Strategies for Enhancing Phytonutrient Content in Plant-Based Foods

Abstract

As for long applied in traditional medicine, plant foods can be reliable sources of therapeutic molecules—phytonutrients—that aid in the avoidance or cure of several diseases. With the modern requirements of healthier and more environmentally friendly plant-based diets, research interest in increasing these molecules in common foods is in the spotlight. This chapter focuses on the description and mode of action of phytonutrients, their application and the main food sources for them, both in human and animal nutrition. Also, an overview of methods to increase phytonutrient content in food, from conventional breeding to genetic modification, is given. Finally, a description of the main technologies, both low and high-throughput, currently utilised to put this in practise.

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Phytonutritional Improvement of Crops, First Edition. Edited by Noureddine Benkeblia.
© 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.
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5.1 Introduction
‘Phytonutrients’ or ‘phytochemical’ are primary or secondary metabolites found in
plants and recognised to have nutritional quality attributes and potential health benefits.
From the etymological point of view, ‘phyto’ refers to the Greek word for plant, while
‘chemical’ refers to a substance in a form of matter that has constant chemical composi-
tion and characteristic properties, while ‘nutrient’ refers to a substance that provides
nourishment essential for the maintenance of life and for growth (Drewnowski and
Gomez‐Carneros 2000).
Beside their protective roles in plants from diseases, disorders and other physiologi-
cal stresses (Agrawal 2007, Appel 1993, Close and McArthur 2002, Harborne 1991),
phytonutrients which include many other minor components in foods, particularly
plant‐derived foods, have been know from the ancient times to elicit biologic responses
in human and animal systems. These elicitors have been shown to reduce the risk of one
or more chronic diseases such as cancer, hypertension, diabetes and others (Beecher
1999, Dillard and German 2000, Percival 1997). Hence this new arisen interest in the
so‐called “healthy foods”, that is based on the intake of fruits, vegetables and many other
plant‐based foods.
The use of plant foods to supress human health and nutrition needs, although ancient
in eastern cultures, has only raised attention for the last decades, and is becoming a
trend in Europe and America, especially since the 1990s (Kochian and Garvin 1999).
Moreover, IARC and WHO have recently made a press release classifying red meat and
processed meat as carcinogenic to humans and recommending to limit intake of meat.
Although a large fraction of consumers believe that phytonutrients are found only in
fruits and green leafy vegetables, many other plant‐based and well‐known crops also
contain these compounds. For example, whole grains, nuts, beans or tea also are plant‐
foods rich in phytonutrients. To date, more than 25,000 different phytonutrients have
been identified, and the most important classes are carotenoids, flavonoids, glucosi-
nolates, phytooestrogens, stillbens and sulphur‐containing compounds (Cassidy and
Strategies for Enhancing Phytonutrient Content
in Plant-Based Foods
Carla S. Santos1, Noureddine Benkeblia2 and Marta W. Vasconcelos1
1 CBQF‐Centro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia, da Universidade Católica Portuguesa/Porto,
Rua Arquiteto Lobão Vital, Apartado 2511, EC Asprela, 4202‐401, Porto, Portugal
2 Department of Life Science, and the Biotechnology Centre, Faculty of Science and Technology|, The University of the West
Indies, Mona Campus, Kingston, Jamaica

Phytonutritional Improvement of Crops
204
Kay 2010). Many researchers report that c.a. 40,000 phytonutrients are found in plant
foods, and extensive literature reports that phytonutrients are recognised as “nutraceu-
ticals” (Rechkemmer 2001), having tremendous impact on the health care system and
therefore, having potential in providing medical health benefits (Gupta and Prakash
2014). Nonetheless, the effects of these phytochemicals depends more on their bioavail-
able dose rather than on the total dose ingested.
Because of the increased incidence of cancer and cardiovascular diseases, as well as
diabetes, one of the most pressing challenges for many governments is to promote a
healthy diet. Therefore, plant biotechnology programmes aiming at phytonutritional
improvement can make significant contributions to human health through the develop-
ment of phytonutrient‐rich plant‐based foods (Grusak and DellaPenna 1999, Kochian
and Garvin 1999, Martin 2013, Martin etal. 2011, Zhao 2007).
To achieve these goals, genetics and metabolic engineering of food crops is one the
ways to make crops improve their contents of specific phytonutrients. With the devel-
opment of omics technologies (Benkeblia 2012) for food science, genomic and prot-
eomic technologies have been used to identify these compounds, as well as genes and
proteins involved in their synthesis. Nowadays, a recent trend has put high‐throughput
tools as a valuable technique in the analysis of phytochemicals (Saito 2013). However,
the application of the data obtained with these approaches in an agricultural context
depends on further information on phenomics, which is the study of plant growth,
performance and composition with regards to the environment.
5.2 What are Phytonutrients?
Literally, a phytonutrient is a plant‐derived nutrient, which comprises proteins, lipids,
carbohydrates and essential minerals and vitamins. Besides these compounds, the term
phytonutrient or phytochemical has also been utilised to describe any organic or inor-
ganic compound in plant foods that is beneficial to human health or nutrition (Kochian
etal. 1999). Hence, the phytonutrient class also includes other secondary plant products
that present characteristics that contribute for health improvement. The phytochemi-
cals can be divided in three main groups, phenolic acids, flavonoids and lignans. The
ones with current preponderance in market and public health are polyphenols, terpe-
noids, resveratrol, flavonoids, isoflavonoids, carotenoids, lycopene, limonoids, phytos-
terols, phytooestrogens, glucosinolates, ω‐3 fatty acids, and anthocyanins (Gupta and
Prakash 2014).
The applications of phytonutrients are extensive, and their efficacy has been tested in
different contexts. Generally, these compounds, especially phenolics (Mathew et al.
2015) and vitamin C, but also tocopherols and tocotrienols, as well as flavonoids and
carotenoids (Liang et al. 2014), have been associated with antioxidant properties.
Antioxidant activity can lower the risk of disease and slow biological ageing, by prevent-
ing chronic degenerative disease. For example, it can greatly improve cardiovascular
disease prevention by reducing cholesterol levels (Riccioni et al. 2012, Constans etal.
2015). In fact, consumption of nutrient‐rich plant foods has been associated with the
prevention of diabetes (Nunes etal. 2014) and hypertension (Rodriguez‐Casado 2016).
The plant‐based diet was shown to have a positive impact in the treatment of fibromy-
algia, a chronic fatigue syndrome, in an 8‐week pilot study (Lamb etal. 2011). Moreover,

5 Strategies forEnhancing Phytonutrient Content inPlant-Based Foods 205
quercetin, which is a flavonol compound also found in fruits and vegetables, has proven
to have an effect over atherosclerosis progression (Hung etal. 2015).
Also due to their antioxidant role, high‐intake of phytonutrients is vastly connoted
with cancer prevention. Early studies have suggested that flavonoids and flavones could
decrease cancer incidence, but with no statistical support (Ronco etal. 1999, Birt etal.
2001). Since then, and through the last decade (Birt 2006, Nakamura etal. 2009, Kang
etal. 2011), new studies have proven the beneficial effect of a combined chemopreven-
tion treatment with dietary phytochemicals intake. Nowadays, substantial proof has
been gathered and effective compounds in cancer treatment/prevention have been
identified (Cardeno etal. 2013, Chaithongyot etal. 2015, Li etal. 2015b, Hosseini and
Ghorbani 2015), even against the most aggressive types (Ham etal. 2015). It is thought
that this protective effect of phytonutrients contained in fruits and vegetables is due to
the modulation of the expression of genes that are important in cancer‐related biologi-
cal and genetic pathways (de Kok etal. 2010).
Polyphenols, alkaloids, terpenes, saponins, amines and carbohydrates also possess
antidepressant activity and could be utilised as an alternative to conventional antide-
pressants (Bahramsoltani etal. 2015). Still on neurological diseases, compounds such as
flavonoids (Thapa and Chi 2015) have been shown to prevent and arrest neurodegen-
eration, impacting neurodegenerative diseases, like Alzheimer’s disease (Venkatesan
etal. 2015, Hügel 2015). Plant secondary metabolites, like ethanol and hexane extracts,
have also been identified as antimicrobial substances (for a recent review please see
Borges etal. 2015) and have been tested for their antimicrobial activity against multi‐
drug resistant bacteria, with positive results (Barreto etal. 2015).
Phytochemicals bioavailability is limited, since besides enduring food processing,
their release from food matrices to the organism is dependent on several factors (Bohn
etal. 2015). Their absorption occurs mainly in the microflora of the intestine and must
resist both liquid and solid phases of the digesta (Bohn et al . 2015). Moreover, it is
important to account that phytonutrients can eventually have toxic effects if consumed
in excess or combined with other incompatible supplements (de Kok etal. 2010).
5.3 Which Plant-Based Foods are the Best Known Sources
of Phytonutrients?
A diet composed of at least 400 g (corresponding to five portions) of fruits and vegeta-
bles a day (WHO 2015), which are rich in protein, micronutrients and dietary fibre, and
low in fat, is highly recommended and is part of the traditional diet in most Oriental
(Lee etal. 2013), Mediterranean and also some Nordic countries (Tennant etal. 2014).
However, most countries do not meet the fruit and vegetables intake recommendations
or even if the recommendations are met, the consumption of a limited number of foods
constraints a diverse phytonutrient intake, as happens in the United States (Murphy
etal. 2012). Some compounds, like vitamin A, vitamin C, vitamin E and ß‐carotene and
the food that provide them are widely known and consumed by the general public.
Carrots are the preferred source for vitamin A; oranges, tomatoes and sweet peppers
are the most common sources of vitamin C; for providing vitamin E, cereals, nuts and
seeds are the most consumed; and for ß‐carotene, green leafy, root and fruiting vegeta-
bles (García‐Closas etal. 2004, Sharma etal. 2014).

Phytonutritional Improvement of Crops
206
The Mediterranean diet has been considered has a health‐promoting diet for a long
time (Corella and Ordovás 2014). A recent review pointed certain fruits and vegetables
that can justify this potential effect in chronic disease control: broccoli, for their high
content in glucosinolate; dandelion, of which young leaves, roots and flower extracts
can be consumed, are rich in phenolics like hydroxycinnamic acid derivatives and in
flavonoids, chlorogenic and chicoric acids, luteolin and quercetin glycosides; garlic,
that not only contains sulphur compounds, but also enzymes, amino acids and miner-
als; and cocoa, which is rich in antioxidants, flavonoids and xanthines (Rodriguez‐
Casado 2016).
Other component of the Mediterranean diet that contributes to its high health
favourable effect is olive oil. This product has a highly diverse phenolic fraction, namely,
simple phenols, secoiridoids, lignans and flavones that possess the ability to decrease
LDL cholesterol and cell oxidation, microbial activity, markers of inflammation and of
platelet function, and increase HDL cholesterol, antioxidant capacity and bone health
(Cicerale etal. 2010), all of these contributing for an antiageing (Vazquez‐Martin etal.
2012) and anticarcinogenic (Coccina etal. 2014) effects.
Berries are another food rich in phytonutrients, particularly phenolics and flavonoids
(Wang and Lewers 2007) and carotenoids (Lashmanova et al. 2012), and have been
shown to have a powerful antioxidant effect (Liu etal. 2002, Wang and Lewers 2007)
and anticancer activity (Neto 2007). A recent study focused on the identification and
quantification of the phytochemicals present in Arbutus unedo berries and concluded
that these berries are a source of ω‐3 and ω‐6 fatty acids, phytosterols and tocopherols
(Fonseca etal. 2015). The main chemical components present in berries and their role
in regulating cellular processes have also been recently reviewed (Bishayee etal. 2015,
Mazzoni etal. 2016) and a putative chemopreventive role for the occurrence of cancers
and other chronic pathologies is suggested.
Certain beverages are widely associated with high phytonutrients content. For more
than two decades that an inverse relationship between red wine intake and the develop-
ment of coronary disease has been shown, and this is mostly due to the phenolic com-
pounds synthesised by red grapes, particularly resveratrol (Visioli etal. 2000).
Early reports showed that red wine has the ability to supress an oestrogen synthetase
(Eng et al. 2001) as well as the damage caused by 7,12‐dimethylbenz[a]anthracene
(Leung et al. 2009), both involved in breast cancer development; that the intake at
regular doses of red wine, expected in the Mediterranean diet, provides the sufficient
phytonutrients amounts to reduce cardiovascular disease incidence (Carluccio et al.
2003); and that grapes have not only high levels of phenylpropanoids but also of
melatonin, which is responsible for the regulation of physiological and pathological
conditions (Iriti and Faoro 2006). Despite all of these proven advantageous aspects of
wine light consumption, recent concern has been raised due to the correlation between
ethanol (independently from the type of beverage) and oral cancer (Varoni etal. 2015).
Other beverage widely associated with disease control is tea. Regarding its fermenta-
tion process, there are three tea types: green, black and oolong tea. Green tea isn’t fer-
mented and has high levels of a characteristic group of polyphenols, catechins. On the
contrary, since black and oolong tea are fermented, the natural simple polyphenols are
converted to more complex polyphenols, decreasing catechins content and increasing
caffeine content (Hayat etal. 2015). Despite the positive impact of black tea theaflavins
on health, for example, in the inhibition of tumour cell proliferation (Mujtaba and Dou

5 Strategies forEnhancing Phytonutrient Content inPlant-Based Foods 207
2012) and in the alleviation of cardiovascular disease (Cheang etal. 2015), the catechin
found in green tea—epigallocatechin gallate (EGCG)—is the most studied for its high
antioxidant capacity (Seeram etal. 2006) and cancer therapeutic properties (Bigelow
and Cardelli 2006; Zeng etal. 2014; Fang etal. 2015). Five to six cups per day of green
tea is the recommended dosage to consume sufficient amount of EGCG to act as a
therapeutic agent (Wolfram 2007). Green tea effectiveness was also shown in Parkinson’s
disease control (Dutta and Mohanakumar 2015), in bone mineral density increase in
elderly women (Devine etal. 2007), in inhibition of atherosclerosis associated inflam-
matory mediators (Cai etal. 2012), and in obesity control and low glycemic parameters
(Vernarelli and Lambert 2013, Lee etal. 2015).
A recent trend showcases edible flowers as essential components in gastronomy: as
ingredients in a recipe, for seasoning or garnish (Xiong etal. 2014). One notable exam-
ple is Moringa oleifera, which is fairly resistant to extreme weather and is native of India,
Pakistan, Asia Minor, Africa and Arabia (Anwar etal. 2007). The flowers from this tree
have high medicinal value and are commonly utilised as a main dish, as part of fruit
juices or as medicinal tea for cold treatment or weight loss (Anwar etal. 2007). The
antioxidant value of different plants was studied and Rosa spp. presented high levels of
polyphenolic content, as well as high antioxidant effect (Youwei etal. 2008). Further
studies with 10 different flowers show that Paeonia suffruticosa also have high antioxi-
dant capacity and phenolic content (Xiong etal. 2014).
On another context, food animals have been for decades treated with growth promot-
ing antibiotics, in order to increase production and as therapeutics or prophylactics.
However, a correlation between the administered antibiotics and foodborne human
pathogens antibiotic resistance has been found, and this is aggravated by the fact that,
as the manure utilised to amend soils is contaminated with antimicrobial resistance, the
environment is also a reservoir for this problem and is part of the transmission cycle
(Yang etal. 2015, Woolhouse etal. 2015). As such, the utilisation of phytonutrients as
animal feed additives is a viable alternative to antibiotics use, since they also have
growth promoting action and antimicrobial effect (Oh etal. 2013). Several plant‐derived
compounds have inclusively been patented as reviewed in Thormar (2012) and an
invitro study showed that specifically saponins, tannins and essential oils can reduce
methane potential by modulating the process of fermentation in the rumen (Cieslak
etal. 2013).
5.4 How Can We Enhance Phytonutrients?
5.4.1 Conventional Breeding
Plant breeding is one of the most used approaches to develop new varieties with specific
agronomic traits and improved nutritional qualities (Farnham etal. 1999, Unnevehr
etal. 2007). However, classical breeding approaches have many limitations because the
crossing can only be done between closely related specie or genus, and therefore it uses
available genetic diversity and existing traits to obtain new varieties. It also uses wild
relative species (WRS) with specific traits and transfers them to the new target varie-
ties although some WRS are not compatible for crossing (Farnham etal. 1999, Lemaux
2008). However, plant breeding and engineering researchers admit that WRS could be

Phytonutritional Improvement of Crops
208
used for potential breeding by introducing traits that have been lost or underutilised
during the domestication and subsequent breeding of these wild species (McCouch
etal. 2013). Despite these limitations, different successful attempts have been reported
to improve crops with phytonutrients. Lago etal. (2014) reported the development of
a new variety of maize rich in anthocyanins and high antioxidant properties, and Juhász
etal. (2014) obtained by preselecting potato individuals prior to breeding high vitamin
C, B5, and B6 contents bred tubers. The flesh of potatoes also contain carotenoids
mainly xanthophylls represented by lutein and zeaxanthin (Andre etal. 2007, Brown
2005, Brown etal. 1993). Nevertheless, screening of advanced breeding lines and varie-
ties of potatoes growing in different locations has shown that different cultivars contain
higher level of phenolics (Navarre etal. 2011), carotenoids (Brown etal. 2006), antho-
cyanins (Brown etal. 2003), as well as a good level of iron (Brown 2008). Other crops
have been improved by breeding to increase their nutritional values such as wheat with
higher proteins and fibres content (Baylan et al. 2013). Indeed, numerous attempts
have been made to enhance the phytochemical levels in crops using traditional breed-
ing strategies (Bouis et al. 2002, Mayer et al. 2006, Nestel etal. 2006). Nonetheless,
these strategies using multi‐generations crossing and back crossing is a time consum-
ing process, requires high trait genetic variations, and heritability as well (McGhie
etal. 2008).
5.4.2 Molecular Breeding
The technique of molecular breeding–called marker‐assisted breeding‐ is considered
one of the most powerful tools in modern biotechnology. Indeed, this technique is
based on the use of polymorphic single genes to facilitate the process of plant breeding,
and was first proposed by Sax in 1923. With the development of molecular biology, This
technique has found wide listeners amongst farmers due to the fact that it relies on
biological breeding processes rather than plant engineering by inserting external gene
in order to change the DNA of plants organism (Johnson 2004, Thompson etal. 2009).
Therefore, by using this technique the molecular marker can be detected in the new
seedlings, and consequently the presence or absence of the desired trait can be deter-
mined in the young plant and not delayed to the mature stage, and then reducing the
number of generation and saving years of time on crossing (Collard and Mackill 2008,
Lande and Thompson 1990).
Different crops have been or are being manipulated to have their phytonutrient con-
tents enhanced using molecular breeding as shown in Table 5.1.
5.4.3 Metabolic Engineering andGenetic Modification
Conventional agricultural approaches have show limitation in enhancing the nutritional
traits of food crops, however, with the development of molecular biology and related
techniques are making the exploitation to engineer crops with enhanced phytonutri-
ents progressing more rapidly (Hirschi 2009).
At the turn of the twenty‐first century, genetic engineering has known a rapid devel-
opment resulting from the progress made in molecular biology and the better under-
standing of the DNA and its functions in living organisms. Genetic engineering aims to
makeup the genome of a living organism in a laboratory using ‘recombinant DNA tech-
nology’ by inserting, altering, removing or switching off specific piece(s) of DNA

5 Strategies forEnhancing Phytonutrient Content inPlant-Based Foods 209
Table 5.1 Examples ofsome breeding programmes performed or being performed toenhance
thephytonutrient contents andmajor bioactive groups ofcompounds.
Crop Compounds with bioactive properties References
Artichoke (Cynara cardunculus
var.scolymus L.)
Phenolics, in particular chlorogenic
acid
Pandino etal. (2012)
Asparagus (Asparagus
officinalisL.)
Phenolics, in particular phenolic acids,
flavonoids, flavanols and ascorbic acid
Lee etal. (2014)
Cabbage and cauliflower
(Brassicaoleracea L.)
Glucosinolates, cartoenoids and
anthocyanins
Padilla etal. (2007)
Carrot (Daucus carota L.) Carotenoids and phenolics, in
particular cholorogenic acid and
anthocyanins
Baranski etal.
(2012)
Celery (Apium graveolens L.) Phenolics Yao etal. (2010)
Cucumber (Cucumis sativus L.) Carotenoids, in particular β‐carotene Navazio and Simon
(2001)
Eggplant (Solanum melongena L.) Phenolics, in particular chlorogenic
acid and antocyanins
Prohens etal. (2007)
Leek (Allium porrum L.) Phenolics, lutein, β‐carotene, ascorbic
acid and vitamin E
Bernaert etal.
(2012)
Lettuce (Lactuca sativa L.) Carotenoids, in particular β‐carotene
and lutein, and anthocyanins
Mou (2005)
Melon (Cucumis melo L.) Carotenoids Harel‐Beja etal.
(2010)
Onion (Allium cepa L.) Phenolics, in particular flavonoids,
flavonols and anthocyanins, and
ascorbic acid
Yang etal. (2012)
Pepper (Capsicum annuum L.) Carotenoids, phenolics, and ascorbic
acid
Rodríguez‐Burruezo
etal. (2011)
Pumpkin, squash and zucchini
(Cucurbita spp.)
Carotenoids, tocopherol, ascorbic acid de Carvalho etal.
(2012)
Spinach (Spinacia oleracea L.) Lutein and phenolics Pandjaitan etal.
(2005)
Table beet (Beta vulgaris subsp.
vulgaris L.)
Betalains Gaertner etal.
(2005)
Tomato (Solanum lycopersicum L.) Carotenoids, in particular lycopene,
phenolics, anthocyanins and ascorbic
acid
Adalid etal. (2010)
Jones etal. (2003)
Watermelon (Citrullus lanatus
(Thunb.) Matsum. & Nakai)
Carotenoids, in particular lycopene,
and ascorbic acid
Yoo etal. (2012)
Mustard (Brassica juncea L.) Glucosinolates, total tocopherols and
oil content
Gupta etal. (2015)
Papaya (Carica papaya L.) Carotenoids and ascorbic acid Wall and Tripathi
(2013)
(Continued)

Phytonutritional Improvement of Crops
210
containing the gene(s) of interest. As results, crops developed through genetic engi-
neering are commonly known as transgenic or genetically modified (GM) crops (Datta
2013, Desmond and Nicholl 1994). GE allows transferring specific and targeted genes
from close or distant related plant species to the targeted species, and therefore obtain-
ing a ‘new’ plant with desired agronomic traits. The two most interesting benefits of GE
are (i) the possibility to obtain a plant with specific agronomic traits difficult to obtain
in the case the trait is not present in the germplasm of the crop, and (ii) the long time
needed to introduce that trait in the targeted crop using conventional breeding
(Desmond and Nicholl 1994).
During the last decade, many commercial GE or GM crops have been made available
for farmers and delivered benefits in crop production and, moreover, a number of other
GM plants are still being developed to enhance their nutritional values including phy-
tonutrients. The many examples of phytonutrients‐enhanced crops are rice with higher
level of beta‐carotene, high‐lysine corn, maize with improved feed value and tomatoes
with high levels of flavonols amongst others. Table 5.2 below shows the different GM
and transgenic crops that have been developed or under development for higher phyto-
nutrient content.
Undoubtedly, gene transfer systems have led to numerous developments and offered
clear insights into the regulation of gene expression and protein function in crops, and
living organisms as well. With the tremendous efforts made in the identification and
isolation of crop genes, a dramatic expansion in our understanding of gene structure
and function at the molecular level have been achieved, and new crops are released
every year.
5.5 Phenotyping for Phytonutrients at Different Levels
5.5.1 Low Throughput Techniques
Certain phytonutrients can be analysed using conventional techniques and the associ-
ated methods are well described (Table 5.3).
Table 5.1 (Continued)
Crop Compounds with bioactive properties References
Maize (Zea mays L.) Anthocyanins Lago etal. (2014)
Brassica spp. Glucoraphanin and other aliphatic
glucosinolates
Stansell etal. (2015)
Sweet corn (Zea mays L.) and
broccoli (Brassica oleracea L. ssp.
italica)
Carotenoids and tocopherols. Ibrahim and Juvik
(2009)
Strawberry (Fragaria × ananassa) Ellagic acid and ascorbic acid Atkinson etal.
(2006)
Pepper (Capsicum annuum L.). Ascorbic acid Geleta and
Labuschagne (2006)
Partly adapted from Plazas etal. (2014).

5 Strategies forEnhancing Phytonutrient Content inPlant-Based Foods 211
Table 5.2 Examples ofGM crops developed or inresearch withenhanced phytonutritional traits.
Crop Compounds with bioactive properties References
Tomato (Solanum lycopersicum) Higher levels of polyamines,
folate, phytoene, β‐carotene, lycopene,
provitaminA
Davuluri etal. (2005)
DellaPenna (2007)
Díaz de la Garza
etal. (2004)
Enfissi etal. (2005)
Fraser etal. (2001)
Mehta etal. (2002)
Neelam etal. (2008)
Rosati etal. (2000)
Sestari etal. (2014)
Apple (Malus pumila) Stilbenes Szankowski etal.
(2003)
Kiwi (Actinidia chinensis) Resveratrol Kobayashi etal.
(2000)
Maize (Zea mays) Carotenoids, vitamin E, vitamin C,
flavonoidsandother phenolic
compounds
Cahoon etal. (2003)
Chen etal. (2003)
Matthews etal.
(2003)
Muzhingi etal.
(2016)
Rocheford etal.
(2002)
Yu etal. (2000)
Soybean (Glycine max) Flavonoids Yu etal. (2003)
Canola (Brassica napus) Vitamin E, β‐carotene Shintani and
DellaPenna (1998)
Shewmaker etal.
(1999)
Potato (Solanum tuberosum)β‐carotene, lutein Ducreux etal. (2005)
Rice (Oryza sativa)β‐carotene, flavonoids and resveratrol Ye etal. (2000)
Shin etal. (2006)
Stark‐Lorenzen etal.
(1997)
Strawberry
(Fragaria × ananassa)
Vitamin C Agius etal. (2003)
Orange and other citrus
(Citrus × sinensis)
β‐carotene, anthocyanins Pons etal. (2014)
Sweetpotato (Ipomoea batatas) Anthocyanins, proanthocyanidin and
total phenolics
Park etal. (2015)
Dutt etal. (2016)
Spinach (Spinacia oleracea) Phytoecdysteroids, and
polyhydroxylated triterpenoids
Cheng etal. (2010)
Lupin (Lupinus angustifolius) Methionine Molvig etal. (1997)

Phytonutritional Improvement of Crops
212
For example, the Folin‐Ciocalteu assay (Folin and Ciocalteau 1927) is widely employed
to determine total phenolic content in different types of samples, which is based in the
reduction of the phosphor‐molybdate heteropoly acids Mo(VI) centre in the heteropoly
complex to Mo(V). Through this method, root, leaf, stem or fruit fraction extracts can
be analysed using a standard curve to quantify the products and measuring samples at
765 nm with an UV‐visible spectrophotometer (Ramirez‐Sanchez etal. 2010, Jing etal.
2014, Chen etal. 2015).
Total flavonoids content can also be analysed using a methanolic extract of leaves or
roots of different plants. The aluminium chloride colourimetric method is often used
using a standard curve and reading the absorbance of the samples at 415 nm (Chang
et al. 2002). This protocol allows the quantification of flavonoids in diverse species,
such as Blumea eriantha (Gore and Desai 2014), Manihot esculenta (Omar etal. 2012)
or in medicinal plants of different genus, like Hibiscus, Premna, Mallotus, Trichosanthus,
Maharanga, Astilbe and Syzygium (Subedi etal. 2014).
For evaluating the antioxidant activity, the 1,1‐Diphenyl‐2‐picryl‐hydrazyl (DPPH)
method is conventionally used (Rivero‐Pérez etal. 2007). This method is also based in
the spectrophotometric measurement of samples (at 515 nm) and a change in the col-
our of the solution occurs when the DPPH free radical is reduced by hydrogen donation
(Omar etal. 2012). It has been widely applied and is an useful test to evaluate the anti-
oxidant capability of flowers, fruits and vegetables (Youwei etal. 2008, Omar etal. 2012,
Gore and Desai 2014, Subedi etal. 2014).
Anthocyanins and carotenoids are photosynthetic pigments, considered phytonutri-
ents, for their antioxidant action. Anthocyanins can be analysed with a spectrophoto-
metric differential pH method and applying a molar extinction coefficient of
pelargonidin‐3‐glucoside (Giusti and Wrolstad 2001, Brown etal. 2003) or of cyani-
din‐3‐glucoside chloride (Anisimoviené et al. 2013). Carotenoids can be quantified
accordingly to the method proposed by Sims and Gamon (2002), using spectrophoto-
metric readings and accounting the concentration of chlorophyll a, chlorophyll b and
anthocyanins (Pereira etal. 2014).
Determining foods’ mineral composition is essential in order to understand its nutri-
tional value. Atomic absorption spectroscopy was shown to be effective at detecting
several metals at parts per million level of concentration (Robinson 1960) and it is still
applied in mineral profiling of, for example, teas (Dambiec etal. 2013), medicinal plants
(Küçükbay and Kuyumcu 2014) or coffee (Oliveira etal. 2015).
Table 5.3 Conventional methods forphytonutrient phenotyping.
Analyte Method Reference
Phenolics Folin‐Ciocalteau Folin and Ciocalteau (1927)
Flavonoids Aluminium chloride Chang etal. (2002)
Antioxidant activity DPPH Rivero‐Pérez etal. (2007)
Anthocyanins pH differential Giusti and Wrolstad (2001)
Carotenoids Tris‐methanol extraction Sims and Gamon (2002)
Minerals Atomic absorption spectrometry Robinson (1960)

5 Strategies forEnhancing Phytonutrient Content inPlant-Based Foods 213
Although these techniques can be useful, their common feature is that the evaluation
of each phytonutrient can be performed at the tissue level, besides being time consum-
ing and having low representativeness. Hence, the increased demand for higher plant
yields and quality calls for the development of modern techniques that allow a more
efficient phenotyping and consequent use of food phytonutrients.
5.5.2 High‐Throughput Techniques
The development of plant phenotyping techniques has been associated with the meas-
uring of plant growth, architecture and composition (Fiorani and Schurr 2013), and
high‐throughput techniques have as main advantage the possibility to rapidly and
simultaneously determine a large profile of analytes, which can be the phytonutrients
themselves or the genes and proteins responsible for their production.
‘Omic’ technologies allow the characterisation of genetic diversity in plant systems
and have an important role in phytonutrient phenotyping. High‐throughput tools
accelerate the discovery of new genes and the functional characterisation of enzymes,
thus elucidating biochemical pathways or mineral trafficking players inside the plant
(Grusak 2002) (Figure 5.1).
Next‐generation sequencing (NGS) helps to establish the link between genotype and
phenotype regarding complex traits. Using NGS, the assembly of new data on genes and
their function/regulation, can reveal new molecular and useful phenotypic markers, for
example, in breeding programmes (Pawełkowicz etal. 2016). Genetic profiling, using
PHYTONUTRIENT prole
GENOTYPING
Genomics Resequencing
Whole Exome Sequencing
RNA Sequencing (RNA Seq)
Nuclear Magnetic Resonance (NMR)
Mass spectrometry (MS) coupled with:
Liquid Chromatography (LC)
Gas Chromatography (GC)
Matrix-assisted laser desorption
ionisation (MALDI)
Exomics
Transcriptomics
Metabolomics
Proteomics
Lipidomics
PHENOTYPING
HIGH
THROUGHPUT
Figure 5.1 High‐throughput techniques relevant in phytonutrient phenotyping. (Seecolor plate
section for the color representation of thisfigure.)

Phytonutritional Improvement of Crops
214
single nucleotide polymorphisms (SNPs), started as a good way to understand absorp-
tion, circulation and the metabolism of phytochemicals, but its high sequencing cost
was a big disadvantage of this technology (de Kok etal. 2012).
Plant genomics gathers high‐throughput tools, like genotype‐by‐sequencing (GBS),
which is the next step in SNP discovery and genotyping, and uses restriction enzymes
to reduce genome complexity, together with multiplex NGS and enables the sequencing
of the whole genome (Elshire etal. 2011). Although GBS does not require a previous
genomic knowledge of the studied species, the obtained reads must be aligned to a ref-
erence genome using alignment tools and, since a large amount of data is produced,
adequate tools for data analysis must be applied. For example, association mapping like
genome‐wide association studies (GWAS) are statistical techniques for measuring the
strength between a marker locus and a natural variation in a target phenotype (Brachi
etal. 2011) and have been used to identify markers underlying natural variations in rice
nutritional quality traits, like phenolic and flavonoid content and antioxidant capacity
(Shao etal. 2011, Yang etal. 2014). Besides being utilised for SNPs discovery, GBS has
been also applied for linkage map construction and QTL identification for agronomi-
cally important traits (Bekele et al. 2013, Verma et al. 2015, Lee et al. 2016), where
GWAS can also be applied.
When compared to whole‐genome sequencing, the study of plant exomics through
exome sequencing allows the identification of protein‐coding genes in specific regions
of the genome, which reduces the time of analysis and the associated costs (Teer and
Mullikin 2010). Although these technologies have been mostly used in biomedical con-
text (Seaby et al. 2015), developing molecular markers to study the allelic variation
behind certain phenotypic traits through whole‐exome sequencing contributes to the
identification of thousands of exome SNPs and holds potential for practical crop
improvement strategies (Singh etal. 2012, Hashmi etal. 2015).
Although SNP discovery is simple and effective in diploid species, the genomic com-
plexity of polyploid plants, that have multiple homologous gene copies, hampers SNP
identification (Chopra et al. 2015). In this scenario, plant transcriptomics can give
information on genetic diversity and analysis. High‐throughput RNA sequencing tech-
nology (RNA‐seq) developed by Illumina has enabled the understanding of biological
pathways, through their transcriptional information given by millions of short sequence
reads. It gathers numerous advantages since RNA‐seq does not depend on genomic
sequence description of the target species, enables gene expression quantification (even
of low‐abundance transcripts), permits the simultaneous identification of different
types of transcripts (isoforms, promoters, transcription start sites and alternative splic-
ing sites), and its output is in the form of base pair resolution (Mata‐Pérez etal. 2015).
RNA‐seq can be used in phytonutrient phenotyping, at the tissue level, since expression
results can provide information on genes relevant in phytonutrient and pathway regula-
tion (Santos etal. 2013, Li and Lan 2015); on transcription factors related to secondary
metabolism pathways, namely the regulation of the biosynthesis of carotenoids in
potato (Li etal. 2015a), and ascorbic acid, carotenoids and flavonoids in tomato (Ye
etal. 2015); and on the identification of transcripts related to pathways involved in the
synthesis of anticancer compounds (Annadurai etal. 2013).
However, understanding the regulation of certain compounds in plants cannot rely
solely on one type of analysis and, nowadays, a combined approach is being adopted.
For example, combining gene expression analysis with metabolite analysis facilitates the

5 Strategies forEnhancing Phytonutrient Content inPlant-Based Foods 215
interpretation between key regulators and compound accumulation profile results
(Wen etal. 2015). As previously mentioned, plant metabolites comprise amino acids,
lipids and carbohydrates (primary metabolites) and polyphenols, alkaloids, terpenes,
polyketides and hormones (secondary metabolites). Hence, plant metabolomics can not
only help to establish the function of transcription factors suspected to control plant
metabolic pathways, but also to identify and quantify plant phytonutrients, contribut-
ing to the characterisation of the potential health benefits of a plant. This has been
achieved using two main techniques, namely, nuclear magnetic resonance (NMR) spec-
troscopy and mass spectrometry (MS) coupled with gas chromatography (GS/MS) or
liquid chromatography (LC/MS) (Guo etal. 2011).
When utilising NMR, the analysis is performed at the plant tissue level and the sam-
ple doesn’t need to be destroyed and can be recovered, resulting in low effort for sample
preparation; also, it is possible to quantify without using internal standard (Wishard
2008). This technique is utilised to analyse the pharmacological properties of medicinal
plants due to the fact that it enables the identification of both primary and secondary
plant metabolites, as well as new metabolites (Holmes etal. 2006). This technique was
applied to study of the primary and secondary metabolites of leaves and seeds of the
papaya plant (Gogna etal. 2015); to identify markers for nutrient deficiency (Lima etal.
2014) and to assess seed metabolomic diversity (Harrigan etal. 2015) in soybean; or to
analyse the unknown phytochemical composition of different plants, such as a highly
consumed Egypt‐native desert palm fruit, Hyphaene thebaica (Farag and Paré 2013)
and the guggul plant, which has proven anti‐inflammatory, antirheumatic, hyppocho-
lesterolemic properties (Bhatia etal. 2015).
The technique most utilised to detect volatile organics is GC/MS. With this type of
technology, in a single sample extract is possible to identify hundreds of metabolites.
Also, by adding time‐of‐flight‐MS to GC (GC‐TOF‐MS) is possible to reach faster scan
times (resolving co‐eluting peaks) and higher sample throughput (Fernie and Schauer
2008). In a recent work, GC‐MS analyses were performed to understand how the differ-
ences in metabolite composition in response to climate factors might influence tea
quality and flavour and detected several chemical families, such as hydrocarbons; oxy-
genated monoterpenes, diterpenes, sesquiterpenes and heterocycles; monoterpene and
sesquiterpene hydrocarbons; aliphatic alcohols, aldehydes, ketones and esters; acids;
and nitrogen‐ and sulphur‐containing compounds (Kowaksick et al. 2014). On the
other hand, when analysing secondary metabolites in which plants are rich, LC/MS
seems to be the most suitable method (Guo etal. 2011), although it does not resolve the
problem for co‐eluting entities (Fernie and Schauer 2008). High‐performance liquid
chromatography (HPLC) is frequently utilised in phytonutrient phenotyping, for exam-
ple, the potential of sea buckthorn as a source of nutrients was evaluated and amongst
the most prevalent phenolic compounds and flavonoids were gallic, caffeic, p‐coumaric,
and ferulic acids and myricetin, kaempferol, naringin, quercetin and isorhamnetin
(Fatima etal. 2015); and also, the variation in secondary metabolites between different
developing stages of pear fruits was studied using this technique, and these data can aid
in the development of phytonutrient enhancement strategies in this crop (Oikawa etal.
2015). Furthermore, when compared to HPLC, ultra‐performance liquid chromatogra-
phy (UPLC) provides better peak separation and higher reproducibility of retention
time (Guo etal. 2011) and, when coupled with Q‐TOF, its sensitivity increases (Farag
and Paré 2013). This technique has been applied in metabolite profiling of Hyphaene

Phytonutritional Improvement of Crops
216
thebaica (Farag and Paré 2013) and Ligustrum lucidum Ait (Guo etal. 2011) fruits, and
it is also being associated with commercial metabolite software packages to automati-
cally profile flavonoids, allowing an automatic determination of the identity of the com-
pounds (Gu etal. 2015).
Mass spectrometry methods also allow analysing a specific part of the metabolome
—the proteins. Plant proteomics can be defined as the high‐throughput study of these
molecules. Proteomic profiling is an advantageous tool in the improvement of crop
nutritional value as, with it, one can investigate the genotypic variability of two closely
related cultivars with regard to: (i) the biochemical composition (Shekhar etal. 2015,
Gupta etal. 2015); (ii) quantifying protein abundances and correlating this factor with
the expression of certain phenotypes (Morton etal. 2016); (iii) or even performing a
targeted analysis using the mass spectrometry technique Selected Reaction Monitoring
(SRM), that enables detection and quantification of preselected peptides (Chawade
etal. 2016).
Lipids are a subset of the metabolome and fewer studies on plant lipidomics are avail-
able (Han and Gross 2003; German etal. 2007). Nowadays, lipidomic studies can be
used in the production of food with optimised composition, through a detailed charac-
terisation of lipids and the gathering of quantitative information on lipid class, head-
group and acyl group combination (González‐Thuillier etal. 2015). For example, this
technique has been applied in the establishment of the lipid profile of functional foods,
like edible macroalgae (Van Ginneken etal. 2011) and olive oils (Alves etal. 2016).
Despite of the advantages that each one of these techniques comprises, research stud-
ies are currently combining them, in order to surpass their individual constraints. For
example, NMR and HPLC‐MS methods were combined in order to identify a larger
number of metabolites in Allium species (Soininen etal. 2014). Both gas‐ and liquid‐
chromatography can be simultaneously applied, as recently performed in rice seeds
where their antioxidant properties were compared to the metabolites content in differ-
ent rice cultivars (Kim etal. 2014) and in red grape berries, to study the influence of
climate changes in metabolite profile (Ayenew etal. 2015). Furthermore, integrating
metabolomics results with transcriptomic and proteomic data gives information about
the function of a certain gene in a given metabolic pathway and also about the tran-
scriptional control of the various enzymatic steps of that pathway (Tohge etal. 2015).
This is being frequently applied, for example, in the study of the carotenoid pathway in
maize kernels engineered for higher carotenoid biosynthesis (Decourcelle etal. 2015);
to understand the transcriptional regulation in phenylpropanoid pathway or in antho-
cyanin metabolism in red grape berries (Ayenew etal. 2015); to correlate the expression
of genes involved in flavonoid biosynthesis with the accumulation of phenolics in sea
buckthorn developing berries (Fatima et al. 2015); and also to identify differentially
expressed proteins and metabolites between two soybean cultivars with contrasting
seed coat colour (Gupta etal. 2015).
5.6 The Future Ahead/Concluding Remarks
Plant phytonutrients are receiving increasing attention by scientists and the general
public alike due to their perception as important anti‐disease agents. As phenotyping
is an emerging field and is being applied mostly in the evaluation of growth, yield and

5 Strategies forEnhancing Phytonutrient Content inPlant-Based Foods 217
plant disease resistance, it is important to explore the tools presented in this chapter
in the study of other less explored features, namely, nutrient use efficiency and phyto-
nutrient characterisation, in order to produce high‐quality food with increased phy-
tonutrient value. The technological development that is being leveraged by the
increased worldwide nutritional need could not be enough facing the predicted chal-
lenges ahead: population increase, drastic climate changes and lack of available arable
land (Carvalho and Vasconcelos 2013).
Supplementation through diet is well accepted, and the use of nutraceuticals (or food‐
derived products with positive effect on health) is increasing. However, it is also largely
associated with lack of information, since these health benefits are not yet fully under-
stood from a scientific point of view and their regulation, regarding legislation and
distribution, is also ambiguous (Ahmad etal. 2011). Combining efforts in order to bet-
ter understand the mechanisms of action of such products and use breeding and new
‘omic techniques to increase food phytonutrient content might hold the answer for the
future in agriculture.
Acknowledgements
This work was supported by National Funds from FCT through projects UID/
Multi/50016/2013 and PTDC/AGRPRO/3972/2014, and PhD scholarship SFRH/
BD/78353/2011.
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