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Carotenoids from fruits and vegetables: Chemistry, analysis, occurrence,

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 Food Research International 76 (2015) 735–750

Contents lists available at ScienceDirect

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 a b s t r a c t

Article history: Fruits and vegetables are generally considered as important contributors to a healthy diet and their intake is ex-
Received 27 May 2015 tremely helpful to reduce the risk of specific diseases like cancers, cardiovascular diseases, neural tube defects,
Received in revised form 23 July 2015 and cataracts. Bioactive constituents from fruits and vegetables, such as carotenoids, folic acid and dietary fiber
Accepted 31 July 2015
appear to play important roles in the prevention of these diseases. Carotenoids and their derivatives are versatile
Available online 3 August 2015
isoprenoids and play a vital role in plants and animals, starting from cellular antioxidant to gene regulation and so
Keywords:
their importance at cellular and molecular level is well established. The most significant aspect of carotenoids in
Carotenoids our diet is the antioxidant and provitamin A activity, and also the color that they impart to our food. The compo-
Lutein sition and bioavailability of carotenoids in food are significantly influenced by processing and other post-harvest
β-Carotene technologies. This review discusses the theoretical aspects and recent developments in structural properties, bio-
Lycopene synthesis and enhancement, processing, methods of analysis, composition in fruits and vegetables, and bioacces-
Biosynthesis sibility and bioavailability of carotenoids. Additionally, future research challenges in this context are identified.
Physiology © 2015 Published by Elsevier Ltd.
Processing

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735
2. Chemistry of carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736
3. Carotenoids in fruits and vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736
4. Post-harvest physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738
5. Biosynthesis of carotenoids in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739
6. Bioavailability and bioaccessibility of carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741
7. Functions, biological activity and recommended daily allowance (RDA) of carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742
8. Genetic engineering of carotenoid pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 743
9. Enhancement of carotenoids with non-GM based approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744
10. Methods of analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744
11. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747

1. Introduction yellow, orange, and red colors in various fruits and vegetables
(Namitha & Negi, 2010). Carotenoids can be classified into two groups
Carotenoids are natural pigments which are metabolized by plants, on the basis of functional groups; xanthophylls, containing oxygen as
algae, and photosynthetic bacteria; which are responsible for the functional group, including lutein and zeaxanthin, and carotenes,
which contain only parent hydrocarbon chain without any functional
⁎ Corresponding authors.
group, such as α-carotene, β-carotene and lycopene. Addition of polar
E-mail addresses: saini_1997@yahoo.com (R.K. Saini), sewpark@konkuk.ac.kr groups (epoxy, hydroxyl and keto) alters the polarity of carotenoids
(S.W. Park). and affects biological functions (Britton, 2008). Fruit and vegetables

http://dx.doi.org/10.1016/j.foodres.2015.07.047
0963-9969/© 2015 Published by Elsevier Ltd.
736 R.K. Saini et al. / Food Research International 76 (2015) 735–750
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). Epide-
miological studies demonstrated that the consumption of diets rich in
carotenoids is associated with a lower incidence of cancer, cardiovascu-
lar diseases, age related macular degeneration and cataract formation
(Meyers et al., 2014; Sharoni et al., 2012). Deficiency of carotenoids re-
sults in clinical signs of conjunctiva and corneal aberrations including,
xerophthalmia, night blindness, keratomalacia, corneal ulceration, scar-
ring, and resultant irreversible blindness (Sommer, 2008). Other than
the above, deficiency of provitamin A carotenoids leads to vision-
disability 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 pharma-
ceutical industries, makes them potential candidates for enhancement
and manipulation. Over the past decades advances in molecular genet-
ics and biotechnological approaches have led to the understanding of
carotenoid biosynthesis phenomena and its enhancement in microor-
ganisms and higher plants (Potrykus, 2001, 2003). This review discusses
the theoretical aspects and recent developments in structural proper-
ties, biosynthesis and enhancement, processing, methods of analysis,
composition in fruits and vegetables, and bioaccessibility and bioavail-
ability 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 togeth-
er in a specific manner that the organization of isoprenoid units is re-
versed 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). Ca-
rotenoids 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 & Mínguez-
Mosquera, 2005). These properties also depend on the kind of fatty
acid bound to the carotenoid molecule. Esterification facilitates caroten-
oid storage, aiding in the integration of these highly lipophilic molecules
Fig. 1. a) Joining style of eight isoprenoid units to form β-carotene, and b) molecular structure of β-carotene chromophore.
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). Caroten-
oids 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 pre-
sented in carotenoid molecule determines the light absorption and
light harvesting properties. Thus, chromophore is the part of a caroten-
oid 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 carot-
enoids by oxidative cleavage, catalyzed by family of carotenoid cleavage
dioxygenases (CCDs). CCDs often exhibit substrate, promiscuity, and cat-
alyze many important reactions with remarkable precision which con-
tributes 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 pig-
ment bixin, and aromatic volatile aroma compounds β-ionone and α-
ionone. 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
 R.K. Saini et al. / Food Research International 76 (2015) 735–750 737

Rodriguez-Amaya, Kimura, Godoy & Amaya-Farfan

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

(2008) Porcu & Rodriguez-Amaya (2006)

Lokesh, Divya, Puthusseri, Manjunatha &

bright red color) is the major constituents of tomatoes and tomato

Delgado-Pelayo, Gallardo-Guerrero &

Faria, Marques & Mercadante (2011)

products. Lutein (nearly 45%) and β-carotene (25–30%) followed by

Campbell & Padilla-Zakour (2013)

Campbell & Padilla-Zakour (2013)

Martínez-Valdivieso et al. (2014)

violaxanthin (10–15%), and neoxanthin (10–15%) are the predominant

Delgado-Pelayo et al. (2014)

forms of carotenoids in green leafy vegetables (Lakshminarayana, Raju,

Khonsarn & Lawan (2012)

Khonsarn & Lawan (2012)
Hornero-Méndez (2014)
Krishnakantha & Baskaran, 2005; Priyadarshani & Jansz, 2014),
although the absolute concentration of each carotenoid varies consider-

Lokesh et al. (2014)

Lokesh et al. (2014)

Freitas et al. (2015)

Bunea et al. (2012)
Neelwarne (2014)
ably among different vegetables. α-Carotene, β-cryptoxanthin, zeaxan-
thin, antheraxanthin, and lutein 5,6-epoxide (luteoxanthin) are also

References
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 α-
carotene can be found in a limited number of fruits and vegetables,
such as sweet potato, carrots, pumpkin, and dark green vegetables,

carotenoids
such as green beans, spinach and broccoli (Khoo, Prasad, Kong, Jiang &

173.00
Total
Ismail, 2011). Knowledge on carotenoid composition in different edible

1.24

0.54
19.7

12.0

185
–

–
–
–
parts and cultivars will be useful to nutritional experts for the selec-
tion of nutrient-rich plants for food fortification and proper diet recom-

Lycopene
mendation. Compositions of major carotenoids in important fruits
and vegetables are given in Tables 1 and 2. In recent years, numerous

7.0
–

–
–
–
–
–

–
underutilized leafy vegetables, such as Moringa oleifera (Drumstick
tree) and Lactuca indica (Indian lettuce) and Oenanthe javanica (Water

β-Carotene
dropwort) were established as exceptionally rich sources of carot-

170.00
enoids (Andarwulan et al., 2012; Kongkachuichai, Charoensiri, Yakoh,

0.17

0.66

0.26
8.38
0.23
0.23
1.4

9.3
9.9
12

–
Kringkasemsee & Insung, 2015; Saini, Shetty & Giridhar, 2014). Among
25 common and less common leafy vegetables, including ama- α-Carotene

ranthus, fenugreek and spinach, the highest content of β-carotene

(19.7 mg/100 g) was recorded in leaves of M. oleifera (Bhaskarachary,

0.18

0.32
8.62
1.1
Rao, Deosthale & Reddy, 1995). Similarly, among underutilized vegeta-
–

–
–
–

–
bles from Indonesia, maximum content of β-carotene (14 mg/100 g)
β-Cryptoxanthin

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

0.003
and O. javanica, respectively (Kongkachuichai et al., 2015). International
0.73
1.1

1.6
non-governmental organizations (NGOs) such as Trees for Life and
–

–
–
–

–
Educational Concerns for Hunger Organization (ECHO) have vigorously
Zeaxanthin

supported Moringa leaves as “natural nutrition for the tropics”. India is

the world's largest producer of Moringa and enabled to provide the low-
0.02

0.01

0.02

14.1
2.7

1.5

cost food for the malnourished population. In the future, detailed 1.1
–

–
–
–

composition of carotenoids in these underutilized vegetables may con-

Lutein

tribute significant information to select nutrient rich plants for food

0.71

0.06
0.09

0.06

0.47
0.39
31.7
237

172
1.3

0.4

0.9

formulation.
–

In plant kingdom, flower stigmas of saffron plant (Crocus sativus)

Cucurbita pepo subsp. pepo

and waxy seed arils of achiote shrub (Bixa orellana) is the richest source
Values are in μg/g fresh weight basis (#values are in μg/g dry weight basis).

of apocarotenoids (Rosati, Diretto & Giuliano, 2009). However, small

contents of apocarotenoids can be found in citrus fruits and paprika
Mangifera indica L.
Prunus armeniaca
Musa paradisiaca

Syzygium cumini
Malpighia glabra

Malus domestica

Malus domestica

Musa acuminate
Musa acuminate

Ananas comosus

vegetables. Agócs et al. (2007) recorded 2–7% red apocarotenoids β-

Prunus persica
Carica papaya
Vitis vinifera

citraurin in pulps of citrus species. Fleshman et al. (2011) recorded

Botanical

1.5% of β-apocarotenoids to the total β-carotene present in cantaloupe

name

(Cucumis melo Reticulatus Group) and Orange Dew (Cucumis melo,

Inodorus Group).
Contents of major carotenoids in selected fruits.

Seed pulp or aril of ripened fruits of Momordica cochinchinensis, com-

#

Apricot (var. hargrand) unpeeled canned

Apple, green (cv. green golden delicious)

Peach (var. redhaven) unpeeled canned

monly known as gac fruit, is the known richest source of carotenoids

Pineapple (core, minimally processed)

plant derived carotenoids. Lycopene and β-carotene are the major ca-
Grapes (white, cv. aromat de Iaşi)

rotenoids in gac fruit, which accounted 408 μg/g and 83.3 μg/g fresh
Banana (cv. nanjangud rasabale)

weigh, respectively (Vuong, Franke, Custer & Murphy, 2006). De Rosso

Acerola (home garden, ripe)

Apple, red (cv. royal gala)#

Banana (red, cv. chandran)

and Mercadante (2007), quantified the major and minor carotenoids

Jambolão fruits (Brazil)
Banana (cv. cavendish)

from native fruits of Amazonia region, including buriti (Mauritia vinif-

Watermelon (Flesh)#

era), 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 caroten-
Papaya
Mango
Table 1

Fruit

oid 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
738 R.K. Saini et al. / Food Research International 76 (2015) 735–750

carotenoids and other bioactive compounds in pulps and by-products of

Kaulmann, Jonville, Schneider, Hoffmann & Bohn (2014)

Carvalho, Smiderle, Carvalho, Cardoso & Koblitz (2014)

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.

Maurer, Mein, Chaudhuri & Constant (2014)

Traditional and non-traditional tropical fruits are considerably rich in

Saini, Shetty, Prakash & Giridhar (2014)

carotenoids and other bioactive compounds, which indicate promising

Divya, Puthusseri & Neelwarne (2012)

perspectives for the utilization of these fruit species and their by-

Niizu & Rodriguez-Amaya (2005)

products in potential commodities.

Lakshminarayana et al. (2005)

Lakshminarayana et al. (2005)

Žnidarčič, Ban & Šircelj (2011)

De Carvalho et al. (2012)

Kaulmann et al. (2014)

Kaulmann et al. (2014)

Kaulmann et al. (2014)

4. Post-harvest physiology
Žnidarčič et al. (2011)

Žnidarčič et al. (2011)

Maurer et al. (2014)

Content and types of carotenoids in plants depend on several pre-

and post-harvesting factors, genotype, ripening time, cultivation meth-
References

od and climatic conditions, processing. Different parts of the same plant

also may contain different types and amounts of carotenoids. For exam-
ple, peel of the fruits is generally richer in carotenoids compared to pulp.
Packaging and processing methods greatly influence the content of
carotenoids

0.746

carotenoids in processed food products (Moura, Miloff & Boy, 2015). Ir-
46.46
39.54

1691.5

1092.6

2386.2
804.8

132.9

236.1

409.9

respective of thermal (heat drying) or non-thermal (e.g., high-pressure,

Total

pulsed electric field, ultrasound drying), processing may significantly

degrade the level of carotenoids in food products. For most vegetables,
Lycopene

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
β-Carotene

were protected from high temperature, air and light. In addition to

0.718

this, thermal processing may cause significant quantitative changes in

11.38

0.22
53.6

73.1

63.4

79.6
44.0
14.9
586.1

231.5
121.3

172.2
365.3
244.2

carotenoid isomers, due to possible trans to cis isomerization of β-

carotene and lutein (Colle, Lemmens, Knockaert, Loey & Hendrickx,
α-Carotene

2015). So, higher retention of cis-isomers was generally recorded in

thermal processed fruits and vegetables compared to trans-isomers. In
67.06
45.0

39.9
2.8

general, cis-isomers of carotenoids exhibit less potent provitamin activ-

ity compare to trans-isomers, which cause further loss of provitamin
activity in processed food products (Castenmiller & West, 1998). Degra-
β-Cryptoxanthin

dation of β-carotene and lutein and formation of cis-isomers (4–40%)

during thermal dehydration studied in a number of fruits and vegeta-
bles including peas, broccoli, kale, spinach, and corn (Saini, Shetty,
11.63
0.15

0.15

0.21
1.7

Prakash & Giridhar, 2014; Updike & Schwartz, 2003). All the available
methods used in processing (dehydration) of fruits and vegetables
Zeaxanthin

cause significant degradation of carotenoids. Amount of degradation is

more in thermal (heat) base methods compared to non-thermal
0.9

6.2
0.8

9.5
0.6
135.4

15.1

methods. However, major food industries in developing countries

are using thermal based method of processing due to high initial set-
0.28
28.05
11.63

65.22
Lutein

up and running cost of other advanced methods. For example, lyophi-

1.5

312.7
596.0

775.8
59.1

13.1
52.5

74.4

13.5

lisation is the best method to preserve the major nutrient, including ca-
rotenoids during dehydration (Saini, Shetty, Prakash & Giridhar, 2014),
Brassica oleracea var. gemmifera

Values are in μg/g fresh weight basis (#values are in μg/g dry weight basis).
Brassica oleracea var. acephala

however, this method is not economical in large scale industries due to

Brassica oleracea var. botrytis
Brassica oleracea var. italica

Trigonella foenum-graecum

high running and initial cost of machineries. Non-thermal based pro-

cessing methods, such as high pressure, high electric field pulse, high-
Taraxacum officinale
Coriandrum sativum

Cucurbita moschata

pressure CO2, are emerging in food processing sector. In the future,

Cucurbita maxima
Cichorium intybus

Spinacia oleracea
Moringa oleifera
Botanical name
Contents of major carotenoids in selected vegetables (μg/g).

these advanced processing methods can play an important role in the

Daucus carota

Lactuca sativa
Eruca sativa

preservation of bioactive compounds. However, additional attention is

Zea mays

Table 3
Relative provitamin A activity of common food carotenoids.
Coriander leaves (var. GS4 Multicut)#

Squash (Landraces A, Aracaju, Brazil)

Carotenoid % provitamin A activity

All-E-β-carotene 100
Broccoli (var. verde calabrese)

Drumstick leaves (cv. Bhagya)

Cauliflower (var. White Rock)

All-E-β-cryptoxanthin 57
13-Z-β-carotene 53
All-E-α-carotene 53
Chicory (cv. Anivip)

All-E- mutatochrome 50
Brussels sprouts

15-E-β-cryptoxanthin 42
Garden rocket

9-E-β-carotene 38
Fenugreek#
Dandelion
Vegetable

9-Z-β-cryptoxanthin 27
Pumpkin
Spinach#
Lettuce

β-Carotene-5,6-epoxide 21
Carrot
Table 2

Corn

Kale

13-Z-α-carotene 16
9-cis-α-carotene 13
 R.K. Saini et al. / Food Research International 76 (2015) 735–750
also required in the development of effective processing units with min-
imum 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 carot-
enoids 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, re-
spectively. 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 ex-
posed to high temperatures and higher sunlight incidence may have en-
hanced the carotenoids biosynthesis, to protect the plant from photo
oxidation.
Fig. 2. Biosynthetic pathway of carotenoids in plants.
739
5. Biosynthesis of carotenoids in plants
Carotenoid biosynthesis starts with the condensation of two
Geranylgeranyl diphosphate (GGPP) molecules which were synthe-
sized in the methylerythritol 4-phosphate (MEP) pathway (Cazzonelli
& Pogson, 2010). The carotenoid biosynthesis pathway in plants is sum-
marized in Fig. 2. In the last few years, almost complete sets of genes
encoding and enzymes required for the biosynthesis of these indispens-
able 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 precur-
sor for several other metabolites, such as terpenes, terpenoids, gibberel-
lins, chlorophylls, ubiquinones and tocopherols. 15-Z-phytoene then
undergoes four sequential reactions to form lycopene through the ac-
tion 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, intro-
duces 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 ac-
tivity (Send & Sundholm, 2007). Thus, lycopene lacks provitamin A ac-
tivity due to the absence of β-ionone ring. α-Carotene is acted upon
by a β-ring hydroxylase to form zeinoxanthin, which is then hydroxyl-
ated by an ε-ring hydroxylase to produce lutein. In another branch, β-
carotene can be hydroxylated to form zeaxanthin, by the action of β-
carotene hydroxylase. Zeaxanthin can be epoxidized to antheraxanthin
740 R.K. Saini et al. / Food Research International 76 (2015) 735–750
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 spe-
cialized 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-esteri-
fied) 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 enzy-
matic degradation in plastids. Plastid-localized carotenoid cleavage
Fig. 3. Carotenoid cleavage dioxygenases (CCD) mediated formation of apocarotenoids in plants.
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 com-
pounds. Carotenoid cleavage dioxygenases (CCDs) mediated the forma-
tion 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. Dur-
ing germination of seeds, carotenoids present in etioplasts are facilitat-
ing 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, Rodríguez-
Concepción & 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,
 R.K. Saini et al. / Food Research International 76 (2015) 735–750
and post-transcriptional feedback regulation (Cazzonelli & Pogson,
2010). Epigenetic regulatory mechanisms (chromatin modifications)
regulate the expression of carotenoid isomerase (CRTISO), which cata-
lyzes cis–trans reactions; 7,9,7′,9′-tetracis-lycopene to all-Z-lycopene.
This epigenetic mechanism is mediated by a chromatin-modifying his-
tone 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 biosynthe-
sis, 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 biosyn-
thesis during ripening. During tomato ripening, chlorophylls are differ-
entiated into chromoplasts, which accumulate lycopene membrane-
bound 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 stud-
ied due to its importance in animal nutrition and plant survival. Our un-
derstanding 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 caroten-
oid esterification can help in understanding the sequestration and stor-
age process of carotenoids. This knowledge will facilitate further
advancement in the field of carotenoid metabolic engineering to im-
prove 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 propor-
tion of ingested carotenoid that is released from the food matrix and in-
corporated into micelles in the gastrointestinal tract, and thus available
for intestinal absorption (Rodriguez-Amaya, 2015). Absorption of carot-
enoids 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 chylomi-
crons 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 deg-
radation 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
741
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 stimu-
late 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 pro-
cessing 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. How-
ever, their results are not conclusive, so, detailed and preciously con-
trolled 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 veg-
etables are comparatively less bioavailable then fruits (Schweiggert
et al., 2014), however, vegetables are the major contributors of caroten-
oids in human diets (Bowen, Stacewicz-Sapuntzakis & Diwadkar-
Navsariwala, 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 ran-
domized 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 ca-
rotenoids may surpass the adverse impact of dietary fibers. Factors re-
lated 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 carot-
enoids. So, detailed interrelated studies are required in identifying the
favorable factors to improve the bioavailability of carotenoids from dif-
ferent 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 is-
sues 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 diges-
tive 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 plan-
ning 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 degener-
ative 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 nu-
trients, such as lycopene, become more bioavailable after cooking and
742 R.K. Saini et al. / Food Research International 76 (2015) 735–750

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 le-
gumes 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 nu-
trition 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 photosyn-
thetic 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
 R.K. Saini et al. / Food Research International 76 (2015) 735–750
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 an-
imals 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 provi-
tamin A activity (Send & Sundholm, 2007). Thus, provitamin A active
carotenoids include β-carotene, α-carotene, γ-carotene, and β-
cryptoxanthin. As, β-carotene contains two β-ionone ring, so it pos-
sesses 100% provitamin A activity, and lycopene lacks provitamin A ac-
tivity 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 caroten-
oids, 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 activ-
ities of carotenoids in animals are summarized in Fig. 6. The biological
activity of carotenoids is assessed by their conversion to retinol equiva-
lents (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). Bi-
ological activity of carotenoids can be stabilized by the addition of
phenyl groups. You, Jeon, Byun, Koo and Choi (2015) prepared synthetic
743
carotenoids containing the aromatic phenyl groups with a para-
substituent 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 toma-
toes 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 hydrox-
ylase from a marine bacterium Brevundimonas sp., and isopentenyl
diphosphate isomerase (IPP isomerases) from a marine bacte-
rium Paracoccus sp., to produce astaxanthin fatty acid. Production of
astaxanthin is not common among higher plants, only the petal of ined-
ible Adonis plants such as A. aestivalis, can produce astaxanthin (Harada
et al., 2013). For commercial application, astaxanthin is produced syn-
thetically. Naturally unicellular green alga, H. pluvialis and Chlorella
zofingiensis represent the most promising producers of natural
astaxanthin. Numerous studies have shown potential antihyperten-
sive, anti-inflammatory, anti-diabetic, anticancer, neuroprotective,
nephroprotective and cardiovascular disease prevention properties of
744 R.K. Saini et al. / Food Research International 76 (2015) 735–750
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 over-
expressing tomato SlAPRR2-like gene driven by the 35S promoter (Pan
et al., 2013). Sweet potato plants overexpressing sweet potato orange-
insertion type (IbOr-Ins) under the control of CaMV 35S promoter in
an anthocyanin-rich purple-fleshed cultivar exhibited increased carot-
enoid 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
requirement and a great challenge to the food scientist to eradicate
the hunger and malnutrition.
9. Enhancement of carotenoids with non-GM based approaches
Non-GM genetic modification (non-GM) based approaches have
also been successfully used in food plants for enhanced carotenoid bio-
synthesis 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). Ex-
ogenous application of 25 ppm methyl jasmonate (MJ) and salicylic acid
(SA) was also shown being beneficial for enhanced production of carot-
enoids 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 abi-
otic elicitors, however, changes in commercial quality, such as appear-
ance, color, aroma, crispness and flavor were not observed.
O'Hare, Fanning and Martin (2015), succeeded to increase zeaxan-
thin concentration in sweet-corn kernels by 10 times at sweet-corn
eating-stage. This was achieved using conventional breeding and selec-
tion 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 bioaccessibi-
lity 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 veg-
etables at pre-harvest and post-harvest stages for quantitative enhance-
ment 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 bio-
availability of health promoting compounds should be analyzed to jus-
tify 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 impor-
tant bioactive compounds can play a potential role in the future to im-
prove 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, spectrophotomet-
ric, fluorometric, paper, open-column and thin-layer chromatography,
high performance liquid chromatography (HPLC), and capillary electro-
phoresis. 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) 735–750 745

Table 4 avoided during quantification of carotenoids to reduce time, solvent and

Molar absorption coefficient and absorption maxima for the visible light spectrum of com- artifact formations in the sample (Kimura & Rodriguez-Amaya, 1999;
mon food carotenoids in light petroleum ether solvent.
Source — Britton (1995)and Hurst (2008).
Rodriguez-Amaya, 2001). Whereas, for carotenoid purification, saponi-
fication is necessary to destroy lipids, chlorophyll and other materials in
Carotenoid Absorption maxima Molar absorption the sample that potentially interfere with chromatographic separations.
λ max (nm) coefficient (E 1% 1 cm)
In detection, carotenoids show absorption in the visible region (400 and
α-Carotene 422 500 nm) due to the long conjugated double-bond system (Britton,
444 2800
1995), but the λmax of individual carotenoids can vary depending on
473
β-Carotene 425 functional groups. The molar absorption coefficient and absorption
449 2592 maxima for the visible light spectrum of common food carotenoids are
476 given in Table 4. A simplified method with different steps used for ex-
δ-Carotene 431 traction and quantification is also given in Table 5. This table summariz-
456 3290
489
ing sampling, extraction, partitioning and chromatographic spearing
γ-Carotene 437 method for extraction and quantification of major carotenoids form
462 3100 fruits and vegetables.
494 Structure, polarity, sequence of elution in reverse-phase column (es-
β-Cryptoxanthin 425
pecially in C30 hydrophobic column) and source of major carotenoids
449 2386
476 from fruits and vegetables are illustrated in Fig. 7. Sequence of elution
Lutein 421 of these major carotenoids may be useful in the identification of respec-
445 2550 tive carotenoids during chromatographic separation. During chromato-
474 graphic separation of carotenoids from leafy vegetables in reverse phase
Lycopene 444
column (especially in C30 hydrophobic column), first peaks are general-
470 3450
502 ly composed of neoxanthin and violaxanthin, followed by lutein, zea-
Neoxanthin 416 xanthin, β-cryptoxanthin, α-carotene and β-carotene. Always central
438 2243 cis isomers elute first then trans isomers, due to high polarity of cis, com-
467
pared to trans isomers (Pendon et al., 2005). However, this sequence of
Violaxanthin 416
440 2250
elution is reversed in C18 reverse phase columns. However, this se-
465 quence of elution is reversed in C18 Monomeric or C18 polymeric re-
Zeaxanthin 424 verse phase columns. Rivera and Canela-Garayoa (2012) reviewed the
449 2348 carotenoid resolution and detection techniques for qualitative and
476
quantitative analysis of carotenoids. The authors compared the perfor-
mance of stationary phases for separation cis- and trans-isomers of ca-
rotenoids, and concluded that both C18 and C30 stationary phases can
Significant diversity of carotenoids (More than 700 types) exists provide good resolution for the separation of geometrical isomers of ca-
in plants, animals, fungi, and micro-organisms, and obtaining and rotenoids with similar polarity.
maintaining pure carotenoid standards is one major problem in quanti- Atmospheric pressure chemical ionization (APCI)-tandem mass
fication. Apart from this, significant qualitative and quantitative compo- spectrometry (MS/MS) in positive ion mode is the most common
sitional variation between plants and between cultivars of the same method to identify and characterize different carotenes and oxygen-
plant, complexity of the food matrices, and susceptibility of carotenoids functionalized carotenoids containing epoxy, hydroxyl, and ketone
to cis and trans isomerization and oxidation during analysis and during groups (Rivera, Christou & Canela-Garayoa, 2014). Mass spectrometry
storage is the major problems in reliable and accurate analysis of carot- is the powerful technique to identify new compounds, even pigments
enoid. A scheme for obtaining standards carotenoids by open column with a very similar structure can be differentiated through their frag-
chromatography from leafy vegetable carotenoids was proposed by mentation pattern. In structural elucidation, mass spectrometry (MS)
Kimura & Rodriguez-Amaya (2002). The strategy described by these au- is generally combined with proton nuclear magnetic resonance (1H
thors is low-cost and provides a constant supply of carotenoid standards NMR) data to obtain the absolute confirmatory results on highly similar
(90–100% purity), including those which cannot be acquired commer- isomers (Qiu, Zhu, Tang, Shi & Gao, 2014). In a recent review, Rivera
cially. Saponification and direct solvent extraction are commonly used et al. (2014) have summarized the positive MS fragmentation data of se-
to extract carotenoids from leafy vegetables. Saponification is generally lected 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
Saponification 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)
Chromatographic separation By RF-HPLC in C30 column
Identification and quantification Confirmation of carotenoids by appropriate standards,
mass spectrometry and absorbance spectrum
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 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
746 R.K. Saini et al. / Food Research International 76 (2015) 735–750
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, com-
plied in this review will be very useful in the analysis of MS data for ca-
rotenoid 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 ex-
traction (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) com-
paratively 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 meth-
ods and industrial applicability. In results, PLE technique showed the
highest selectivity for carotenoids compared to LPSE techniques. How-
ever, 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 α-
carotene and 4 mg of lutein could be isolated from 100.2 mg crude car-
rot 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 carot-
enoid analytical methods from food has also recently been reviewed
by Rodriguez-Amaya (2015). Identification and quantification of carot-
enoids are still difficult in many labs due to unavailability of standards
at normal price. So, advance techniques need to be developed for puri-
fication of carotenoids. In addition to food industries, purified caroten-
oids 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, α-
carotene, β-carotene and lycopene are studied in fruits and vegetable.
So, in the future, investigations on extraction, quantification and biolog-
ical 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 se-
lectivity (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
 R.K. Saini et al. / Food Research International 76 (2015) 735–750
a two-dimensional chromatogram in the form of two or three-
dimensional plot. The electiveness of LC × LC separations has been dem-
onstrated 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 es-
sential 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 mecha-
nism of action are needed to be investigated further. The process of ca-
rotenoid 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, struc-
tural barriers in food (matrix, cell wall integrity, bio-encapsulation), ad-
dition 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. Bio-
logical activity and significance of several new carotenoids, such as
deepoxyneoxanthin, mimulaxanthin, and strigolactones are also need-
ed 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.
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