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Chemical composition and antioxidant capacity of lettuce: Comparative study

of regular-sized (Romaine) and baby-sized (Little Gem and Mini Romaine)
types

Article  in  Journal of Food Composition and Analysis · January 2013

DOI: 10.1016/j.jfca.2013.10.001

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 Journal of Food Composition and Analysis 33 (2014) 39–48

Contents lists available at ScienceDirect

Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Research Article

Chemical composition and antioxidant capacity of lettuce:

Comparative study of regular-sized (Romaine) and baby-sized
(Little Gem and Mini Romaine) types
Alicia López a, Garcı́a-Alonso Javier b, Jose Fenoll a, Pilar Hellı́n a, Pilar Flores a,*
a
Dpto. Calidad y Garantı´a Alimentaria, IMIDA, Murcia, Spain
b
Dpto. Nutrición y Bromatologı´a, Facultad de Veterinaria, Universidad de Murcia, Spain

A R T I C L E I N F O A B S T R A C T

Article history: The aim of this study was to assess the differences in sugars, organic acids and main antioxidant
Received 30 July 2012 compounds, as well as nitrate concentration, between different lettuce cultivars belonging to three main
Received in revised form 24 September 2013 types: one regular-sized type (Romaine) and two baby-sized types (Little Gem and Mini Romaine),
Accepted 28 October 2013
usually consumed as whole-head and fresh-cut lettuces. Overall, in the studied cultivars, chlorogenic
acid and caffeic derivates were the major compounds among the free and bound phenolics, respectively.
Keywords: As regards folates, only 5-methyl tetrahydrofolate (5-MTHF) was detected in the monoglutamic form,
Food analysis
whereas the hydrolysis of polyglutamatic forms released further 5-MTHF and tetrahydrofolate (THF).
Food composition
HPLC
The major carotenoid found was b-carotene followed by lutein, lactucaxanthin, violaxanthin and
Quality neoxanthin. Romaine type showed the highest content of total sugars, phenolic compounds, vitamin C
Canonical discriminant analysis and folates. Mini Romaine showed the highest content of organic acids, carotenoids and chlorophylls.
Fresh-cut Finally, Little Gem presented the highest nitrate content, which can be considered a negative
Ready-to-eat characteristic of this lettuce type. The differences found in colour and metabolite and nitrate
Bioactive concentrations could be attributed to the lettuce head structure and size that determine the penetration
Antioxidants of the sunlight and therefore the synthesis of light-dependent metabolites and the nitrogen assimilation.
ß 2013 Elsevier Inc. All rights reserved.

1. Introduction antioxidants (Joshi et al., 2001; Rezk et al., 2003) since they show
activity values comparable to those of vitamin C and E
Lettuce (Lactuca sativa L.) is one of the most popular vegetables (Gliszczyńska-Świgło, 2007); finally, dietary chlorophyll and its
for human consumption, especially in salad, and is considered a derivatives are known to have antioxidant capacity through their
good source of health-promoting compounds such as phenolics, ability to reduce free radicals (Ferruzzi et al., 2002).
vitamin C, folates, carotenoids and chlorophylls (Nicolle et al., Lettuce is marked in two ways, as whole-heads and as fresh-cut
2004). The protective effect of these compounds is largely due to lettuce. Indeed, in the fresh-cut industry, fresh-cut lettuce is one of
their antioxidant effects. Phenolics have been suggested to be the the most important products. However, the physical damage or
main compounds responsible for the antioxidant capacity of wounding caused by preparation increases the rate of biochemical
plants, based on their ability to donate hydrogen atoms to free reactions responsible for changes in visual quality (colour, texture
radicals. In addition, they possess ideal structural properties for and browning) and phytochemicals (vitamin C content and
free radical scavenging properties (Sulaiman et al., 2011); in phenolic compounds) (Saltveit, 2003).
addition to the pro-vitamin A role of b-carotene, a-carotene and b- According to Martı́nez-Sánchez et al. (2012), baby leaf lettuce
cryptoxanthin, most of the carotenoids that we eat possess has several advantages as fresh-cut material compared with
antioxidant capacity (Paiva and Russell, 1999); vitamin C is regular-sized lettuces. These benefits were partly attributed to the
involved in several biochemical mechanisms and it also has a minimal oxidation and lower bruising incurred during processing,
recognized antioxidant effect by reducing oxidative free radicals since baby leaves need little or no further preparation because the
both in vivo and in vitro (Duarte and Lunec, 2005); several authors entire leaf is consumed. However, baby leaves presented certain
have suggested that folates (water-soluble vitamin B) may act as disadvantages with regard to the shelf-life since they are immature
leaves with a softer texture and higher respiration rates than those
of regular-sized lettuces. Apart from these characteristics that
* Corresponding author. Tel.: +34 968 366804; fax: +34 968 366792. make different lettuce types more suitable for the fresh-cut or
E-mail address: mpilar.fl[email protected] (P. Flores). whole-head market, depending on their shelf-life, differences in

0889-1575/$ – see front matter ß 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jfca.2013.10.001
40 A. López et al. / Journal of Food Composition and Analysis 33 (2014) 39–48

the phenolic, vitamin C and carotenoid contents between baby and Aliquots of 5 g of powered sample were homogenized with 5 mL of
regular lettuces in have been reported (de Azevedo-Meleiro and phosphate buffer (pH = 7.5) in a Polytron (PT-MR 3100, Littau-
Rodriguez-Amaya, 2005; Martı́nez-Sánchez et al., 2012). Luzern, Switzerland). Ethyl acetate (10 mL) was added and the
The variability in the composition and quantity of phytochem- mixture was homogenized again. The aqueous fraction was used
icals in lettuce indicate the importance of choosing certain types or for analysing soluble sugars, organic acids and antioxidant capacity
cultivars. However, little information is available about the of the water-soluble fraction (W-ABTS and W-FRAP), while the
concentration of secondary metabolites in different lettuce ethyl acetate fraction was used for analysis of the antioxidant
cultivars and further studies are needed to improve the nutrient capacity of the organic fraction (EA-ABTS and EA-FRAP). Soluble
and antioxidant intake in the diet. In addition to these health- sugars were analysed in the aqueous phase using a Hewlett-
promoting compounds, other components with the potential to Packard model 1100 HPLC system liquid chromatograph (Wald-
adversely affect health should be taken into account when bronn, Germany) equipped with a refraction index detector. The
comparing cultivars. Thus, leafy green vegetables constitute the separation was performed in a 300  7.8 mm i.d., CARBOSep CHO-
major dietary intake source of nitrate. A low nitrate content in 682 LEAD column (Transgenomic, Omaha, NE, USA) with ultra-
vegetables is very important for human health, since nitrate can be pure water as mobile phase at a flow rate of 0.4 mL/min. Standard
reduced to nitrite and then combined with secondary amines, solutions of sucrose, glucose and fructose (purity >99.5%) from
increasing the risk of gastrointestinal cancer (Hord et al., 2009). Sigma (Steinheim, Germany) were injected at concentrations of 1–
The aim of this study was to compare lettuce cultivars 10 g L1 to obtain the linearity of the detector response and the
belonging to regular-sized (Romaine) and baby-sized (Little detection limits of the studied sugars. Organic acids were analysed
Gem and Mini Romaine) types, usually marketed as whole lettuce according to Flores et al. (2012) using a liquid chromatograph
heads or fresh-cut processing. Morphologically, these types differ (Agilent Series 1200, Santa Clara, CA, USA) equipped with a
in the head structure and size. Romaine type presents long leaves Prontosil C18 analytical column of 250 mm  3 mm and 3 mm
20–26 cm long, entire margins, and very broad midrib and open particle size (Bischoff, Leonberg, Germany). The injected sample
head. Mini Romaine is similar to Romaine type but smaller gauge volume was 20 mL, while the mobile phase was 0.1% (v/v) formic
and about 18–20 cm long. Finally, Little Gem is a long leaf lettuce acid at a flow rate of 0.4 mL/min. The mass spectral analysis was
that forms a compact and closed head 10–15 cm long and 8–10 cm performed on a G6410A triple quadrupole mass spectrometer from
in diameter. Agilent equipped with an ESI interface operating in negative ion
These lettuce types would also be expected to present mode. The effect of co-eluting matrix components, which can
differences in the chemical composition and metabolite profiling cause ion suppression or enhancement of the analyte, was
but, in spite of being widely consumed worldwide, little eliminated following the standard additions approach. For that,
information is available for these green lettuce types. Studying standard solutions of glutamic, tartaric, a-ketoglutaric, malic,
the chemical composition and the main antioxidant compounds shikimic, succinic, fumaric, citric, malonic and quinic acids (purity
they contain (polyphenols, vitamin C, folates, carotenoids and 99%) from Sigma–Aldrich (Steinheim, Germany) were added to the
chlorophylls) could provide valuable information about their matrix at concentrations 0.05–2000 mg mL1 for each organic acid.
advantages and disadvantages with regard to their quality and Free phenolics were extracted (3 g of fresh powdered sample)
potential health-promoting properties, as well as their suitability with methanol:formic acid (97:3) according to Cantos et al. (2002).
to be used as whole heads or fresh-cut lettuce. For the determination of bound compound, extraction and
simultaneous hydrolysis were performed according to Luthria
2. Materials and method et al. (2006) with 2 N NaOH containing 10 mM EDTA and 1%
ascorbic acid as antioxidants, and subsequent acidification with
2.1. Experimental design HCl. Afterwards, free phenolic acids were extracted twice with
ethyl acetate, the organic layer was evaporated to dryness under
Lettuce plants (Lactuca sativa L.) belonging to three different vacuum and the residue was re-dissolved in methanol. Both
types of regular (Romaine), intermediate (Mini-romaine) and low extracts (methanolic and hydrolysed) were injected in a liquid
(Little gem) size were cultivated in greenhouse conditions. A total chromatograph (Agilent Series 1200, Santa Clara, CA, USA)
of sixteen cultivars were studied in Romaine (Carrascoy, Espuña, equipped with a triple quadrupole mass spectrometer detector
Aitana, Collado, Alhama, Isasa, Ar-29213), Little gem (Ricote, Petra, and ESI interface operating in negative ion mode, using N2 as
Etna, Urbion, Sandra, Maite, Ferro) and Mini Romaine (Marta and nebulizer and drying gas and the following operation parameters:
Ar-29232) lettuces. There were four replicates per cultivar in a 2000 V capillary voltage, 60 psi nebulizer pressure, 13 L/min
randomized design. Plants were irrigated with a nutritive solution drying gas flow and 350 8C drying gas temperature. Separation
containing (mM): 6 N, 7 K, 4 Ca, 1 Mg, 1 P, 1 S; and (mM) 2.24 Fe, was achieved in a Lichrosphere C18 analytical column of
0.11 Mn, 0.27 B, 0.13 Zn, 0.032 Cu and 0.05 Mo. Sixty days after 250 mm  4 mm and 5 mm particle size (Agilent Technologies,
transplanting, plants were harvested and washed with deionised Waldbronn, Germany) with 5% formic acid (solvent A) and
water. The colour of each sample was measured by reflectance in acetonitrile (solvent B) as the mobile phase at a flow rate of
the inner and outer leaves, using a Minolta CR-200 (Minolta, 1 mL/min. The gradient began with 5% B, reaching 10% B in 9 min,
Ramsey, NJ, USA) colorimeter through direct reading in three 30% B in 50 min, increased to 100% in 2 min and held at 100% B for
different areas of the surface of randomly selected leaves, taking an additional 3 min, returning to initial conditions in 1 min and
the mean of the three measurements as the definitive. Colour data remaining isocratic for 6 min. Standards of protocatechuic acid,
are provided as hue angle (Hue = tan1 b*/a*) and chroma chlorogenic acid, caffeic acid, p-coumaric acid, rutin (quercetin 3-
[Chr = (a*2 + b*2)1/2]. Finally, the samples (500 g per replicate) O-rutinoside), phloridzin (phloretin 60 -O-glucoside), luteolin,
were powdered with liquid N2 and frozen at 80 8C until apigenin and kaempferol were purchased from Sigma–Aldrich
subsequent analysis. (Steinheim, Germany) (purity >99%). Phenolic compounds in the
samples were identified by comparison of retention times and
2.2. Analysis of metabolites and nitrate quantifier transition with those of the corresponding standard.
Quantification was performed in single reaction monitoring (SRM)
An overall extraction of water- and ethyl acetate-soluble mode at m/z 153!109 (protocatechuic acid), m/z 353!191
compounds was performed according to Cano et al. (2002). (chlorogenic acid), m/z 179!135 (caffeic acid), m/z 163!119
 A. López et al. / Journal of Food Composition and Analysis 33 (2014) 39–48 41

(p-coumaric acid), m/z 609!300 (rutin), m/z 435!273 (phlor- Steinheim, Germany). Lactucaxanthin was estimated using
idzin), m/z 285!117 (luteolin), m/z 269!117 (apigenin) and m/z neoxanthin as reference.
285!117 (kaempferol). The antioxidant capacity was estimated in both fractions
For the extraction of vitamin C (ascorbic and dehydroascorbic), (water- and ethyl acetate-soluble) using ABTS [2,2-azinobis(3-
3 g of powdered lettuce samples were homogenised with EDTA, ethylbenzothiazoline-6-sulfonic acid)] and FRAP (ferric reducing
0.05% (w/v). Vitamin C, measured as the sum of ascorbic acid and ability of plasma) assays. The antioxidant capacity by reducing the
dehydroascorbic acid, was analysed using liquid chromatography radical ABTS was determined following the method described by
equipped with a triple quadrupole mass spectrometer detector Cano et al. (2002). FRAP assay was carried out in both fractions
(Agilent Series 1200, Santa Clara, CA, USA) according to the according the procedure reported by Benzie and Strain (1996). For
methodology developed by Fenoll et al. (2011). For accurate analysing the antioxidant capacity in the organic fraction by the
quantification of the samples, the standard addition approach was FRAP assay, ethyl acetate extract was previously evaporated to
performed. Ascorbic acid standard was purchased from Sigma– dryness and re-dissolved in methanol. For both ABTS and FRAP
Aldrich (Steinheim, Germany) (purity >99%). assays, the antioxidant assay was carried out in triplicate for each
Folates from lettuce samples (4 g of powdered sample) were extract using a spectrophotometer Shimadzu UV-2401PC (Kyoto,
determined following the procedure described by Vahteristo et al. Japan) and was expressed as mmoles Trolox equivalents (TE)/g
(1996). Samples were homogenized with 25 mL of extraction fresh weight.
buffer (50 mM CHES, 50 mM HEPES, containing 2% sodium For nitrate determination, aliquots of powered samples were
ascorbate and 10 mM 2-mercaptoethanol, pH = 7.85) under a previously freeze-dried in a lyophiliser Christ Alpha 1-4 LSC
nitrogen atmosphere. In order to determine mono- and poly- (Osterode, Germany). Nitrate was extracted by stirring 50 mg of
glutamate forms, samples were treated both with and without freeze-dried samples in 10 mL of water for 30 min (Abbas et al.,
folate conjugase prepared from fresh pig kidneys as described by 1991) and determined by an ion chromatograph Dionex ICS-
Gregory et al. (1984). Then, samples were purified in strong anion- 3000DC (Sunnyvale, CA, USA), equipped with an ASRS-ULTRAII 4-
exchange (SAX) cartridges (LiChrolut1, Merck, Darmstadt, mm suppressor (75 mA) (Sunnyvale, CA, USA).
Germany) according to Jastrebova et al. (2003). Samples were
analysed in a liquid chromatograph (Merck-Hitachi, Tokyo, Japan) 2.3. Statistical analysis
equipped with a 7485 scanning fluorescence detector. Separations
were achieved in a LiChrocart1250-4 with a LiChrocart14-4, Data were analysed statistically using a statistical significance
LiChrospher1 100 RP-18 (5 mm) guard columns (Merck, Darm- level of 0.05 with the SPSS 18.0 software package, by ANOVA for
stadt, Germany), following the method described by Vahteristo the main effect (lettuce type), Duncan’s Multiple Range Test to
et al. (1996). The column temperature was maintained at 30 8C and determine differences between means, Pearson’s test to detect
the flow rate at 0.9 mL/min. The initial conditions of the mobile significant bivariate correlations and canonical discriminant
phase gradient were 6% acetonitrile and 94% phosphate buffer, analysis (CDA) to classify the different lettuce types with respect
which remained isocratic for the first 6 min. Subsequently, the to the measured parameters.
concentration of acetonitrile was increased linearly up to 25% for
20 min. The excitation and emission wavelengths were set at 280 3. Result and discussion
and 350 nm, respectively. Quantification was performed using
tetrahydrofolate (purity 95%) and 5-methyltethrahydrofolate Colour is one of the most important attributes affecting
(purity 98%), purchased from Dr. B. Schircks Laboratories (Jona, consumer perception of quality. As such, it plays a key role in
Switzerland) as standards. The purity of the standards was checked food preference and acceptability and may even influence taste
as described by Vahteristo et al. (1996) using molar extinction thresholds and sweetness perception (Clydesdale, 1993). When
coefficient and the concentration of all standard stock solutions comparing the colour of the outer leaves, Mini Romaine had more
was corrected for purity. greenish (higher hue) but less intense (lower chroma) colour than
Carotenoids and chlorophyll were extracted from powdered Romaine and Little Gem (Fig. 1A). However, some exceptions were
lettuce samples (3 g) according to the method described by observed in Romaine cultivars as cv. Aitana and cv. Alhama showed
Böhm (2001) with methanol/tetrahydrofuran (1/1, v/v) contain- a similar hue (equal greener) but higher chroma (higher colour
ing 0.1% BHT and using b-apo-80 -carotenal as internal standard. intensity) and cv. Carrascoy showed similar chroma but lower hue
Carotenoids and chlorophylls were determined using a Hewlett- than Mini Romaine. As regards the colour of the inner leaves,
Packard mod. 1100 HPLC system (Waldbronn, Germany) Romaine lettuces were more greenish (higher hue) than Mini
equipped with a photodiode array UV/vis detector. Separation Romaine and Little Gem but no differences in colour intensity were
was achieved in a 250 mm  4.6 mm i.d., 3 mm Prontosil C30 detected between lettuce types (Fig. 1B).
column (Bischoff, Leonberg, Germany) operating at 25 8C with Among the primary metabolites found in vegetables, soluble
methanol (solvent A) and methyl tert-butyl ether (solvent B) as sugars and organic acids are important components, and both
mobile phase. The gradient procedure was as follows: 1) initial greatly contribute to their flavour characteristics and nutritional
conditions 15% solvent B and 85% solvent A, 2) a 10-min linear value (Luning et al., 1994). Moreover, carbohydrates promote
gradient to 15% solvent B, 3) a 20-min linear gradient to 90% ascorbic acid stability, and thus enhance the vitamin content (Birch
solvent B. Elution was performed at a solvent flow rate of 1.3 mL/ and Pepper, 1983). The soluble sugars detected in lettuce were
min with an injection volume of 20 mL and detection at 450 nm glucose and fructose, which were found in similar concentrations,
for b-carotene, 444 nm for lutein, 461 nm for internal standard and sucrose at a 2 to 5-fold lower concentration than glucose
and chlorophyll a and 438 nm for neoxanthin, violaxanthin, (Table 1). On average, Romaine lettuces showed the highest soluble
lactucaxanthin and chlorophyll b. Identification of compounds sugar concentrations followed by Mini Romaine and Little Gem
was achieved by comparing the retention times and spectra (Fig. 2), the last mentioned showing the lowest concentrations,
obtained from photodiode array detection using external except for sucrose, whose values were similar to those of Mini
standards. Quantification was performed using commercially Romaine. Some deviations from the general tendency were
available external standard solutions of b-carotene, lutein, observed in the Romaine cv. Carrascoy and the Little Gem cv.
neoxanthin and violaxanthin (DHI LAB, Hoersholm, Denmark) Petra whose glucose and fructose contents were similar to those of
and chlorophyll standards (purity 90%) (Sigma–Aldrich, Mini Romaine lettuce. The analysis of organic acids in lettuce
42 A. López et al. / Journal of Food Composition and Analysis 33 (2014) 39–48

A B

118 106
119 108

110 55
120
45

50
121 112
40 45

40
122 114
35
35

123 30 116 30
45 40 35 30 55 50 45 40 35 30

Fig. 1. Chroma-hue plot of inner (A) and outer leaves (B) from Romaine (circular symbols), Mini Romaine (square symbols) and Little Gem (triangular symbols) lettuce
cultivars.

samples revealed the presence of malic, citric, fumaric, glutamic, showing similar values to Mini Romaine cultivars. As regards other
tartaric, quinic, a-ketoglutaric, succinic, shikimic and malonic organic acids, Mini Romaine type recorded the highest concentra-
acids (Table 2). Organic acids also play an important role in the tions (fumaric, glutamic and a-ketoglutaric) or at least similar
perception of flavour by modifying the taste of sugars. For example, concentrations than Romaine (tartaric, succinic and shikimic) or
citric acid masks the perception of sucrose (Bonnans and Noble, Little Gem (citric). Malonic acid concentration was similar for the
1993; Schifferstein and Fritjers, 1990) and fructose (Pangborn, three studied lettuce types.
1963), while malic acid seems to enhance the perception of sucrose Phenolic are believed to extend shelf life and increase the stress
(Fabian and Blum, 1943). In all the lettuce cultivars, the tolerance of plants, leading to lower postharvest losses (Hodges
concentration of malic acid was substantially higher than the and Forney, 2003; Meir et al., 1995). Such a property is based on
concentration of other organic acids. The highest concentration of their ability to scavenge reactive oxygen species that are known to
malic acid and consequently of total organic acids (Fig. 2) was be involved in leaf senescence and in the plant antioxidant defence
observed in Mini Romaine type, whereas Little Gem and Romaine system. Phenolic acids may occur in multiple conjugated forms
type had similar but lower concentrations. However, a wide with sugars, acids and other phenolic compounds (Robbins, 2003).
variation was observed in malic concentration, especially in The analysis of bound phenolics usually involves alkaline or acidic
Romaine lettuce with some cultivars (Alhama and AR-29213) hydrolytic procedures that result in the loss of phenolic acids,
especially when acidic hydrolysis is used (Krygier et al., 1982).
However, the addition of ascorbic acid and ethylenediaminete-
Table 1
Soluble sugar concentrations (mg g1) of regular and baby-sized lettuce types.
traacetic acid (EDTA) has been shown to totally prevent the loss of
phenolic acids during alkaline hydrolysis (Nardini et al., 2002). This
TYPE Sucrose Glucose Fructose study was designed to investigate the profiles of total phenolics,
ROMAINE including both free and bound forms, through extraction and base
CARRASCOY 1.9  0.2 3.6  0.2 4.5  0.2 digestion with addition of EDTA-ascorbate. The free phenolic
ESPUÑA 2.0  0.2 4.7  0.1 6.1  0.2
compounds detected in the methanolic extract were protocate-
AITANA 2.4  0.2 4.7  0.3 6.2  0.6
COLLADO 1.9  0.1 4.0  0.4 5.1  0.5
chuic, chlorogenic, caffeic and p-coumaric acids kaempferol,
ALHAMA 2.6  0.2 5.3  0.4 6.5  0.3 luteolin, apigenin and phloridzin (Table 3). Rutin was also found
ISASA 1.9  0.3 4.6  0.5 5.3  0.4 in the methanolic extract. Chlorogenic acid was the major
AR-29213 2.3  0.1 5.1  0.4 6.1  0.4 compound among the free phenolics. In general, Romaine lettuces
MEAN 2.2b 4.6c 5.7c
showed the highest concentration of chlorogenic and caffeic acids
LITTLE GEM although great variability was observed among cultivars. Whereas
RICOTE 1.7  0.3 2.7  0.6 3.8  0.6 chlorogenic acid concentrations were far higher in cvs. Espuña,
PETRA 1.7  0.2 3.5  0.2 4.6  0.3
Aitana and AR-29213 than in baby leaf lettuce, cv. Collado and cv.
ETNA 1.1  0.2 2.9  0.3 3.4  0.4
URBIÓN 1.2  0.1 2.8  0.2 3.4  0.2 Carrascoy showed lower concentrations than those observed in
SANDRA 1.2  0.1 3.2  0.2 3.9  0.1 some Little Gem or Mini Romaine cultivars. Moreover, Romaine
MAITE 1.3  0.1 3.4  0.4 4.1  0.4 lettuce together with Mini Romaine, showed the highest p-
FERRO 1.1  0.1 3.1  0.3 3.9  0.3
coumaric acid, luteolin and phloridzin values. No significant
MEAN 1.3a 3.1a 3.9a
differences in rutin, kaempferol, protocatechuic and apigenin
MINI ROMAINE content were observed between lettuce types. Bound phenolic
MARTA 1.9  0.1 3.7  0.2 4.7  0.2
compounds were quantified as the difference between free
AR-29232 1.3  0.3 4.2  0.7 5.0  0.8
MEAN 1.6a 3.9b 4.8b phenolics determined in the methanolic extract and those
*** *** *** determined in the hydrolyzed fraction. The most abundant bound
*** Significant differences between means at 0.1% level of probability at P = 5%.
phenolic compounds were caffeic and p-coumaric acids derivates
Different letters in the same column indicate significant differences between means (Table 4), which represented between 43 and 92% of the total
according to Duncan’s test. Values are means  standard error (n = 4). phenolic content, depending on the cultivar studied. Caffeic acid
 A. López et al. / Journal of Food Composition and Analysis 33 (2014) 39–48 43

Fig. 2. Total soluble sugars and organic acids, expressed as mg g1 FW, and total phenolic compounds, folates and carotenoids, expressed as mg g1 FW, in regular and baby-
sized lettuce types. Values were calculated as sum of individual compounds.

derivates concentrations were 15 to 34-fold higher than those of p- 2004). In this study, vitamin C (AA plus DHAA) showed an average
coumaric derivates. Romaine and Mini Romaine showed the concentration of 78 mg g1 FW which is in the range previously
highest concentrations of both bound phenolic compounds and reported for lettuce (Nicolle et al., 2004). Differences in vitamin C
total phenolic compounds (Fig. 2). According to the results, concentration were found among lettuce cultivars and, although
Romaine and Mini Romaine presented higher antioxidant value Romaine lettuce recorded the highest average concentration, large
(related to the phenolic content) and potentially higher shelf life inter-cultivars variability were observed, as it was for other
when consumed as whole-head lettuce than Little Gem types. On metabolites (Table 4).
the contrary, the same lettuce types would present disadvantages Natural folates in food are mainly found in polyglutamate forms
with regard to their shelf-life when marketed as fresh-cut lettuce (80%) and the rest as monoglutamates (Vahteristo et al., 1996).
since phenolic compounds act as a substrate of polyphenolox- Polyglutamic forms can not be assimilated by the human body and
idases and would therefore promote browning (Martı́nez-Sánchez need to be hydrolyzed to monoglutamates to be absorbed in the
et al., 2012). intestine (Chandler et al., 1986). The need for this previous step has
Vitamin C concentration, measured as the sum of ascorbic acid generally been considered as a limitation to the bioavailability of
and dehydroascorbic acid, is used as an index of the health-related polyglutamic forms (Gregory, 1995; Melse-Boonstra, 2003) and,
quality of vegetables. The major active form of vitamin C is ascorbic knowing the content of mono- and polyglutamic forms in foods,
acid (AA) although its oxidized form, dehydroascorbic acid (DHAA), provides relevant information about the suitability of the food as a
also exhibits biological activity since it is easily converted into source of bioavailable folates. Vegetables rich in folates include
ascorbic acid in the human body; hence, the interest in the spinach (1.7 mg g1 FW), chard (1.5 mg g1 FW) and escarole
simultaneous analysis of AA and DHAA (Lee and Kader, 2000). (1.0 mg g1 FW), followed by lettuce (0.6 mg g1 FW) which can
Lettuce is a relatively poor source of vitamin C compared with be considered a moderate source of folates (Konings, 1999). In the
other vegetables such as broccoli (750 mg g1 FW), cauliflower studied lettuce cultivars, only 5-methyl tetrahydrofolate (5-MTHF)
(500 mg g1 FW) or pepper (350 mg g1 FW) (Bahorun et al., was detected in monoglutamic form (Table 4). On average, Little

Table 2
Concentration of malic, citric, fumaric, glutamic and tartaric acids (expressed as mg g1) and quinic, a-ketoglutaric (a-keto), succinic, shikimic (shikim) and malonic acids
(expressed as mg g1) in regular and baby-sized lettuce types.

TYPE Malic Citric Fumaric Glutamic Tartaric Quinic a-Keto Succinic Shikim Malonic

ROMAINE
CARRASCOY 15.9  1.4 0.84  0.48 0.95  0.08 0.32  0.02 0.15  0.03 59.0  11.6 46.0  5.9 36.0  4.4 13.8  0.4 10.2  1.0
ESPUÑA 14.4  0.7 0.62  0.14 0.89  0.04 0.34  0.01 0.09  0.01 77.4  7.4 36.9  5.1 20.9  2.2 14.8  0.7 10.8  0.3
AITANA 17.0  0.3 0.94  0.11 0.92  0.03 0.38  0.03 0.12  0.01 89.0  1.8 38.1  2.5 24.9  1.6 16.9  0.6 11.4  0.5
COLLADO 21.4  2.0 2.05  0.25 1.13  0.12 0.57  0.06 0.21  0.02 53.2  4.3 56.4  7.0 28.6  3.8 16.2  1.1 12.3  0.9
ALHAMA 23.1  0.8 1.57  0.12 1.25  0.04 0.52  0.05 0.19  0.01 70.4  4.1 68.6  3.4 49.7  2.0 18.1  1.5 10.6  0.3
ISASA 21.9  1.8 1.06  0.43 1.11  0.10 0.51  0.04 0.18  0.02 79.3  19.6 43.2  7.8 24.7  5.3 19.5  1.0 12.2  0.4
AR-29213 25.3  0.8 1.72  0.23 1.24  0.08 0.70  0.04 0.21  0.02 77.8  5.5 61.9  6.0 36.5  3.4 16.7  1.0 13.0  0.4
MEAN 19.8a 1.26a 1.07b 0.48a 0.16b 73.0b 49.9b 31.7b 16.6b 11.5

LITTLE GEM
RICOTE 17.0  0.9 1.88  0.28 0.77  0.12 0.52  0.10 0.09  0.01 48.7  5.4 31.5  3.7 28.0  2.3 12.5  0.6 11.9  0.9
PETRA 18.8  1.2 1.48  0.18 0.89  0.05 0.56  0.05 0.12  0.01 50.0  6.8 34.0  2.0 21.1  0.9 14.5  0.7 11.2  0.4
ETNA 17.5  1.5 1.18  0.15 0.90  0.08 0.58  0.02 0.10  0.01 51.2  4.5 38.0  3.8 19.0  2.6 12.5  0.7 11.3  0.9
URBIÓN 20.4  0.8 1.88  0.22 0.88  0.03 0.62  0.04 0.11  0.01 51.6  7.0 35.5  2.0 21.2  2.6 13.9  0.4 11.5  0.1
SANDRA 18.9  0.6 1.61  0.13 0.93  0.02 0.66  0.05 0.12  0.01 57.0  2.9 41.2  2.9 21.8  0.5 14.3  0.5 10.9  0.4
MAITE 20.4  1.2 2.37  0.32 0.97  0.05 0.65  0.10 0.11  0.01 61.4  5.6 40.1  1.6 22.1  1.3 13.7  0.5 11.6  0.8
FERRO 21.3  0.9 1.75  0.25 0.97  0.07 0.60  0.04 0.10  0.01 57.8  8.6 35.2  2.6 20.7  1.6 14.7  0.4 10.9  0.5
MEAN 19.2a 1.73b 0.90a 0.60b 0.12a 53.9a 36.5a 22.0a 13.7a 11.3

MINI ROMAINE
MARTA 24.5  0.9 1.60  0.13 1.23  0.04 0.71  0.03 0.15  0.01 72.5  4.1 62.5  2.1 34.7  2.9 14.5  0.6 11.7  0.3
AR-29232 26.0  1.8 2.12  0.28 1.34  0.10 0.68  0.05 0.18  0.02 61.2  7.5 74.6  6.4 39.5  1.7 16.6  1.4 13.0  0.8
MEAN 25.3b 1.86b 1.29c 0.70c 0.17b 66.9b 68.6c 37.1b 15.6b 12.3
*** ** *** *** *** *** *** *** *** n.s.

**, *** Significant differences between means at 1 or 0.1% level of probability respectively; n.s., non-significant at P = 5%. Different letters in the same column indicate
significant differences between means according to Duncan’s test. Values are means  standard error (n = 4).
44 A. López et al. / Journal of Food Composition and Analysis 33 (2014) 39–48

Table 3
Concentration of chlorogenic acid, rutin and kaempferol (kaempf) (expressed as mg g1) and p-coumaric acid (p-Cou) and caffeic acid, phloridzin (phlorid), protocatechuic
acid (protocat), luteolin and apigenin (expressed as mg kg1) in regular and baby-sized lettuce types.

TYPE Chlogenic Rutin Kaemf p-Cou Caffeic Phlorid Protocat Luteolin Apigenin

ROMAINE
CARRASCOY 4.0  1.2 0.04  0.01 0.20  0.02 80.9  1.3 36.8  4.6 47.0  0.5 18.5  1.2 13.6  2.1 0.75  0.12
ESPUÑA 72.9  7.3 0.29  0.22 0.25  0.01 82.2  0.6 76.5  18.5 48.8  0.4 25.5  2.6 16.9  1.9 0.75  0.08
AITANA 50.2  3.1 0.28  0.14 0.23  0.02 81.0  1.3 60.8  15.5 48.0  0.6 25.8  1.5 16.5  1.2 1.01  0.20
COLLADO 7.9  1.9 0.26  0.21 0.20  0.03 80.4  0.4 45.4  6.5 47.4  0.2 22.3  2.6 10.8  1.1 0.59  0.13
ALHAMA 25.3  4.2 0.25  0.14 0.25  0.01 82.5  0.7 58.0  8.5 48.7  0.4 26.1  2.3 19.1  3.8 0.79  0.13
ISASA 17.4  4.8 0.05  0.01 0.23  0.02 81.3  0.6 91.4  20.1 47.4  0.4 26.8  1.9 11.9  0.9 0.81  0.06
AR-29213 56.8  9.0 0.14  0.08 0.24  0.01 81.0  0.9 55.6  11.5 47.9  0.8 24.7  1.2 13.6  1.1 0.62  0.05
MEAN 34.1b 0.19 0.23 81.3b 60.7b 47.9b 22.4 14.6b 0.76

LITTLE GEM
RICOTE 14.0  5.1 0.10  0.06 0.24  0.02 78.6  0.3 32.2  2.8 46.6  0.2 19.9  1.2 11.7  0.7 0.56  0.15
PETRA 5.6  0.9 0.44  0.28 0.24  0.01 79.1  0.9 34.3  3.9 46.4  0.2 23.0  1.7 11.1  0.6 0.68  0.02
ETNA 14.3  3.9 0.48  0.24 0.26  0.03 78.2  1.5 46.3  6.8 46.2  0.6 21.9  1.8 11.6  0.9 0.66  0.06
URBIÓN 3.6  0.9 0.14  0.06 0.25  0.02 79.4  0.5 34.9  4.8 47.1  0.3 21.5  1.0 12.3  1.1 0.64  0.13
SANDRA 8.0  0.8 0.77  0.10 0.30  0.04 78.1  0.6 36.9  1.1 46.9  0.2 24.2  2.6 13.3  1.1 0.56  0.11
MAITE 13.5  2.6 0.07  0.01 0.25  0.01 79.1  1.1 44.1  5.5 46.6  0.4 23.1  1.8 11.5  0.5 0.66  0.03
FERRO 14.6  7.2 0.19  0.14 0.26  0.02 77.7  1.1 44.1  8.4 46.0  0.4 19.4  1.5 12.3  0.8 0.48  0.08
MEAN 11.0a 0.31 0.26 78.6a 39.0a 46.5a 21.9 12.0a 0.61

MINI ROMAINE
MARTA 19.6  5.3 0.07  0.02 0.22  0.02 80.8  0.7 50.8  5.5 47.4  0.2 22.5  2.2 13.5  1.8 0.53  0.11
AR-29232 8.5  1.8 0.34  0.27 0.25  0.01 81.4  1.0 38.9  4.1 48.1  0.5 26.1  0.8 12.8  0.5 0.76  0.06
MEAN 14.0a 0.20 0.24 81.1b 44.9a 47.8b 24.3 13.2ab 0.64
*** n.s. n.s. *** *** *** n.s. ** n.s.

**, *** Significant differences between means at 1 or 0.1% level of probability respectively; n.s., non-significant at P = 5%. Different letters in the same column indicate
significant differences between means according to Duncan’s test. Values are means  standard error (n = 4).

Gem showed the highest concentration of 5-MTHF whereas which folate is present in the polyglutamate form, shows similar
Romaine and Mini Romaine showed similar values. The addition amounts of monoglutamate and polyglutamate forms. In our
of conjugase for determine polyglutamatic forms released comparative study, Romaine lettuce had the highest 5-MTHF
further 5-MTHF and tetrahydrofolate (THF), as previously concentration with some exceptions (cv. Isasa), whereas THF
described in lettuce (Scott et al., 2000). These polyglutamic content was not influenced by lettuce type. The total folate
forms accounted from 25% to 65% of the total folates content. This content, calculated as the sum of mono- and polyglutamate
proportion agrees with the results previously reported by Wang forms, was also higher for Romaine type than for the baby-sized
et al. (2010) for lettuce, which, unlike most of vegetables in lettuce types (Fig. 2)

Table 4
Concentration of bound caffeic and p-coumaric acids (p-Cou), vitamin C, monoglutamate and polyglutamate folates, expressed as mg g1, and nitrate (mmol kg1) in regular
and baby-sized lettuce types.

TYPE Caffeic p-Cou Vitamin C Monoglut Polyglutamate Nitrate

5-MTHF 5-MTHF THF

ROMAINE
CARRASCOY 39.3  4.4 2.4  0.6 75.4  5.0 0.22  0.01 0.24  0.03 0.07  0.02 185.6  69.0
ESPUÑA 48.9  1.1 2.9  0.2 104.5  9.2 0.21  0.02 0.20  0.02 0.08  0.01 19.7  4.3
AITANA 76.3  6.1 2.2  0.5 100.5  5.6 0.21  0.02 0.33  0.06 0.07  0.02 19.0  4.3
COLLADO 46.5  2.8 1.6  0.6 90.5  14.0 0.15  0.01 0.11  0.02 0.07  0.02 115.8  35.1
ALHAMA 46.7  5.1 2.1  0.5 93.6  5.5 0.18  0.04 0.17  0.08 0.04  0.01 30.1  5.2
ISASA 60.1  10.4 4.1  2.8 69.8  11.9 0.19  0.02 0.05  0.01 0.03  0.03 221.4  49.9
AR-29213 44.4  6.8 2.8  0.7 109.2  4.2 0.20  0.02 0.21  0.05 0.08  0.03 161.2  39.3
MEAN 51.8b 2.5b 92.0b 0.20a 0.19b 0.06 100.5a

LITTLE GEM
RICOTE 31.3  5.8 1.1  0.2 59.9  0.4 0.26  0.03 0.10  0.06 0.04  0.02 733.3  71.8
PETRA 19.8  3.8 1.1  0.2 63.9  1.8 0.27  0.02 0.10  0.02 0.03  0.01 783.3  58.9
ETNA 23.8  7.1 0.9  0.1 131.9  13.0 0.16  0.03 0.04  0.01 0.01  0.00 395.9  62.2
URBIÓN 24.5  4.3 1.2  0.2 78.8  4.6 0.24  0.03 0.02  0.01 0.09  0.08 630.2  99.6
SANDRA 44.0  12.1 1.5  0.5 81.2  5.1 0.28  0.05 0.05  0.02 0.08  0.03 755.8  189.3
MAITE 30.6  5.9 1.6  0.3 60.8  4.4 0.24  0.03 0.22  0.07 0.05  0.02 571.6  185.2
FERRO 33.6  4.3 1.3  0.1 35.8  10.2 0.20  0.02 0.03  0.01 0.06  0.01 402.3  77.4
MEAN 29.9a 1.2a 73.2a 0.24b 0.08a 0.05 626.6b

MINI ROMAINE
MARTA 53.5  7.2 2.0  0.5 73.4  12.0 0.19  0.02 0.07  0.06 0.09  0.05 152.2  24.6
AR-29232 35.4  4.8 1.9  0.2 61.0  7.2 0.25  0.03 0.08  0.05 0.07  0.02 297.8  72.0
MEAN 44.4b 2.0b 67.2a 0.22a 0.07a 0.07 214.6a
*** *** ** * ** n.s. ***

*, **, *** Significant differences between means at 5, 1 or 0.1% level of probability respectively; n.s., non-significant at P = 5%. Different letters in the same column indicate
significant differences between means according to Duncan’s test. Values are means  standard error (n = 4).
 A. López et al. / Journal of Food Composition and Analysis 33 (2014) 39–48 45

Table 5 incident light energy exceeds that needed for photosynthesis; the
Antioxidant activity measured by the ABTS and FRAP assays in the water-soluble
carotenoids that compose the xanthophyll cycle dissipate excess
(W-ABTS and W-FRAP) and ethyl acetate-soluble (EA-ABTS and EA-FRAP) fractions,
expressed as mmoles of trolox equivalents per 100 g in regular and baby-sized energy, thus avoiding damage to the photosynthetic system
lettuce types. (Demmig-Adams and Adams, 1996; Krause, 1988). In addition to
the differences in head compactness between Little Gem and the
TYPE W-ABTS EA-ABTS W-FRAP EA-FRAP
other lettuce types, the head of Mini Romaine lettuces is smaller
ROMAINE than that of Romaine cultivars and thus they have proportionally
CARRASCOY 31.8  2.1 19.5  2.9 46.1  5.9 5.2  0.5
more outer green leaves, explaining the higher carotenoid content
ESPUÑA 53.4  4.5 21.0  3.9 63.9  5.1 6.8  0.5
AITANA 60.9  7.4 34.0  5.0 71.2  8.5 6.7  1.1 observed in Mini Romaine type.
COLLADO 37.7  4.4 20.5  5.1 49.7  6.4 6.0  0.9 Two assays, ABTS and FRAP, were used as complementary
ALHAMA 42.7  2.9 29.3  1.2 56.4  4.0 7.6  0.6 methods to evaluate the potential antioxidant capacity of the
ISASA 38.8  4.3 20.6  2.4 52.2  5.0 2.6  0.4
lettuce samples. In both assays, the highest antioxidant capacity
AR-29213 49.5  6.4 28.5  3.7 55.0  5.9 5.0  0.5
MEAN 44.9a 24.8a 56.3b 5.7c
was obtained in the water-soluble fraction but they did not detect
the same differences between lettuce types (Table 6). This
LITTLE GEM
indicates that the aqueous fraction contained varying proportions
RICOTE 31.8  2.1 19.5  2.4 40.5  1.9 2.5  0.6
PETRA 53.4  4.5 18.8  2.3 32.2  2.2 2.2  0.5 of antioxidants, with different activities in the FRAP and ABTS
ETNA 60.9  7.4 16.7  4.7 34.3  3.4 2.8  0.7 assays. Antioxidants deactivate radicals by two major mecha-
URBIÓN 37.7  4.4 19.0  1.7 34.3  2.8 2.4  0.6 nisms, single electron transfer (SET) and hydrogen atom transfer
SANDRA 42.7  2.9 21.5  3.7 30.6  2.1 2.7  0.8 (HAT) (Prior et al., 2005). Whereas FRAP is a SET-based method,
MAITE 38.8  4.3 26.6  0.9 34.9  3.0 4.4  0.2
FERRO 49.5  6.4 20.5  1.3 36.2  4.4 2.1  0.3
ABTS assay utilizes both HAT and SET mechanisms. The response
MEAN 46.9a 20.4a 34.7a 2.7a of the different antioxidants to these assays depends on their
ability to quench free radicals by hydrogen donation and/or their
MINI-ROMAINE
MARTA 58.1  2.1 33.5  3.0 52.3  2.3 5.6  0.7
ability to transfer one electron to reduce any compound. The
AR-29232 57.0  5.5 31.7  2.5 48.5  4.9 3.4  0.7 antioxidant capacity measured in the water-soluble fraction by
MEAN 57.8b 32.6b 50.4b 4.5b the ABTS assay (W-ABTS) was higher in Mini Romaine than in the
* *** *** *** other types, while no differences were found between Romaine
*, *** Significant differences between means at 5, or 0.1% level of probability and Little Gem. Using the FRAP assay (W-FRAP), the highest
respectively at P = 5%. Different letters in the same column indicate significant antioxidant capacities were observed in Mini Romaine and
differences between means according to Duncan’s test. Values are means  standard Romaine. According to Gil et al. (2000), the major organic acids
error (n = 4).
present in lettuce, malic and citric acids, do not show antioxidant
capacity when they are evaluated with the FRAP assay. This may
explain the different results observed in both methods, Mini
Green leaves are known to have a strikingly constant carotenoid Romaine type showing higher antioxidant capacity with the ABTS
pattern, as follows: lutein (45%), b-carotene (25–30%), violax- method due to their high organic acids content, whereas Romaine
anthin (15%) and neoxanthin (15%) (Britton, 1991). Unlike other type showed higher antioxidant capacity with the FRAP method,
green leaf vegetables, lettuce also contains lactucaxanthin; its probably due to their higher phenolic compound content. As
neoxanthin concentration is lower than that of violaxanthin, and regard the ethyl acetate-soluble fraction, the antioxidant capacity
b-carotene concentration is higher than that of lutein (Kimura and measured by ABTS assay (EA-ABTS) was higher in Mini Romaine
Rodriguez-Amaya, 2003). According to this carotenoid pattern, the than in the other types and no differences were found between
major carotenoid found in all the lettuce cultivars was b-carotene, Romaine and Little Gem. In the FRAP ethyl acetate-soluble fraction
making up on average 50% of total carotenoids followed by lutein (EA-FRAP), Romaine reached the highest values followed by Mini
(20%), lactucaxanthin (13%), violaxanthin (11%) and neoxanthin Romaine and finally by Little Gem. The antioxidant capacity in the
(6%) (Table 5). Previous reports have demonstrated the influence of organic fraction measured by the FRAP assay (EA-FRAP) showed
the cultivar on the carotenoid content of lettuce (Mou, 2005). For lower antioxidant values than was observed with the ABTS assays
example, Caldwell and Britz (2006) found that all the carotenoids (EA-ABTS). Although both the FRAP and ABTS assay show low
varied significantly among cultivars of lettuces that belonged to chemical reactivity of cyclic carotenes and their carbonyl
the same type. In the current study, Mini Romaine showed the substituted derivatives due to steric hindrance, the ABTS assay
highest concentration (Fig. 2) and Little Gem recorder the lowest is able to recognize, at least in part the antioxidant properties of all
content. As was the case for carotenoids, Mini Romaine showed the carotenoids (Müller et al., 2011; Pellegrini et al., 2003). Therefore
highest chlorophyll (a and b) concentrations (Table 5). the ABTS assay would identify higher antioxidant capacity with
Differences observed in the nutritional and bioactive composi- the carotenoid content.
tion of the studied lettuce types could be explained in part by Finally, the nitrate content was determined as a compound with
differences in the head structure and size. Opener lettuce heads a potential to adversely affect health. Vegetables, especially leafy
such as those of Romaine and Mini-Romaine lettuces have a higher ones, represent the major sources of dietary nitrate intake, owing
photosynthetic area, which would contribute to increasing to their nitrate accumulation capacity. A low nitrate content in the
chlorophylls and sugars as well as other related metabolites edible part of the plants is very important for human health, due to
(Mou, 2009; Ozgen and Sekerci, 2011). In contrast, in Little Gem its potential transformation to nitrites, which are much more likely
cultivars with closer heads than Romaine or Mini Romaine, the to interact with haemoglobin and so affect blood oxygen
penetration of sunlight would be obstructed, leading to a lower transportation (Santamarı́a, 2006). In addition, nitrate may
metabolite content. According to Mou (2005), the influence of head combine with secondary amines to yield nitrosamines, increasing
structure on carotenoid content is especially relevant. b-carotene the risk of gastrointestinal cancer (Hord et al., 2009). In spite of the
plays a role in photosynthesis as an accessory pigment that absorbs great variability among cultivars of the same type (especially in
at different wavelengths from the chlorophylls and transfers the Romaine) significantly higher nitrate content was found in Little
absorbed light energy to the chlorophylls (Mou, 2009). Besides Gem lettuce compared with the other types, with concentrations
their function as light-harvesting pigments, carotenoids also 3- and 6-fold higher than those of Mini Romaine and Romaine
protect cells against the actions of oxygen free radicals when types, respectively (Table 4). According to Burns et al. (2004),
46 A. López et al. / Journal of Food Composition and Analysis 33 (2014) 39–48

Table 6
Concentration of b-carotene, lutein, lactucaxanthin, violaxanthin, neoxanthin, chlorophyll a (chl a) and chlorophyll b (chl b), expressed as mg g1, in regular and baby-sized
lettuce types.

TYPE b-Carotene Lutein Lactucaxanthin Violaxanthin Neoxanthin Chl a Chl b

ROMAINE
CARRASCOY 26.4  3.0 11.7  0.9 6.8  0.3 5.9  0.3 3.4  0.3 100.9  9.0 39.6  3.4
ESPUÑA 24.6  5.0 10.0  2.1 5.9  1.1 5.5  1.0 3.2  0.6 82.1  16.2 32.5  6.8
AITANA 34.9  2.3 13.9  1.2 8.2  0.7 7.5  0.5 4.6  0.4 121.4  10.9 49.8  4.8
COLLADO 32.0  0.9 11.6  0.2 8.1  0.7 5.3  0.3 3.5  0.1 96.1  7.8 23.71  7.2
ALHAMA 33.9  2.8 13.4  1.0 8.6  0.8 6.2  0.4 4.1  0.3 105.5  12.4 37.9  4.6
ISASA 20.1  1.3 7.7  0.5 4.0  0.6 5.0  1.2 2.3  0.3 59.8  5.3 19.5  2.8
AR-29213 33.0  2.2 14.1  1.1 9.3  0.8 6.9  0.7 4.5  0.6 114.8  9.6 35.4  6.8
MEAN 29.3b 11.8b 7.3b 6.0b 3.7b 97.2b 34.0a

LITTLE GEM
RICOTE 20.5  1.0 7.8  0.3 5.0  0.7 4.5  0.3 2.6  0.2 71.8  9.1 26.8  3.9
PETRA 19.5  1.0 7.8  0.7 4.7  0.2 4.6  0.1 2.5  0.2 60.2  4.6 23.4  3.2
ETNA 18.8  2.2 8.4  0.7 5.8  0.6 4.3  0.3 2.6  0.2 67.8  11.9 29.8  3.3
URBIÓN 20.6  0.5 8.4  0.4 5.3  0.5 4.8  0.3 2.7  0.1 76.5  5.5 25.5  1.9
SANDRA 20.7  4.1 8.7  1.3 4.8  0.7 4.9  0.6 2.7  0.4 72.9  17.2 28.6  5.9
MAITE 22.7  2.9 9.7  1.3 6.5  0.5 5.3  0.7 3.1  0.3 90.5  11.4 31.8  3.3
FERRO 20.3  3.4 7.8  1.1 4.6  0.6 4.2  0.5 2.5  0.4 69.0  9.7 23.4  3.2
MEAN 20.5a 8.4a 5.2a 4.7a 2.7a 72.7a 27.0a

MINI ROMAINE
MARTA 41.8  5.9 14.9  2.0 11.1  0.7 7.5  0.6 5.0  0.7 122.3  13.9 47.3  6.5
AR-29232 33.3  1.4 12.7  0.4 8.4  0.3 6.2  0.3 3.9  0.1 112.0  9.5 51.9  4.3
MEAN 37.6c 13.8c 9.7c 6.8b 4.5c 117.2c 49.6b
*** *** *** *** *** *** ***

*** Significant differences between means at 0.1% level of probability at P = 5%. Different letters in the same column indicate significant differences between means according
to Duncan’s test. Values are means  standard error (n = 4).

nitrate accumulation in leaves increases when there is a reduction cross-validation procedure was applied to assess the reliability of
in solar radiation, or when parts of the lettuce head become the statistical model. The dataset consisted of the 64 individual
subjected to shading due to increased canopy density. Therefore, samples (28 Romaine, 28 Little Gem and 8 from Mini Romaine).
limiting light availability in closer heads such as those of Little Gem Two canonical discriminant functions resulted from entering all
lettuces could lead to lower nitrate assimilation, resulting in a the variables and the predictor variables selected by the CDA were
higher nitrate content. fructose (with a canonical function coefficient of 0.265), citric acid
Canonical discriminant analysis (CDA) was carried out to (0.110), vitamin C (0.105), W-FRAP (0.293), nitrate (0.348) and
assess the parameters that allow the three lettuce types (Romaine, hue angle in the inner leaves (0.380) for the first function and a-
Little Gem and Mini Romaine) to be classified according to the ketoglutaric acid (0.488), b-carotene (0.464), W-ABTS
many parameters measured in this study related with their (0.210) and lightness in outer leaves (0.382) for the second
chemical composition and antioxidant capacity. To this end, a function. The model allowed 100% of the grouped cases (by cross
stepwise analysis was performed to reduce data over-fitting and a validation) to be correctly classified according to the discriminant
distribution depicted in Fig. 3.

4 4. Conclusions

This study was performed to assess differences in the chemical

2
composition and antioxidant capacity between three lettuce types
(Romaine, Little Gem and Mini Romaine). Romaine type showed
2
1 the highest content of phenolic compounds, vitamin C and folates,
with their recognized beneficial effects on human health. Phenolic
Function 2

0
compounds also have the capacity to extend shelf life when the
lettuce is consumed in whole-head form, although with the
potential disadvantage when marketed as fresh-cut lettuce of
-2
promoting enzymatic browning. Mini Romaine showed the highest
content of organic acids and lipophilic compounds including
carotenoids and chlorophylls, both with recognized antioxidant
-4 3
1=Romaine
properties. Finally, Little Gem presented the highest nitrate
2=Little Gem content, which can be considered a negative characteristic of this
3= Mini Romaine lettuce type.
-6 Group Centroid The differences found in metabolite and nitrate concentra-
tions could be attributed to the lettuce head structure and size of
-8 -6 -4 -2 0 2 4 6
the different types which determine the penetration of sunlight
Function 1 and therefore the synthesis of light-dependent metabolites and
the assimilation of nitrogen. Canonical discriminant analysis
Fig. 3. CDA Histograms for lettuce cultivars from regular and baby-sized types,
using fructose, citric acid, vitamin C, W-FRAP, nitrate, inner leaves hue angle, a- (CDA) allowed the three lettuce types to be classified (100% of
ketoglutaric acid, b-carotene, W-ABTS and outer leaf lightness as predictor cases) using as predictor variables fructose, citric acid, vitamin C,
variables. W-FRAP, nitrate and inner leaf hue angle for the first function and
 A. López et al. / Journal of Food Composition and Analysis 33 (2014) 39–48 47

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