthesis phenomena and its enhancement in microorganisms and higher plants (Potrykus, 2001, 2003). This review discusses the theoretical aspects and recent developments in structural properties, biosynthesis and enhancement, processing, methods of analysis, composition in fruits and vegetables, and bioaccessibility and bioavailability of carotenoids. Additionally, future research challenges in this context are identified. 2. Chemistry of carotenoids Carotenoids are C40 tetraterpenoid pigments, biosynthesized by the linkage of two C20 Geranylgeranyl diphosphate molecules (Namitha & Negi, 2010). Carotenoids consist of eight isoprenoid units joined together in a specific manner that the organization of isoprenoid units is reversed at the center of the molecule so that the nonterminal methyl groups are in a 1,5-position and remaining two central methyl groups are in a 1,6-position relationship (Fig. 1a) (Britton & Khachik, 2009). Carotenoids in plants can be found in free form or esterified with fatty acids. Though, esterification does not alter the chromophore properties of the carotenoid, it does modify the chemical and biological properties by changing its immediate environment (Pérez-Gálvez & MínguezMosquera, 2005). These properties also depend on the kind of fatty acid bound to the carotenoid molecule. Esterification facilitates carotenoid storage, aiding in the integration of these highly lipophilic molecules
within lipid rich plasto-globules (Howitt & Pogson, 2006). It has been anticipated that esterification is the natural biological mechanism to protect triacylglycerols, unsaturated lipids, and other light sensitive compounds from photooxidation (Cazzonelli & Pogson, 2010). Carotenoids can be found in green leaves and fruits along with chlorophylls, and also in many other parts of the plant such as red, yellow, and orange flowers, roots (such as carrots and cassava), and seeds (such as maize, annatto). In nature, majority of carotenoids exist in more stable trans isomeric, compared to cis isomeric forms. Gul et al. (2015) studied the various aspects concerning the chemistry, encapsulation for enhanced stability and health benefits of important carotenoids. The natural functions and properties of carotenoids are determined by the molecular structure. The conjugated polyene chromophore presented in carotenoid molecule determines the light absorption and light harvesting properties. Thus, chromophore is the part of a carotenoid molecule responsible for its color and photoprotective actions. The color arises when a chromophore absorbs particular wavelengths of visible light and transmits or reflects others. The molecular structure of β-carotene chromophore is shown in Fig. 1b; the eleven conjugated double bonds form the chromophore of the molecule, which absorb light in a visible range of electromagnetic spectrum (400–500 nm) (Britton, 2008). Apocarotenoids are another class of carotenoids, derived from carotenoids by oxidative cleavage, catalyzed by family of carotenoid cleavage dioxygenases (CCDs). CCDs often exhibit substrate, promiscuity, and catalyze many important reactions with remarkable precision which contributes to the diversity of apocarotenoids found in plants, animals and microorganisms (Harrison & Bugg, 2014). Biologically and commercially important apocarotenoids include vitamin A, retinoids, retinol and retinoic acid, plant hormone abscisic acid and strigolactone, annatto pigment bixin, and aromatic volatile aroma compounds β-ionone and αionone. Interestingly, properties associated with these CCD catalytic products emphasize their role in flavor, fragrances, signaling and many aspects of plant growth development (Auldridge, McCarty & Klee, 2006). Traditionally, carotenoids were named by original discoverer and derived usually from the biological source from which they are isolated, such as carotene from carrot, zeaxanthin from Zea mays and lutein from Macula lutea. Generally, these trivial names are not useful in describing the chemical structure and properties of the carotenoids. So, systematic scheme has been devised that allows carotenoids to be named and in a way that defines and describes their structure, for example, — carotene is correctly referred to as β, β-carotene, and α-carotene as α, ε-carotene (Gul et al., 2015). 3. Carotenoids in fruits and vegetables The type and availability of carotenoids in fruits and vegetable can be predicted by their color, such as yellow-orange vegetables and fruits are
Fig. 1. a) Joining style of eight isoprenoid units to form β-carotene, and b) molecular structure of β-carotene chromophore.
737
185 7.0 172 Values are in μg/g fresh weight basis (#values are in μg/g dry weight basis).
12.0 9.3 9.9 – 1.6
–
0.54 19.7 – – – 0.26 8.38 0.23 0.23 – 0.32 8.62 – – – – – – 0.003 –
Musa acuminate Musa acuminate Vitis vinifera Syzygium cumini Mangifera indica L. Carica papaya Prunus persica Ananas comosus Cucurbita pepo subsp. pepo Banana (cv. cavendish) Banana (red, cv. chandran) Grapes (white, cv. aromat de Iaşi) Jambolão fruits (Brazil) Mango Papaya Peach (var. redhaven) unpeeled canned Pineapple (core, minimally processed) Watermelon (Flesh)#
0.06 0.9 0.47 0.39 31.7 237 –
0.18
– – – 0.02 1.5 14.1 1.1
– – – – –
–
–
0.17 170.00 0.66 –
– 0.73 – 0.01 2.7 – 0.06 0.09 0.4 Malus domestica Prunus armeniaca Musa paradisiaca
Apple, green (cv. green golden delicious)
Apple, red (cv. royal gala)# Apricot (var. hargrand) unpeeled canned Banana (cv. nanjangud rasabale)
– – 1.4 – – 0.02 0.71 Malus domestica
– 12 1.1 1.1 – 1.3 Malpighia glabra
#
Acerola (home garden, ripe)
– 173.00 1.24
Rodriguez-Amaya, Kimura, Godoy & Amaya-Farfan (2008) Porcu & Rodriguez-Amaya (2006) Delgado-Pelayo, Gallardo-Guerrero & Hornero-Méndez (2014) Delgado-Pelayo et al. (2014) Campbell & Padilla-Zakour (2013) Lokesh, Divya, Puthusseri, Manjunatha & Neelwarne (2014) Lokesh et al. (2014) Lokesh et al. (2014) Bunea et al. (2012) Faria, Marques & Mercadante (2011) Khonsarn & Lawan (2012) Khonsarn & Lawan (2012) Campbell & Padilla-Zakour (2013) Freitas et al. (2015) Martínez-Valdivieso et al. (2014)
Total carotenoids Lycopene β-Carotene α-Carotene β-Cryptoxanthin Zeaxanthin Lutein Botanical name Fruit
Table 1 Contents of major carotenoids in selected fruits.
generally rich in β-carotene and the α-carotene. α-Cryptoxanthin and zeinoxanthin can be found in orange fruits, such as mandarin, orange, and papaya. Similarly, lycopene pigment (responsible for bright red color) is the major constituents of tomatoes and tomato products. Lutein (nearly 45%) and β-carotene (25–30%) followed by violaxanthin (10–15%), and neoxanthin (10–15%) are the predominant forms of carotenoids in green leafy vegetables (Lakshminarayana, Raju, Krishnakantha & Baskaran, 2005; Priyadarshani & Jansz, 2014), although the absolute concentration of each carotenoid varies considerably among different vegetables. α-Carotene, β-cryptoxanthin, zeaxanthin, antheraxanthin, and lutein 5,6-epoxide (luteoxanthin) are also recorded in green leafy vegetables in minor concentrations. In most of the fruits and vegetables, β-carotene is generally dominating compared to its geometric isomer α-carotene. Significant high contents of αcarotene can be found in a limited number of fruits and vegetables, such as sweet potato, carrots, pumpkin, and dark green vegetables, such as green beans, spinach and broccoli (Khoo, Prasad, Kong, Jiang & Ismail, 2011). Knowledge on carotenoid composition in different edible parts and cultivars will be useful to nutritional experts for the selection of nutrient-rich plants for food fortification and proper diet recommendation. Compositions of major carotenoids in important fruits and vegetables are given in Tables 1 and 2. In recent years, numerous underutilized leafy vegetables, such as Moringa oleifera (Drumstick tree) and Lactuca indica (Indian lettuce) and Oenanthe javanica (Water dropwort) were established as exceptionally rich sources of carotenoids (Andarwulan et al., 2012; Kongkachuichai, Charoensiri, Yakoh, Kringkasemsee & Insung, 2015; Saini, Shetty & Giridhar, 2014). Among 25 common and less common leafy vegetables, including amaranthus, fenugreek and spinach, the highest content of β-carotene (19.7 mg/100 g) was recorded in leaves of M. oleifera (Bhaskarachary, Rao, Deosthale & Reddy, 1995). Similarly, among underutilized vegetables from Indonesia, maximum content of β-carotene (14 mg/100 g) was recorded in leaves of Moringa pterygosperma (syn. M. oleifera) (Andarwulan et al., 2012). In a survey of indigenous Thai vegetables, maximum contents of β-carotene and lutein were recorded in L. indica and O. javanica, respectively (Kongkachuichai et al., 2015). International non-governmental organizations (NGOs) such as Trees for Life and Educational Concerns for Hunger Organization (ECHO) have vigorously supported Moringa leaves as “natural nutrition for the tropics”. India is the world's largest producer of Moringa and enabled to provide the lowcost food for the malnourished population. In the future, detailed composition of carotenoids in these underutilized vegetables may contribute significant information to select nutrient rich plants for food formulation. In plant kingdom, flower stigmas of saffron plant (Crocus sativus) and waxy seed arils of achiote shrub (Bixa orellana) is the richest source of apocarotenoids (Rosati, Diretto & Giuliano, 2009). However, small contents of apocarotenoids can be found in citrus fruits and paprika vegetables. Agócs et al. (2007) recorded 2–7% red apocarotenoids βcitraurin in pulps of citrus species. Fleshman et al. (2011) recorded 1.5% of β-apocarotenoids to the total β-carotene present in cantaloupe (Cucumis melo Reticulatus Group) and Orange Dew (Cucumis melo, Inodorus Group). Seed pulp or aril of ripened fruits of Momordica cochinchinensis, commonly known as gac fruit, is the known richest source of carotenoids plant derived carotenoids. Lycopene and β-carotene are the major carotenoids in gac fruit, which accounted 408 μg/g and 83.3 μg/g fresh weigh, respectively (Vuong, Franke, Custer & Murphy, 2006). De Rosso and Mercadante (2007), quantified the major and minor carotenoids from native fruits of Amazonia region, including buriti (Mauritia vinifera), mamey (Mammea americana), marimari (Geoffrola striata), peach palm (Bactris gasipaes), physalis (Physalis angulata), and tucuma (Astrocaryum aculeatum). In results, a total of 60 different carotenoids were identified, among these, all-E-β-carotene was the major carotenoid in all fruits, and the total carotenoid content ranged from 38 μg/g in marimari to 514 μg/g in buriti. Silva et al. (2014) analyzed the
References
R.K. Saini et al. / Food Research International 76 (2015) 735–750
R.K. Saini et al. / Food Research International 76 (2015) 735–750
4. Post-harvest physiology
236.1 2386.2 409.9
132.9
804.8 1092.6
1691.5
46.46 39.54
0.746
Kaulmann, Jonville, Schneider, Hoffmann & Bohn (2014) Kaulmann et al. (2014) Maurer, Mein, Chaudhuri & Constant (2014) Kaulmann et al. (2014) Žnidarčič, Ban & Šircelj (2011) Divya, Puthusseri & Neelwarne (2012) Maurer et al. (2014) Žnidarčič et al. (2011) Saini, Shetty, Prakash & Giridhar (2014) Lakshminarayana et al. (2005) Žnidarčič et al. (2011) Kaulmann et al. (2014) Niizu & Rodriguez-Amaya (2005) Carvalho, Smiderle, Carvalho, Cardoso & Koblitz (2014) Lakshminarayana et al. (2005) De Carvalho et al. (2012)
carotenoids and other bioactive compounds in pulps and by-products of tropical fruits. On dry weight basis, the highest content of β-carotene was recorded in pulp of Acerola (26.23 μg/g), followed by papaya (20.24 μg/g) and Surinam cherry (Eugenia uniflora; 15.64 μg/g) pulp. Traditional and non-traditional tropical fruits are considerably rich in carotenoids and other bioactive compounds, which indicate promising perspectives for the utilization of these fruit species and their byproducts in potential commodities.
67.06
39.9
Values are in μg/g fresh weight basis (#values are in μg/g dry weight basis).
15.1 775.8
0.21
6.2 0.8 135.4 9.5 0.6 13.1 52.5 312.7 596.0 74.4 65.22 13.5
0.9
1.7
0.15
2.8
45.0
63.4 231.5 121.3 79.6 44.0 14.9 172.2 365.3 244.2
11.38 0.718 53.6 0.22 73.1 586.1 0.15 11.63
Brassica oleracea var. italica Brassica oleracea var. gemmifera Daucus carota Brassica oleracea var. botrytis Cichorium intybus Coriandrum sativum Zea mays Taraxacum officinale Moringa oleifera Trigonella foenum-graecum Eruca sativa Brassica oleracea var. acephala Lactuca sativa Cucurbita maxima Spinacia oleracea Cucurbita moschata Broccoli (var. verde calabrese) Brussels sprouts Carrot Cauliflower (var. White Rock) Chicory (cv. Anivip) Coriander leaves (var. GS4 Multicut)# Corn Dandelion Drumstick leaves (cv. Bhagya) Fenugreek# Garden rocket Kale Lettuce Pumpkin Spinach# Squash (Landraces A, Aracaju, Brazil)
28.05 11.63 1.5 0.28 59.1
Botanical name Vegetable
Table 2 Contents of major carotenoids in selected vegetables (μg/g).
Lutein
Zeaxanthin
β-Cryptoxanthin
α-Carotene
β-Carotene
Lycopene
Total carotenoids
References
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Content and types of carotenoids in plants depend on several preand post-harvesting factors, genotype, ripening time, cultivation method and climatic conditions, processing. Different parts of the same plant also may contain different types and amounts of carotenoids. For example, peel of the fruits is generally richer in carotenoids compared to pulp. Packaging and processing methods greatly influence the content of carotenoids in processed food products (Moura, Miloff & Boy, 2015). Irrespective of thermal (heat drying) or non-thermal (e.g., high-pressure, pulsed electric field, ultrasound drying), processing may significantly degrade the level of carotenoids in food products. For most vegetables, drying resulted in 10–20% loss of carotenoids (Saini, Shetty, Prakash & Giridhar, 2014), with the increased surface area of dried or powdered products leading to further losses (through autoxidation) unless they were protected from high temperature, air and light. In addition to this, thermal processing may cause significant quantitative changes in carotenoid isomers, due to possible trans to cis isomerization of βcarotene and lutein (Colle, Lemmens, Knockaert, Loey & Hendrickx, 2015). So, higher retention of cis-isomers was generally recorded in thermal processed fruits and vegetables compared to trans-isomers. In general, cis-isomers of carotenoids exhibit less potent provitamin activity compare to trans-isomers, which cause further loss of provitamin activity in processed food products (Castenmiller & West, 1998). Degradation of β-carotene and lutein and formation of cis-isomers (4–40%) during thermal dehydration studied in a number of fruits and vegetables including peas, broccoli, kale, spinach, and corn (Saini, Shetty, Prakash & Giridhar, 2014; Updike & Schwartz, 2003). All the available methods used in processing (dehydration) of fruits and vegetables cause significant degradation of carotenoids. Amount of degradation is more in thermal (heat) base methods compared to non-thermal methods. However, major food industries in developing countries are using thermal based method of processing due to high initial setup and running cost of other advanced methods. For example, lyophilisation is the best method to preserve the major nutrient, including carotenoids during dehydration (Saini, Shetty, Prakash & Giridhar, 2014), however, this method is not economical in large scale industries due to high running and initial cost of machineries. Non-thermal based processing methods, such as high pressure, high electric field pulse, highpressure CO2, are emerging in food processing sector. In the future, these advanced processing methods can play an important role in the preservation of bioactive compounds. However, additional attention is
Table 3 Relative provitamin A activity of common food carotenoids. Carotenoid
% provitamin A activity
All-E-β-carotene All-E-β-cryptoxanthin 13-Z-β-carotene All-E-α-carotene All-E- mutatochrome 15-E-β-cryptoxanthin 9-E-β-carotene 9-Z-β-cryptoxanthin β-Carotene-5,6-epoxide 13-Z-α-carotene 9-cis-α-carotene
100 57 53 53 50 42 38 27 21 16 13
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also required in the development of effective processing units with minimum initial set-up and running cost. Thirteen tomato cultivars (breeding lines) with distinct color and were recorded with significant difference in total carotenoid content, varies from 120.50 to 278.00 μg/g DW (Li, Deng, Liu, Loewen & Tsao, 2013). Valcarcel, Reilly, Gaffney and O'Brien, (2014), studied the carotenoids content in among 60 varieties of potato, including rare, heritage and commercial varieties. Higher levels of total carotenoids were found in the skin of tubers, compared to flesh, with variety ‘Burren’ showed maxima values of 28 and 9 mg/kg dry weight (DW) in skin and flesh, respectively. In another study, Song, Li, He, Chen and Liu (2015) analyzed the carotenoids content in corn (Z. Mays) genotypes, and found high content of total carotenoids in sweet corn compared to waxy corn grains. In the study of Saini, Shetty, and Giridhar (2014), significant difference was observed in carotenoid content among eight cultivars of M. oleifera, the fresh leaves of cultivar Bhagya (KDM-1) was recorded for the maximum amount of all-E-zeaxanthin, all-E-β-carotene and total carotenoids. Climatic conditions, growing locations also significantly influence the content of carotenoids in plants. Increased temperature produces more carotenoids in tomato fruit, and its enhanced production depends on its developmental stage (Hernández, Hellín, Fenoll & Flores, 2015). Cândido, Silva and Agostini-Costa (2015) comparatively studied the content of carotenoids in buriti fruits (Mauritia flexuosa) grown in Cerrado and Amazon biomes. Amazon region is characterized by higher temperatures and humidity and dense forest vegetation, compared to Cerrado. Result showed significantly high total carotenoids content (52.86 mg/100 g) in fruits grown in amazon region, compared to fruits grown in Cerrado (31.13 mg/100 g). These studies suggest that fruits exposed to high temperatures and higher sunlight incidence may have enhanced the carotenoids biosynthesis, to protect the plant from photo oxidation.
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5. Biosynthesis of carotenoids in plants Carotenoid biosynthesis starts with the condensation of two Geranylgeranyl diphosphate (GGPP) molecules which were synthesized in the methylerythritol 4-phosphate (MEP) pathway (Cazzonelli & Pogson, 2010). The carotenoid biosynthesis pathway in plants is summarized in Fig. 2. In the last few years, almost complete sets of genes encoding and enzymes required for the biosynthesis of these indispensable pigments have been identified. Carotenoid biosynthesis starts with the condensation of two Geranylgeranyl diphosphate (GGPP) molecules which was synthesized in the methylerythritol 4-phosphate (MEP) pathway (Cazzonelli & Pogson, 2010), to form 15-Z-phytoene, a reaction catalyzed by phytoene synthases (PSY). GGPP is also serving as a precursor for several other metabolites, such as terpenes, terpenoids, gibberellins, chlorophylls, ubiquinones and tocopherols. 15-Z-phytoene then undergoes four sequential reactions to form lycopene through the action of two desaturases and two isomerases: phytoene desaturase (PDS), zeta-carotene desaturase (ZDS), carotenoid isomerase (CRTISO) and zeta-carotene isomerase (Z-ISO). Further, cyclization of lycopene with lycopene ε-cyclases (ε-LCY) and lycopene β-cyclases (β-LCY) to form δ-carotene and γ-carotene, respectively, is the key branch-point in carotenoid biosynthesis. In one branch, a single enzyme, β-LCY, introduces a β-ionone ring at both ends of lycopene to form β-carotene. However in the other branch, leading to lutein, requires both ε-LCY and β-LCY to introduce one β- and one ε-ionone ring into lycopene to form α-carotene. β-Ionone ring is required to provide provitamin A activity (Send & Sundholm, 2007). Thus, lycopene lacks provitamin A activity due to the absence of β-ionone ring. α-Carotene is acted upon by a β-ring hydroxylase to form zeinoxanthin, which is then hydroxylated by an ε-ring hydroxylase to produce lutein. In another branch, βcarotene can be hydroxylated to form zeaxanthin, by the action of βcarotene hydroxylase. Zeaxanthin can be epoxidized to antheraxanthin
Fig. 2. Biosynthetic pathway of carotenoids in plants.
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and violaxanthin, through the action of zeaxanthin and antheraxanthin epoxidase, respectively. Violaxanthin is further to converted to neoxanthin by the action of neoxanthin synthase. A numerous of other carotenoids with unique biosynthetic routes exist in some algae and plants. For instance, some higher plants, such as Adonis aestivalis, and unicellular green alga Haematococcus pluvialis, accumulate the ketocarotenoid astaxanthin, which is derived from zeaxanthin oxidation; a reaction catalyzed by β-carotene ketolase (Fig. 2). However, A. aestivalis is reported to show toxicity in animals (Woods, Puschner, Filigenzi, Woods & George, 2011). The red pepper (Capsicum annuum) accumulates two keto-xanthophylls, capsanthin and capsorubin, which account for the bright red color of the fruit. A single bi-functional enzyme, capsanthin–capsorubin synthase (CCS) catalyzes the conversion of the antheraxanthin and violaxanthin, into capsanthin and capsorubin, respectively. Following their biosynthesis, major carotenoids accumulate in specialized plastids, chromoplasts, chloroplasts or leucoplasts. These are differentiated from pro-plastids or pre-existing mature plastids during flower maturation and fruit ripening. Most prevalent carotenoids in the chromoplasts are the xanthophylls, which are esterifies with fatty acid. Esterification facilitates the accumulation of xanthophylls within the chromoplast. A recent study demonstrates that most xanthophylls present in tomato flower petals exist as mixtures of free (non-esterified) and esterified forms. Over 85% of the xanthophylls in yellow flower petals are present as esterified forms, consisted of neoxanthin and violaxanthin esterified with myristic and/or palmitic acids (Ariizumi et al., 2014). Esterification promotes the sequestration of xanthophylls and also prevents its degradation. Pale yellow petal (PYP1) is recently identified from tomato petals, which plays a vital role in the production of xanthophyll esters (Ariizumi et al., 2014). Besides biosynthesis, carotenoid levels are also modulated by enzymatic degradation in plastids. Plastid-localized carotenoid cleavage
dioxygenases (CCDs) possibly play fundamental roles in carotenoid degradation in Arabidopsis and other plants, by negatively regulating the carotenoid content (Gonzalez-Jorge et al., 2013). In potato plant, down-regulation of the carotenoid cleavage dioxygenase 4 (CCD4) led to elevated carotenoid contents, specially violaxanthin and lutein (Bruno, Beyer & Al-Babili, 2015). In Brassica species, disruption of a CCD 4 gene converts flower color from white to yellow (Zhang et al., 2015). Various classes of CCDs are identified in plant, which catalyze the important steps in the biosynthesis of apocarotenoid, carlactone, strigolactones, abscisic acid and many other flavor and aroma compounds. Carotenoid cleavage dioxygenases (CCDs) mediated the formation of major apocarotenoids in plants which is illustrated in Fig. 3. These compounds play crucial roles in plant–environment crosstalk, e.g. attraction of pollinators, herbivore deterrence and defense against pathogen (Walter, Stauder & Tissier, 2015). Carotenoid biosynthesis is modulated by developmental, signaling and epigenetic mechanism including pre- and post-transcriptional regulation (Ruiz-Sola & Rodríguez-Concepción, 2012). Light signaling plays an important role in the regulation of carotenoids in aerial parts of the plant. Carotenoid biosynthetic genes, including those of the MEP pathway, are upregulated during light-triggered deetiolation. During germination of seeds, carotenoids present in etioplasts are facilitating the greening of soil-emerging seedlings, which perceive the light and de-etiolate. Light-dependent changes in plastid differentiation also influence the accumulation of carotenoids in plants (Fuentes et al., 2012). Salt stress triggers the enhanced production of carotenoids in roots which contributes to fuel hormone production; specially abscisic acid (ABA) (Ruiz-Sola, Arbona, Gómez-Cadenas, RodríguezConcepción & Rodríguez-Villalón, 2014). Phytoene synthase (PSY) is the primary regulatory enzyme in carotenoid biosynthesis pathway, which responds to developmental and stress signals, such as ABA, high light intensity, temperature, salt and drought stress, photoperiod,
Fig. 3. Carotenoid cleavage dioxygenases (CCD) mediated formation of apocarotenoids in plants.
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and post-transcriptional feedback regulation (Cazzonelli & Pogson, 2010). Epigenetic regulatory mechanisms (chromatin modifications) regulate the expression of carotenoid isomerase (CRTISO), which catalyzes cis–trans reactions; 7,9,7′,9′-tetracis-lycopene to all-Z-lycopene. This epigenetic mechanism is mediated by a chromatin-modifying histone methyltransferase enzyme (carotenoid chloroplast regulatory 1, CCR1 or set domain group 8, SDG8) required for CRTISO expression (Cazzonelli et al., 2009). The CCR1 mutation alters the modification of chromatin associated with the CRTISO gene, impairing lutein biosynthesis, accumulation of cis-carotenes and increased shoot branching by limiting strigolactone biosynthesis (Cazzonelli et al., 2009). Carotenoid biosynthesis is significantly altered during fruit development to match the prevailing developmental requirements. Tomato fruit is actually one of the best studied systems for the regulation of carotenoid biosynthesis during ripening. During tomato ripening, chlorophylls are differentiated into chromoplasts, which accumulate lycopene membranebound crystals, causing the fruit color to change from green to red. Several genetic and hormonal regulatory mechanisms are involved in the accumulation of lycopene during tomato fruit ripening (Pesaresi, Mizzotti, Colombo & Masiero, 2014). Auxin-ethylene balance is the key regulator of tomato fruit ripening and carotenoid accumulation by regulating genes involved in ethylene and auxin signaling (Su et al., 2015). The biosynthesis of carotenoids in plants has been extensively studied due to its importance in animal nutrition and plant survival. Our understanding of carotenoid biosynthesis, regulation, and roles of various carotenoid derivatives for plant and animals is still not well established. Detailed investigations into biochemical process leading to the carotenoid esterification can help in understanding the sequestration and storage process of carotenoids. This knowledge will facilitate further advancement in the field of carotenoid metabolic engineering to improve nutritional quality and nutrient density in food crops. 6. Bioavailability and bioaccessibility of carotenoids Bioavailability refers to the portion of the carotenoid which is absorbed in the body, enters in systemic circulation and becomes available for utilization in normal physiological functions or for storage in the human body. Whereas, the bioaccessibility refers to the proportion of ingested carotenoid that is released from the food matrix and incorporated into micelles in the gastrointestinal tract, and thus available for intestinal absorption (Rodriguez-Amaya, 2015). Absorption of carotenoids involves release of carotenoids from food matrix, diffusion in lipid emulsion, solubilization into pancreatic lipases and bile salts and formation of mixed micelles, movement across the microvilli, uptake of carotenoids by intestinal mucosal cells, incorporated into chylomicrons and enters in the lymphatic system and circulation (Fig. 4) (Donhowe & Kong, 2014). The bioavailability of β-carotene from plant sources is generally low (10–65%), due to resistance of carotene– protein complexes, fibers and the plant cell walls to digestion and degradation to achieve adequate release of carotenoids (Rein et al., 2013). Thus, soluble proteins have been shown to inhibit the incorporation of β-carotene into the gastric emulsion and bile salts, indicating that the interfacial characteristics of the gastric emulsion determine the extent of carotenoid absorption in intestine. Additionally, a factor that is often not fully considered is the mass transfer phenomenon, which is the most crucial and rate limiting in this context (Lemmens et al., 2014). Mass transfer phenomenon is the transport of the carotenoids from the aqueous environment in the fruit and vegetable matrices to the lipid phase of the food. The food matrix plays a significant role on bioavailability because release from the food matrix is a primary factor limiting bioavailability of carotenoids (Palafox-Carlos, Ayala-Zavala & González-Aguilar, 2011). Release of carotenoids depends on the level of digestion and degradation of the food matrix, which may be assisted by mechanical processing prior to digestion. Mechanical processing helps in reducing particle size, results in greater surface area to come
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into contact with pancreatic lipases and bile salts to improve digestion and release (Palafox-Carlos et al., 2011). Absorption of carotenoids occurs only when they are mixed with micelles, so factors affecting the micelle formation, also affect the bioavailability of carotenoids. Since lipids are required for incorporation into micelles and also stimulate release of bile to facilitate micelle formation, addition of dietary fat improves the bioavailability of carotenoids (Lemmens et al., 2014). The amounts of naturally occurring lipids are rather low in most fruits and vegetables, so it is obvious that the addition of extra lipids during processing and/or digestion can play a key role in this context. Addition of oil is more beneficial for non-polar carotenoids (carotenes) compared to polar carotenoids (xanthophylls) (Victoria-Campos, Ornelas-Paz, Yahia & Failla, 2013). Addition of long chain fatty acids, such as oleic acid (C18:1) is also more beneficial compared to short chain fats (Colle et al., 2013). Some studies are also conducted to study the degree of unsaturation of fatty acids on the bioavailability of carotenoids. However, their results are not conclusive, so, detailed and preciously controlled studies are required in this context. Comparative bioavailability of carotenoids from fruits and vegetables is illustrated in Fig. 5. As discussed earlier, natural or synthetic carotenoids in oil form are highly bioavailable, followed by carotenoids from fruits. Carotenoids from vegetables are comparatively less bioavailable then fruits (Schweiggert et al., 2014), however, vegetables are the major contributors of carotenoids in human diets (Bowen, Stacewicz-Sapuntzakis & DiwadkarNavsariwala, 2015). Dietary fiber and pectin, which are the principle components of fruits and vegetables, also possibly inhibit the micelle formation and decrease bioavailability of carotenoids. In a human randomized cross-over study, carotenoids from papaya were found more bioavailable than from tomato and carrot (Schweiggert et al., 2014). In fact, in this study, the papaya test meal was containing substantially more dietary fiber than from the tomato and carrot test meals. Thus, it suggests that some other factors that increased the bioavailability of carotenoids may surpass the adverse impact of dietary fibers. Factors related to bioaccessibility such as thermal processing, structural barriers in food (matrix, cell wall integrity, bio-encapsulation), addition of lipids are the most crucial aspects in determining the bioavailability of carotenoids. So, detailed interrelated studies are required in identifying the favorable factors to improve the bioavailability of carotenoids from different foods. This will help in the development of specialized food with potential bioavailability. Bioavailability is best determined by controlled studies on human subjects. However, such studies are laborious and resource intensive, thus, limiting their use to a few number of samples. Moreover, ethical issues are the major constraint in the use of humans and other animals for trials. In the last decade, simple, inexpensive, rapid and reproducible in vitro methods have been developed for initial screening of carotenoid bioaccessibility and bioavailability (Rodriguez-Amaya, 2015). Currently, in vitro digestion models coupled with Caco-2 cell are thoroughly used as high throughput methods to study the bioaccessibility of carotenoids. Using in vitro methods, detailed investigation of food related factors in a variety of foods can be studied and also a large number of samples can be analyzed within a short time (Rodriguez-Amaya, 2015). Recently, an in vitro dynamic gastrointestinal model was used to investigate the digestive stability and bioaccessibility of carotenoids from boiled, fried, and scrambled eggs. In their observations, bioaccessibility but not digestive stability was significantly affected by the method of cooking (Nimalaratne, Savard, Gauthier, Schieber & Wu, 2015). Detailed analysis of effects of food related factors on bioavailability would help in planning dietary strategies to improve the bioavailability of carotenoids and other important nutrients. Consumption of wide variety bioactive phytochemicals from fruits and vegetables is the best way to minimize the occurrence of degenerative diseases and maintain the health. Content and bioavailability of these phytochemicals are significantly affected by cooking. For example, cooking (heating) can degrade most nutrients in food, while, certain nutrients, such as lycopene, become more bioavailable after cooking and
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Fig. 4. Process of absorption of carotenoids from food matrix. 1, 2) Disruption of food matrix, 3) release of bile salts from common bile duct, 4) uptake of carotenoid molecule in lipid droplet and formation of micelle, 5) uptake of carotenoid molecule in enterocyte, and 6) release of carotenoid molecule in blood circulation.
thermal processing (Colle et al., 2013). Cooking is also helpful to reduce polyphenols, tannins, oxalates and phytates compounds found in legumes and cereals that can interfere with absorption of certain minerals (Khandelwal, Udipi & Ghugre, 2010). So, while deciding between raw and cooked foods, personal choice is ultimately important. The most nutrition experts suggest that eat your fruits and vegetables the way you prefer them and how they taste best to you.
7. Functions, biological activity and recommended daily allowance (RDA) of carotenoids Carotenoids are essential structural components of the photosynthetic apparatus which protect cells against photo-oxidative damage and also play an ecological role of attracting pollinators and seed dispersers due to distinct colors in leaves, flowers and fruits. Some
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Fig. 5. Comparative bioavailability chart of carotenoids from fruits and vegetables.
carotenoids (neoxanthin and violaxanthin) also act as precursors for the biosynthesis of the plant hormones abscisic acid (ABA) and strigolactone (Matusova et al., 2005; Parry & Horgan, 1991). Higher animals are incapable of biosynthesizing carotenoid, so these pigments are essentially indigested through the diet as precursors for retinol (vitamin A) biosynthesis (O'Byrne & Blaner, 2013). More than 700 naturally occurring carotenoids have been identified from plants, animals, fungi, and micro-organisms and nearly 50 are recorded for provitamin A activity (Hurst, 2008). Provitamin A activity is the ability of carotenoids to form vitamin A (retinol and retinal) by the action of carotene dioxygenase (Von Lintig & Vogt, 2000). Any carotenoid containing at least one unmodified β-ionone ring may be cleaved to provide provitamin A activity (Send & Sundholm, 2007). Thus, provitamin A active carotenoids include β-carotene, α-carotene, γ-carotene, and βcryptoxanthin. As, β-carotene contains two β-ionone ring, so it possesses 100% provitamin A activity, and lycopene lacks provitamin A activity due to the absence of β-ionone ring. Provitamin A activity of major carotenoids is illustrated in Table 3. Structurally, retinol is essentially one half of the molecule of β-carotene with an added molecule of water. Although provitamin A activity is the major function of carotenoids, potent antioxidant activity of carotenoids through singlet oxygen quenching and deactivation of free radicals plays important roles in the prevention of certain types of cancer, cardiovascular diseases, and macular degeneration (Müller et al., 2015). Apart from this, carotenoids play important roles in cellular and organelle function. Biological activities of carotenoids in animals are summarized in Fig. 6. The biological activity of carotenoids is assessed by their conversion to retinol equivalents (RE; 1 RE = 1 μg of retinol). Percent absorption and conversion of carotenoids are taken into account by the relationship of 1 RE = 6 μg of β-carotene and 12 μg for other carotenoids, considering 50% biological activity (Eitenmiller, Landen & Ye, 2007). RDA of carotenoids is 1000 RE for adult men, 800 RE for adult and pregnant women and 1300 RE for lactating women (Eitenmiller et al., 2007). International unit (IU) is the other measure of biological activity of carotenoids; 1 IU is equal to 0.3 μg all-Z-retinol and 0.6 μg of β-carotene (Thus, 1RE = 3.3 IU). Biological activity of carotenoids can be stabilized by the addition of phenyl groups. You, Jeon, Byun, Koo and Choi (2015) prepared synthetic
carotenoids containing the aromatic phenyl groups with a parasubstituent at C-13 and C-13′ position in order to overcome a structural instability of carotenoid. These stabilized carotenoids exerted stronger radical scavenging activity than β-carotene in DPPH and ABTS assays. 8. Genetic engineering of carotenoid pathway A genetic engineering approach has been successfully used in plants to enhance the carotenoid content in staple food, fruits and vegetables. The most successful example is the “golden rice” (Potrykus, 2001, 2003), with a significant high amount of β-carotene. This approach has also been successfully applied to “golden potatoes” to obtain higher amounts of β-carotene and lutein (Diretto et al., 2006; Ducreux et al., 2005), carrot plants for novel ketocarotenoid production (Jayaraj, Devlin & Punja, 2008), and tomatoes with enhanced β-carotene levels with the overexpression of lycopene β-cyclase (Guo, Zhou, Zhang, Xu & Deng, 2012). Lutein and δ-carotene contents were also enhanced in tomato by constitutive expression of lycopene ε-cyclase-encoding gene (Giorio, Yildirim, Stigliani & D'Ambrosio, 2013). Transgenic tomatoes were also developed for high-yield production of astaxanthin by co-expressing algal β-carotene ketolase and β-carotene hydroxylase, leading to massive accumulations of mostly free astaxanthin in leaves (3.12 mg/g) and esterified astaxanthin in fruits (16.1 mg/g) (Huang, Zhong, Liu, Sandmann & Chen, 2013). Recently, the plastid genome of lettuce was site-specifically modified with the addition of three transgenes, which encoded β-carotene ketolase and β-carotene hydroxylase from a marine bacterium Brevundimonas sp., and isopentenyl diphosphate isomerase (IPP isomerases) from a marine bacterium Paracoccus sp., to produce astaxanthin fatty acid. Production of astaxanthin is not common among higher plants, only the petal of inedible Adonis plants such as A. aestivalis, can produce astaxanthin (Harada et al., 2013). For commercial application, astaxanthin is produced synthetically. Naturally unicellular green alga, H. pluvialis and Chlorella zofingiensis represent the most promising producers of natural astaxanthin. Numerous studies have shown potential antihypertensive, anti-inflammatory, anti-diabetic, anticancer, neuroprotective, nephroprotective and cardiovascular disease prevention properties of
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requirement and a great challenge to the food scientist to eradicate the hunger and malnutrition. 9. Enhancement of carotenoids with non-GM based approaches
Fig. 6. Biological activities and properties of carotenoids in animals.
astaxanthin (Hussein, Sankawa, Goto, Matsumoto & Watanabe, 2006; Yuan, Peng, Yin & Wang, 2011). Oral administration of astaxanthin is generally recommended with omega-3 rich seed oils to improve the bioavailability and other benefits (Ambati, Phang, Ravi & Aswathanarayana, 2014). Multigene engineering has been used to modify many different metabolic pathway genes simultaneously to produce maize seeds with enhanced levels of carotenoids, folate and ascorbic acid. This strategy fascinates the development of nutritionally enriched staples food crops providing adequate amounts of several unrelated nutrients (Farré et al., 2011). Metabolic engineering is another method applied for enhancing leaf carotenoids and abiotic stress tolerance in plants (Marco et al., 2015). Recently, increases in the plastid number, area, and carotenoid content were observed in transgenic tomato fruits overexpressing tomato SlAPRR2-like gene driven by the 35S promoter (Pan et al., 2013). Sweet potato plants overexpressing sweet potato orangeinsertion type (IbOr-Ins) under the control of CaMV 35S promoter in an anthocyanin-rich purple-fleshed cultivar exhibited increased carotenoid levels (up to 7-fold) in their storage roots compared to wild type plants (Park et al., 2015). Nowadays, many researches are targeting the staple food crop such as wheat to improve the carotenoid content in wheat grains (Wang et al., 2014). The production of transgenic plants for enhanced production of novel carotenoids and other bioactives is expected to contribute significantly in human health (Davies & Espley, 2013). Due to increasing population to agricultural land ratio, production of nutrient dense food through biofortification is prime
Non-GM genetic modification (non-GM) based approaches have also been successfully used in food plants for enhanced carotenoid biosynthesis and accumulation. Application of methyl jasmonate (abiotic elicitor) is reported to enhance carotenoid content in romaine lettuce (Kim, Fonseca, Choi & Kubota, 2007). Similarly, application of salicylic acid is recorded to enhance carotenoid contents in wheat and moong (Vigna radiata) seedlings (Moharekar et al., 2003). In addition to the use of elicitor molecules, foliar application of fertilizers (K, N, Mg) is also reported to promote the biosynthesis of carotenoids in muskmelon fruit (Lester, Jifon & Rogers, 2005) and carrot (Smoleń & Sady, 2009). Exogenous application of 25 ppm methyl jasmonate (MJ) and salicylic acid (SA) was also shown being beneficial for enhanced production of carotenoids through differential regulation (more than 6-fold up-regulation) of carotenoid biosynthetic genes in red algae (Gao et al., 2012; Lu et al., 2010). Foliar administration of biotic elicitors, carboxy-methyl chitosan and chitosan, and signaling molecules, methyl jasmonate (MJ) and salicylic acid (SA) are also found beneficial for the enhancement of major carotenoids in leaves of field grow trees of M. oleifera (Saini, Prashanth, Shetty & Giridhar, 2014). Złotek, Świeca and Jakubczyk (2014) quantified the higher accumulation of flavonoids and phenolic acids in butter lettuce (Lactuca sativa), following the application of abiotic elicitors, however, changes in commercial quality, such as appearance, color, aroma, crispness and flavor were not observed. O'Hare, Fanning and Martin (2015), succeeded to increase zeaxanthin concentration in sweet-corn kernels by 10 times at sweet-corn eating-stage. This was achieved using conventional breeding and selection approach by redirecting carotenoid synthesis towards the β-arm of the pathway where zeaxanthin is synthesized. In addition to enhanced content, conventional breeding can be used to improve the bioaccessibility and bioavailability of bioactives. Berni, Chitchumroonchokchai, Canniatti-Brazaca, De Moura and Failla (2014) recorded the significant bioaccessibility of carotenoids from conventionally bred clone cassava (Manihot esculenta), compared to their respective parental varieties. Various biotic and abiotic elicitors have been used in fruits and vegetables at pre-harvest and post-harvest stages for quantitative enhancement of bioactive compounds, antioxidant activity and economic yield (Baenas, García-Viguera & Moreno, 2014; Saini, Akitha Devi, Giridhar & Ravishankar, 2013); however information about functional efficiency of elicitors is scarce. Thus, the effect elicitors on bioaccessibility and bioavailability of health promoting compounds should be analyzed to justify the functional benefits of elicitation. GM based approach is limited to a few crops and also having societal and regulatory barriers. So, the development of eco-friendly method to enhance nutritionally important bioactive compounds can play a potential role in the future to improve the nutritional quality of food plants at pre- and post-harvest conditions. 10. Methods of analysis Many methods were used for the identification and quantification of carotenoids from food matrix including colorimetric, spectrophotometric, fluorometric, paper, open-column and thin-layer chromatography, high performance liquid chromatography (HPLC), and capillary electrophoresis. HPLC coupled with nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (positive mode atmospheric pressure chemical ionization; APCI+ mode) are often used for characterization and identification studies. HPLC is the gold standard for preparatory step for characterization and analysis of carotenoids in biological and food samples, which can resolve cis-isomers from all-trans isomers (Gupta, Sreelakshmi & Sharma, 2015).
R.K. Saini et al. / Food Research International 76 (2015) 735–750 Table 4 Molar absorption coefficient and absorption maxima for the visible light spectrum of common food carotenoids in light petroleum ether solvent. Source — Britton (1995)and Hurst (2008). Carotenoid
Absorption maxima λ max (nm)
α-Carotene
422 444 473 425 449 476 431 456 489 437 462 494 425 449 476 421 445 474 444 470 502 416 438 467 416 440 465 424 449 476
β-Carotene
δ-Carotene
γ-Carotene
β-Cryptoxanthin
Lutein
Lycopene
Neoxanthin
Violaxanthin
Zeaxanthin
Molar absorption coefficient (E 1% 1 cm) 2800
2592
3290
3100
2386
2550
3450
2243
2250
2348
Significant diversity of carotenoids (More than 700 types) exists in plants, animals, fungi, and micro-organisms, and obtaining and maintaining pure carotenoid standards is one major problem in quantification. Apart from this, significant qualitative and quantitative compositional variation between plants and between cultivars of the same plant, complexity of the food matrices, and susceptibility of carotenoids to cis and trans isomerization and oxidation during analysis and during storage is the major problems in reliable and accurate analysis of carotenoid. A scheme for obtaining standards carotenoids by open column chromatography from leafy vegetable carotenoids was proposed by Kimura & Rodriguez-Amaya (2002). The strategy described by these authors is low-cost and provides a constant supply of carotenoid standards (90–100% purity), including those which cannot be acquired commercially. Saponification and direct solvent extraction are commonly used to extract carotenoids from leafy vegetables. Saponification is generally
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avoided during quantification of carotenoids to reduce time, solvent and artifact formations in the sample (Kimura & Rodriguez-Amaya, 1999; Rodriguez-Amaya, 2001). Whereas, for carotenoid purification, saponification is necessary to destroy lipids, chlorophyll and other materials in the sample that potentially interfere with chromatographic separations. In detection, carotenoids show absorption in the visible region (400 and 500 nm) due to the long conjugated double-bond system (Britton, 1995), but the λmax of individual carotenoids can vary depending on functional groups. The molar absorption coefficient and absorption maxima for the visible light spectrum of common food carotenoids are given in Table 4. A simplified method with different steps used for extraction and quantification is also given in Table 5. This table summarizing sampling, extraction, partitioning and chromatographic spearing method for extraction and quantification of major carotenoids form fruits and vegetables. Structure, polarity, sequence of elution in reverse-phase column (especially in C30 hydrophobic column) and source of major carotenoids from fruits and vegetables are illustrated in Fig. 7. Sequence of elution of these major carotenoids may be useful in the identification of respective carotenoids during chromatographic separation. During chromatographic separation of carotenoids from leafy vegetables in reverse phase column (especially in C30 hydrophobic column), first peaks are generally composed of neoxanthin and violaxanthin, followed by lutein, zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene. Always central cis isomers elute first then trans isomers, due to high polarity of cis, compared to trans isomers (Pendon et al., 2005). However, this sequence of elution is reversed in C18 reverse phase columns. However, this sequence of elution is reversed in C18 Monomeric or C18 polymeric reverse phase columns. Rivera and Canela-Garayoa (2012) reviewed the carotenoid resolution and detection techniques for qualitative and quantitative analysis of carotenoids. The authors compared the performance of stationary phases for separation cis- and trans-isomers of carotenoids, and concluded that both C18 and C30 stationary phases can provide good resolution for the separation of geometrical isomers of carotenoids with similar polarity. Atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry (MS/MS) in positive ion mode is the most common method to identify and characterize different carotenes and oxygenfunctionalized carotenoids containing epoxy, hydroxyl, and ketone groups (Rivera, Christou & Canela-Garayoa, 2014). Mass spectrometry is the powerful technique to identify new compounds, even pigments with a very similar structure can be differentiated through their fragmentation pattern. In structural elucidation, mass spectrometry (MS) is generally combined with proton nuclear magnetic resonance (1H NMR) data to obtain the absolute confirmatory results on highly similar isomers (Qiu, Zhu, Tang, Shi & Gao, 2014). In a recent review, Rivera et al. (2014) have summarized the positive MS fragmentation data of selected carotenoids obtained using various ionization techniques and
Table 5 Common steps used in extraction and quantification of major carotenoids from fruits and vegetables. Steps
Procedure
Important point to consider
Sample peroration
Preparation of homogenous samples
Extraction
Extraction of carotenoid in cold acetone
Partitioning
Partitioned to the petroleum ether 40–60 °C containing 10% (v/v) diethyl ether and washed with water to remove the traces of acetone Saponify overnight with 10% KOH (potassium hydroxide) in methanol (w/v), wash with water to remove the alkali, and dried in vacuum rotavapor (T ≤ 35 °C) (Optional) By RF-HPLC in C30 column
Volume reduction procedure should be followed to minimize the variation between sample samples Minimum 1:10 ratio of sample and acetone should be used. Repeat the extraction until samples became colorless Avoid micelle formation during addition of water
Saponification
Chromatographic separation Identification and quantification
Confirmation of carotenoids by appropriate standards, mass spectrometry and absorbance spectrum
Saponification step can be eliminated to routine chromatographic analysis. chances of artifact formation are more during saponification C30 carotenoid column can separate geometrical (-cis and -trans) isomers of carotenoids Purified carotenoids are very sensitive to oxidation and light induced degradation, check the purity time to time
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Fig. 7. Structure, polarity, sequence of elution in reverse-phase column and source of major carotenoids from fruits and vegetables.
matrices. Fragmentation pattern and intensity fragment ratios, complied in this review will be very useful in the analysis of MS data for carotenoid identification. C30-HPLC–APCI–MS in combination with NMR spectroscopy, gel permeation chromatography (GPC), and UV/Vis spectroscopy techniques are recently employed by Qiu et al. (2014) to separate nine various isomers of canthaxanthin. Accelerated solvent extraction (ASE) is recently applied for the extraction of carotenoids from orange carrot and identified that solvent properties and temperature are the most important factors affecting the ASE extraction (Saha, Walia, Kundu, Sharma & Paul, 2015). Cardenas-Toro et al. (2015) comparatively studied the soxhlet extraction (LPSE–SOX), percolation (LPSE–PE) and pressurized liquid extraction (PLE) for the recovery of carotenoid-rich extracts from pressed palm fiber (PPF) in terms of yield, carotenoid profile and economic viability to evaluate the methods and industrial applicability. In results, PLE technique showed the highest selectivity for carotenoids compared to LPSE techniques. However, the lowest cost of manufacturing was obtained for LPSE compared to PE. A carotenoid purification method with dual-mode countercurrent chromatography (CCC) for β-carotene, α-carotene and lutein from a fresh carrot extract was recently developed by Englert, Hammann and Vetter (2015). With this method, 51 mg of β-carotene, 32 mg of αcarotene and 4 mg of lutein could be isolated from 100.2 mg crude carrot extract in a short time and with high purities of 95–99% by using dual-mode CCC.
Significant research has been conducted in the advancement of carotenoid extraction and quantification methods. The status of carotenoid analytical methods from food has also recently been reviewed by Rodriguez-Amaya (2015). Identification and quantification of carotenoids are still difficult in many labs due to unavailability of standards at normal price. So, advance techniques need to be developed for purification of carotenoids. In addition to food industries, purified carotenoids are also having enormous applications in cosmetic industries (Anunciato & da Rocha Filho, 2012). So far, only few carotenoids, such as neoxanthin, violaxanthin, lutein, zeaxanthin, β-cryptoxanthin, αcarotene, β-carotene and lycopene are studied in fruits and vegetable. So, in the future, investigations on extraction, quantification and biological activity of different isomers and epoxy carotenoids can be made. In the advancement of HPLC, comprehensive two-dimensional liquid chromatography (LC × LC) with normal phase (NP) or reverse phase (RP) separation is employed for the separation of carotenoids (Cacciola et al., 2012; Dugo et al., 2006; Dugo et al., 2008). In LC × LC, combination of two independent separation steps with orthogonal selectivity (change in pH, solvent, and column) is used, in which a primary column is connected to one or more secondary columns. The fractions of the effluent from this first column are injected onto a second column with the help of specialized switched valve, which provides a much faster separation, typically with an analysis time of a minute or less. The results from LC × LC are combined into a matrix and displayed as
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a two-dimensional chromatogram in the form of two or threedimensional plot. The electiveness of LC × LC separations has been demonstrated in several works, where size exclusion chromatography (SEC) in the first dimension, and with HPLC in the second dimension, able to separate the molecules on the basis of size, hydrophobicity and polarity. In the first analysis of carotenoids in citrus by LC × LC, fifty-seven peaks were detected in the two-dimensional space for the sweet orange essential oil and 59 peaks in the red orange juice. In total, 75 carotenoids were detected in the two samples, 37 of which were identified (Dugo et al., 2006). In another study, thirty-three carotenoid belonging to ten different chemical classes were identified in red chili peppers by using comprehensive normal-phase × reversed-phase (NP-LC × RP-LC) liquid chromatography (Cacciola et al., 2012). 11. Conclusions In general, vegetables are comparatively richer source of carotenoids than fruits; however carotenoids from fruits are more bioavailable than from vegetables. Different pre- and post-harvesting factors related to carotenoid bioaccessibility and bioavailability should be completely studied to increase the intake of carotenoids in animals with optimum diet. The biosynthesis of carotenoids in plants has been extensively studied; however their importance for plants and animals is still not well established. The role of carotenoids as antioxidants and its mechanism of action are needed to be investigated further. The process of carotenoid esterification in plants is also not established. This knowledge will facilitate further advancement in the field of carotenoid metabolic engineering to improve nutritional quality and nutrient density in food crops. Enhanced production of carotenoids in staple food crop such as wheat is the most appropriate measure to improve the intake of carotenoids in populations of developed and developing countries. Protection of carotenoids from degradation during processing is also equally important to maintain its required level in human diet. Advance and cost affective processing methods need to be developed to preserve the carotenoids and other bioactive compounds. Factors related to bioaccessibility such as thermal processing, structural barriers in food (matrix, cell wall integrity, bio-encapsulation), addition of lipids are the most crucial in determining the bioavailability of carotenoids. So, detailed interrelated studies are required in identifying the favorable factors to improve the bioavailability of carotenoids from different foods. This will help in the development of specialized food with potential bioavailability. Although, a great advancement has been achieved in the analysis of carotenoids, focused studies are required to explore the carotenoids from underutilized fruits and vegetables. Biological activity and significance of several new carotenoids, such as deepoxyneoxanthin, mimulaxanthin, and strigolactones are also needed to be evaluated. Conflict of interest The authors have declared that there is no conflict of interest. Acknowledgements This paper was supported by the KU Research Professor Programme of Konkuk University, Seoul, South Korea. Also, financial support from the Export Promotion Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, South Korea is highly acknowledged. References Agócs, A., Nagy, V., Szabó, Z., Márk, L., Ohmacht, R., & Deli, J. (2007). Comparative study on the carotenoid composition of the peel and the pulp of different citrus species. Innovative Food Science & Emerging Technologies, 8(3), 390–394. http://dx.doi.org/ 10.1016/j.ifset.2007.03.012.
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