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Journal of Agricultural Science; Vol. 8, No. 11; 2016
ISSN 1916-9752 E-ISSN 1916-9760
Published by Canadian Center of Science and Education
1
Nutritional Value of Crisphead ‘Iceberg’ and Romaine Lettuces
(Lactuca sativa L.)
Moo Jung Kim
1,2
, Youyoun Moon
1
, Dean A. Kopsell
3
, Suejin Park
1
, Janet C. Tou
2
& Nicole L. Waterland
1
1
Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV, USA
2
Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV, USA
3
Department
of Plant Sciences, The University of Tennessee, Knoxville, TN, USA
Correspondence: Nicole L. Waterland, Division of Plant and Soil Sciences, West Virginia University,
Morgantown, WV 26506, USA. Tel: 1-304-293-2969. E-mail: nicole.waterland@mail.wvu.edu
Received: August 16, 2016 Accepted: September 20, 2016 Online Published: October 15, 2016
doi:10.5539/jas.v8n11pxx URL: http://dx.doi.org/10.5539/jas.v8n11pxx
The research is financed by the West Virginia University Foundation and West Virginia University Agriculture
and Forestry Experimental Station (Hatch Grant WVA00665 and WVA00640). Scientific Article No. 3282 of the
West Virginia Agricultural and Forestry Experiment Station, Morgantown.
Abstract
Lettuce (Lactuca sativa L.) is one of the most popular vegetables worldwide, but is often viewed as low in
nutritional value. However, lettuce contains health-promoting nutrients and biosynthesis of such phytochemicals
varies depending on cultivar, leaf color and growing conditions. Studies of such parameters on the nutritional
value have not been conclusive because the lettuce samples were collected from heterogeneous growing
conditions and/or various developmental stages. In our study nutritional composition was evaluated in the two
most popular lettuce types in Western diets, romaine and crisphead ‘Iceberg’, with red or green leaves grown
under uniform cultivating conditions and harvested at the same developmental stage. In the investigated lettuce
cultivars, insoluble fiber content was higher (P ≤ 0.05) in romaine than crisphead lettuces. α-linolenic acid
(omega-3 polyunsaturated fatty acid) was the predominant fatty acid and was higher in romaine than crisphead
Iron and bone health-promoting minerals (Ca, Mg and Mn) were significantly higher (P ≤ 0.001) in romaine. The
content of β-carotene and lutein in romaine (668.3 µg g
-1
) was ~45% higher than in crisphead (457.3 µg g
-1
dry
weight). For leaf color comparison, red cultivars provided higher amount of minerals (Ca, P, Mn and K), total
carotenoids, total anthocyanins and phenolics than green cultivars. Based on our study results, romaine was
generally higher in nutrients analyzed, especially red romaine contained significantly higher amount of total
carotenoids, total anthocyanins and phenolics. Therefore, romaine type lettuces with red rather than green leaves
may offer a better nutritional choice.
Keywords: carotenoid, dietary mineral, fatty acid, insoluble fiber, lettuce, phenolic compound
1. Introduction
Epidemiological studies have reported a correlation between fresh vegetable consumption and reduced risk of
chronic diseases (Rodriguez-Casado, 2016). Lettuce (Lactuca sativa L.) is one of the most popular vegetables
worldwide. In countries with Western diets such as the United States (US), the most serious public health threat
is chronic diseases partially due to poor dietary habits (Cordain et al., 2005). Even though salad consumption is
increasing, lettuce is regarded as low in nutritional value. However, nutritional value of lettuce greatly varies
with lettuce types depending on lettuce type, and nutrient composition can be equivalent to other “nutritious”
vegetables (Kim, Moon, Tou, Mou, & Waterland, 2016).
Lettuce contains several dietary minerals important for human health such as iron (Fe), zinc (Zn), calcium (Ca),
phosphorus (P), magnesium (Mg), manganese (Mn), and potassium (K) and other health-promoting bioactive
compounds (Kim et al., 2016). Yet, few human clinical studies have investigated lettuce consumption in disease
prevention. A case-control study reported an inverse association between colorectal cancer and lettuce
consumption (Fernandez, LaVecchia, Davanzo, Negri, & Franceschi, 1997). Investigating potential nutrients in
lettuce contributing to reduced colorectal cancer found no relationship to Ca, vitamin E, and folate, while
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β-carotene showed a significant relationship. Beneficial health properties of lettuce have mainly been attributed
to carotenoids and other phytochemicals such as phenolic compounds (López, Javier, Fenoll, Hellin, & Flores,
2014). High quantities of carotenoids (i.e. β-carotene and lutein) were reported for several lettuce types including
crisphead, butterhead, romaine, green and red leaf lettuces (Mou, 2005; Nicolle et al., 2004). Phenolic acids and
anthocyanins were reported in red and green butterhead, crisphead (subtype Batavia), and green and red oak leaf
lettuces (García-Macías et al., 2007; Nicolle et al., 2004). In these studies, carotenoids, phenolic acids, and
anthocyanin contents varied depending on lettuce types. Although low in fat, α-linolenic acid, omega-3
polyunsaturated fatty acid, was found to be the major fatty acid in lettuce (Le Guedard, Schraauwers, Larrieu, &
Bessoule, 2008; Pereira, Li, & Sinclair, 2001) but to date, fatty acid composition in different lettuce types has not
been well investigated.
In the US, crisphead and romaine are the most popular lettuce types, however, their nutritional value has not
been extensively evaluated. The United States Department of Agriculture (USDA) nutrient database (2015)
includes crisphead and romaine lettuce; however, few bioactive compounds were measured and cultivars with
different leaf colors (i.e. green and red) were not specified. Nutritional composition is greatly affected by
environmental (light, fertilizers, growing conditions) and biological (cultivar, leaf color, developmental stage)
factors (Kim et al., 2016). In many studies samples were prepared from lettuces grown under different growing
conditions and/or various developmental stages. Thus, the evaluation of the nutritional value in lettuces among
different researchers have been often inconsistent. Particularly, the influence of type/cultivar and leaf
pigmentation on nutritional value merits further investigation.
Our hypothesis is that genetic difference controlling characteristics of lettuce (type/cultivar) and pigmentation of
leaf (red or green) affect phytochemical contents. Although a number of studies reporting nutritional
composition of lettuce are available, there are only a few studies comparing both essential and non-essential
nutrients of popular lettuce types that are grown under the same cultural conditions. Therefore the objective of
this study was to investigate the effect of type/cultivar and leaf color on nutritional value. In our study nutritional
composition, including fatty acid, essential dietary minerals, phytonutrients carotenoids and phenolic compounds,
and insoluble dietary fiber was evaluated in two most popular lettuce types in the Western diets, crisphead
‘Iceberg’, and romaine, with red or green leaves grown under uniform conditions.
2. Materials and Methods
2.1 Plant Materials and Growing Conditions
Seeds of green and red cultivars of crisphead (L. sativa L. var. capitata , ‘Ithaca’ and ‘Red Iceberg’) and romaine
(L. sativa L. var. longifolia , ‘Coastal Star’ and ‘Outredgeous’) lettuces were sown to plug trays filled with
commercial media (Sunshine Mix #1, Sun Gro Horticulture, Agawam, MA), and germinated under a misting
system in a greenhouse. Three-week old seedlings were transplanted to 15-cm pots and grown for additional 60 d.
A total of four plants were grown for each cultivar with one plant as an individual biological replication. Plants
were grown under natural sunlight with supplemental high pressure sodium lighting when the light intensity was
below 50 W m
-2
with 14 h d
-1
of photoperiod.
Mean greenhouse temperature was 22.6/18.0 ± 1.7/2.5 °C (mean ± SD) day/night, and plants were irrigated as
needed with a 20% Hoagland modified nutrient solution #2 (PhytoTechnology Lab., Shawnee Mission, KS).
After harvest plants were freeze-dried (VirTis Freezemobile 12SL, SP Scientific, Warminster, PA) and ground to
powder, and then stored at -80 °C until analyses.
2.2 Insoluble Fiber Content
Insoluble fiber content was analyzed using neutral detergent method to determine cellulose, hemicellulose, and
lignin following the method of Goering and Soest (1970) with modifications. Freeze-dried powdered lettuce (0.5
g) sample were placed into individual fiber filter bag (ANKOM Technology, Macedon, NY) and sealed. Bags
were placed in a fiber analyzer (ANKOM
200
Fiber Analyzers, ANKOM Technology, Macedon, NY) and agitated
with 2 L of neutral detergent solution (ANKOM Technology, Macedon, NY) and 4 mL of heat stable α-amylase
at 100 °C for 1 h. Neutral detergent solution was drained and samples were washed four times with boiling water
(2 L) for 5 min. Bags were rinsed with 100% acetone and then dried in a drying oven at 100 °C overnight.
Individual bags were weighed to quantify total insoluble fiber. Each sample was analyzed in duplicate.
2.3 Fatty Acids Analysis
For lipid extraction, a ground sample (50 mg) was mixed with 5 mL of Triz/EDTA buffer and an internal
standard (50 µL of nonadecanoic acid) (Nu-Chek Prep, Inc., Elysian, MN). Then, samples were mixed with 20
mL chloroform:methanol:acetic acid solution (2:1:0.015, v/v/v) and centrifuged at 900 g
n
for 10 min at 10 °C.
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The chloroform layer was collected and filtered through a pre-rinsed 1-phase separation filter paper (Whatman
1-PS, GE Healthcare Bio-Sciences, Pittsburgh, PA). Samples were re-extracted with 10 mL of
chloroform:methanol solution (4:1, v/v). Extracted lipids were transmethylated into fatty acid methyl esters
(FAMEs) according to Fritsche and Johnston (1990). Briefly, samples were dried under a nitrogen gas at 55 °C
and extracted fatty acids were methylated by adding 4 mL of 4% H
2
SO
4
in anhydrous methanol and incubated at
90 °C for 1 h. After samples were cooled to room temperature, 3 mL of deionized distilled water and 8 mL of
chloroform were added. Samples were centrifuged at 900 g
n
for 10 min at 10 °C and chloroform layer was
filtered through Na
2
SO
4
. Samples were dried under a nitrogen
gas at 55 °C then dissolved in 2 mL of iso-octane.
To analyze FAMEs, a gas chromatography (Varian CP-3800 GC system, Agilent Technologies, Inc., Santa Clara,
CA) equipped with an autosampler, a flame ionization detector, and a CP-Sil 88 capillary column (Agilent
Technologies, Inc., Santa Clara, CA) was used. Initial temperature was 140 °C and held for 5 min and then
increased 4 °C per min to the final temperature of 220 °C. Total running time was 85 min per sample. Carrier gas
was nitrogen at 0.4 mL min
-1
of flow rate. Fatty acids were identified by retention time and quantified using a
standard curve made with external standards (16A, Nu-Chek Prep, Inc., Elysian, MN).
2.4 Mineral Content
Lettuce powder (0.5 g) was ashed at 550 °C overnight. Ashed samples were dissolved in 2% nitric acid and
adjusted to total volume of 20 mL with deionized distilled water. Mineral content of samples was analyzed by
inductively coupled plasma mass spectrometry (Optima 2100DV, Perkin Elmer Corp., Waltham, MA), and each
sample was analyzed in triplicate.
2.5 Carotenoid Analysis
Carotenoids were analyzed according to Kopsell, Barickman, Sams, and McElroy (2007). Freeze-dried samples
(0.1 g) were re-hydrated in 0.8 mL of ultrapure water for 10 min. Ethyl-β-8’-apo-carotenoate (Carotenature,
Lupsingen, Switzerland) was added as an internal standard (0.8 mL). Tetrahydrofuran (THF) (2.5 mL) stabilized
with 2,6-di-tert-butyl-4-methoxyphenol (25 mg L
-1
). Samples were homogenized then centrifuged at 500 g
n
for 3
min. The supernatant was collected and samples were re-extracted in 2 mL of THF as described above. The
combined supernatants were evaporated and total volume was adjusted to 5 mL with acetone. Final extracts of 2
mL was filtered through a 0.2-µm polytetrafluoroethylene filter (Model Econofilter PTFE 25/20, Agilent
Technologies, Wilmington, DE). A high performance liquid chromatography (Agilent 1200 series, Agilent
Technologies, Palo Alto, CA) equipped with a photodiode array detector was used for carotenoid analysis.
Carotenoids were identified and quantified using an external standard (ChromaDex Inc., Irvine, CA).
2.6 Total Anthocyanin and Phenolic Analysis
Total anthocyanin and phenolic compounds were extracted using the method of Nicolle et al. (2004) and
analyzed following the method of Olsen, Aaby, and Borge (2010) with modifications. Absorbance was measured
at 510 and 700 nm using a spectrophotometer (Spectronic
®
Genesys
TM
5, Thermo Fisher Scientific Inc., Waltham,
MA). The total anthocyanin content was expressed as cyanidin 3-glucoside equivalent.
To determine total phenolic content, 0.3 mL of extract or gallic acid standard was mixed with 1.5 mL of 0.5 N
Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO) and the mixture was incubated for 5 min. Then 1.5 mL
of sodium carbonate solution (0.5 M) was added and the mixture was incubated for 1 h at room temperature in
the dark before measuring absorbance at 765 nm using a spectrophotometer (Spectronic
®
Genesys
TM
5, Thermo
Fisher Scientific Inc., Waltham, MA). The total phenolic content was determined using a gallic acid standard
curve.
2.7 Statistical Analysis
The experimental design was a randomized complete block with four replications (n = 4) per cultivar. Each
cultivar within a block was randomly placed. All results were expressed as mean ± standard error. Two-way
analysis of variance (ANOVA) was conducted using SAS (SAS 9.2, SAS Institute, Inc., Cary, NC, USA) with
lettuce type (crisphead vs. romaine) and leaf color (red vs. green) as the main factors. Fisher’s least significant
difference test (LSD) was performed at the 95% significance level for mean separation.
3. Results and Discussion
3.1 Insoluble Fibers
Fiber consumption is low in the US (Dahl & Stewart, 2015). Fresh lettuce (100 g) can provide up to 10% of the
daily recommended intake of fiber for adults (Institute of Medicine, 2002). Eichholz, Förster, Ulrichs, Schreiner,
and Huyskens-Keil (2014) reported the dietary fiber in lettuce consisted mainly of insoluble fiber, with 80% as
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cellulose. In our study, insoluble fiber content depended on lettuce type (Figure 1, Table 1). Insoluble fiber
content was higher (P ≤ 0.05) in the romaine than in the crisphead cultivars. The USDA (2015) reported fiber
content of 21 mg g
-1
FW (420 mg g
-1
DW) in romaine lettuce and 12 mg g
-1
FW (240 mg g
-1
DW) in iceberg
lettuce, but total fiber, rather than insoluble fiber, was measured and leaf color of these cultivars were not
mentioned. Eichholz et al. (2014) reported lower fiber content in red butterhead ‘Teodore’ than in green
butterhead ‘Wiske’, suggesting leaf color can affect fiber content depending on lettuce type. Our study showed
no significant difference in insoluble fiber content between green and red pigmented cultivars of romaine and
crisphead lettuces. Based on the results, fiber intake in the diet may be increased by choosing romaine lettuce
types over others. Among lettuce cultivars examined in our study, insoluble fiber content was higher in romaine
than crisphead lettuce types. This is significant since crisphead (iceberg) is the most commonly used lettuce in
fast food restaurants (Mulabagal et al., 2010).
Cultivar
Coastal Star Outredgeous Ithaca Red Iceberg
Neutral detergent fi ber content
(mg g
-1
DW)
0
40
80
120
160
200
aa
b
b
Green
Red
Green
Red
Romaine
Crisphead
Figure 1. Neutral detergent fiber content in green and red cultivars of romaine (L. sativa L. var longifolia) and
crisphead (L. sativa L. var capitata) lettuces
Note. Vertical bars are standard errors of the means with four replications (n = 4). Different letters indicate
significant difference at P ≤ 0.05 by Fisher’s LSD.
Table 1. ANOVA results for neutral detergent fiber, total anthocyanin and total phenolic contents in green and red
cultivars of romaine (L. sativa L. var longifolia) and crisphead (L. sativa L. var capitata) lettuces
Source of variation Insoluble fiber Total anthocyanin Total phenolic
Type (T) * *** ***
Color (C) NS *** ***
T × C NS *** ***
Note.
NS
,
*
,
***
Nonsignificant or significant at P ≤ 0.05 or 0.001, respectively.
3.2 Fatty Acid Profile
Major fatty acids present in romaine and crisphead lettuces were the essential fatty acids, α-linolenic acid (ALA)
and linoleic acid (LA), which comprise over 75% of the total fatty acids (Table 2). Similarly, the major fatty
acids reported for ‘Trocadéro’ butterhead lettuce (Le Guedard et al., 2008) and Cos romaine lettuce (Pereira, Li,
& Sinclair, 2001) were ALA and LA. In our study, lettuce type, but not leaf color influenced total fatty acids
content. Green romaine had greater (P ≤ 0.01) ALA content than crisphead cultivars. The USDA (2015) reported
higher ALA for romaine compared to crisphead (iceberg). However, leaf color of these types were not identified.
It has been reported that low omega-3 to omega-6 PUFA intake contributes to the increased burden of chronic
diseases (Innis, 2014). Modern Western diets typified by the US are low in omega-3 PUFAs. Based on our results,
green romaine lettuce provides a higher amount of omega-3 PUFA due to its higher ALA content. Although
lettuce provides only a minor contribution to omega-3 PUFA intake in the human diet due to its low fat content,
consumption of green romaine would fulfill some of the omega-3 fatty acid requirement.
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Table 2. Fatty acid composition of green and red cultivars of romaine (L. sativa L. var longifolia) and crisphead
(L. sativa L. var capitata) lettuces
Type Color (Cultivar)
α-Linolenic acid
(18:3n-3)
Linoleic acid
(18:2n-6)
Palmitic acid
(16:0)
Stearic acid
(18:0)
Oleic acid
(18:1n-9) Total
------------------------------------------------------mg g
-1
DW--------------------------------------------------------
Romaine Green 12.9±0.4a
z
4.5±0.2a 3.7±0.1a 0.8±0.0a 0.5±0.1a 22.2±0.7a
(Coastal Star) (58.0%)
y
(20.3%) (16.5%) (3.5%) (1.7%)
Red 11.3±1.0ab 4.1±0.3a 3.1±0.2a 0.5±0.1a 0.3±0.1a 19.3±1.2a
(Outredgeous) (58.4%) (21.4%) (15.8%) (2.8%) (1.7%)
Crisphead Green 7.3±1.1c 4.2±0.3a 3.0±0.4a 0.5±0.2a 0.3±0.1a 15.2±1.8a
(Ithaca) (47.1%) (28.1%) (19.4%) (3.0%) (2.5%)
Red 8.7±2.1bc 3.9±0.6a 2.7±0.4a 0.3±0.1a 0.4±0.1a 16.0±3.0a
(Red Iceberg) (53.4%) (24.8%) (17.1%) (2.2%) (2.5%)
Significance
Type (T) ** NS NS NS NS *
Color (C) NS NS NS NS NS NS
T × C NS NS NS NS NS NS
Note.
z
Data are presented as mean ± standard error (n = 4). Mean separation within columns by Fisher’s LSD at
P ≤ 0.05;
y
Relative ratio of each fatty acid to the total fatty acid content;
NS
,
*
,
**
Nonsignificant or significant at
P ≤ 0.05 or 0.01, respectively.
3.3 Dietary Minerals
Inadequate intake of Zn and Fe is a concern in vegetarian diets (Hunt, 2003), in part due to a relatively low
bioavailability of these minerals in plants compared to meat. Lettuce (100 g fresh weight) provided only 1-4% of
the recommended Zn intake of 8-11 mg/day for adults (Kim et al., 2016). In our study, there was no significant
difference in Zn content regardless of lettuce type and leaf color (Table 3). Fe content was higher (P ≤ 0.001) in
the romaine than the crisphead cultivars. Similarly, the USDA (2015) reported higher Fe content in romaine (9.7
µg g
-1
FW) compared to crisphead (iceberg) lettuce (4.1 µg g
-1
FW). Mou and Ryder (2004) also reported higher
Fe content in romaine than in crisphead. Fresh lettuce (100 g) depending on lettuce type can provided up to 18%
of recommended Fe intake of 8-18 mg/day for adults (Institute of Medicine, 2005; Kim et al., 2016).
Vegetarians may be at greater risk of low bone density and bone fracture due to low Ca in vegetarian diets
(Tucker, 2014). The Ca content was higher (P ≤ 0.05) in romaine than crisphead lettuces (Table 3). There was an
interaction of lettuce type and leaf color with higher (P ≤ 0.05) Ca content in the green than red crisphead
cultivars, but not romaine lettuce cultivars. Another important mineral for bone health, P was higher (P ≤ 0.01) in
red than in green romaine and crisphead cultivars. Koudela & Petříková (2008) analyzed five cultivars of leaf
lettuces with different leaf colors and reported a significant influence of cultivar on Ca content; however,
influence of pigmentation on Ca content was not analyzed. Good-quality vegetarian diets are high in Mg and Mn
which are also important to bone health (Tucker, 2014). In our study, romaine had significantly higher Mg
content than crisphead lettuces. Red ‘Outredgeous’ romaine had higher Mn content than crisphead lettuces
(Table 3). Based on our results, minerals important for bone health were generally higher in the romaine than the
crisphead lettuces studied.
In our study, sodium (Na) content in lettuces ranged from 0.4-0.8 mg g
-1
DW (20-40 µg g
-1
FW) which is low
based on the daily recommended intake of Na of 1.2-1.5 g/day for adults (Kim et al., 2016). Reducing Na and
increasing K intake lowers the risk of hypertension (Adrogué & Madias, 2014). Red ‘Outredgeous’ romaine had
the highest (P ≤ 0.05) K content among the different lettuce cultivars evaluated. And mineral analysis within the
same lettuce types revealed significantly higher amount of K as well as P content in red than green cultivars.
While the role of leaf pigmentation on mineral content has not been intensively studied, our result indicated that
leaf color might be associated with certain mineral compositions of romaine and crisphead lettuces. The mineral
content of our study was generally lower than the result by Baslam, Morale, Garmendia, and Goicoechea (2013)
or similar or higher in Zn and Mg content, respectively, reported by Lyons, Goebel, Tikai, Stanley, and Taylor
(2015), indicating that mineral content in lettuce can greatly vary depending on lettuce type, cultivar, and
cultural conditions.
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Table 3. Mineral content in green and red cultivars of romaine (L. sativa L. var longifolia) and crisphead (L.
sativa L. var capitata) lettuces
Type Color (Cultivar) Zn Fe Ca P Mg Mn Na K
---------µg g
-1
DW-------- ----------------mg g
-1
DW-------------- --µg g
-1
DW-- --mg g
-1
DW--
Romaine Green 24.6±2.7a
z
38.8±2.1a 9.2±0.3a 2.5±0.1bc 5.5±0.1a 19.0±0.5ab 0.8±0.2a 21.9±0.8b
(Coastal Star)
Red 25.1±0.7a 37.6±4.1a 9.1±0.3a 3.0±0.3a 5.8±0.2a 21.8±1.5a 0.7±0.1a 28.3±0.7a
(Outredgeous)
Crisphead Green 22.5±3.0a 26.8±2.2b 8.1±0.4b 2.4±0.2c 5.0±0.2b 14.9±0.6c 0.5±0.1a 18.9±0.2c
(Ithaca)
Red 26.1±4.9a 27.9±2.0b 6.5±0.1c 2.9±0.2ab 4.6±0.1b 16.5±0.9bc 0.4±0.0a 21.9±0.6b
(Red Iceberg)
Significance
Type (T) NS *** *** NS *** *** * ***
Color (C) NS NS * ** NS * NS ***
T × C NS NS * NS * NS NS *
Note.
z
Data are presented as mean ± standard error (n = 4). Mean separation within columns by Fisher’s LSD at
P ≤ 0.05;
NS
,
*
,
**
,
***
Nonsignificant or significant at P ≤ 0.05, 0.01 or 0.001, respectively.
3.4. Carotenoids
Several carotenoids including β-carotene have been studied for their ability to reduce risk of chronic diseases.
Oxygenated carotenoids (xanthophylls) have been studied for its protective effects against macular degeneration
(Cooper, Eldridge, & Peters, 1999). Some carotenoids such as β-carotene have a provitamin A activity with
β-carotene to retinol (animal form of vitamin A) conversion rate ranging 3.6-28.1 by weight in humans (Tang,
2010). In our study β-carotene, lutein, and antheraxanthin were the major carotenoids ranging from 63.2 % of
total carotenoids in green romaine ‘Coastal Star’ to 70.6 % in red crisphead ‘Red Iceberg’ (Table 4). The
β-carotene and lutein were higher (P ≤ 0.01) in red ‘Outredgeous’ romaine compared to the crisphead lettuces.
According to the USDA database (2015), romaine contained a higher amount of β-carotene (52.3 µg g
-1
FW)
compared to crisphead (iceberg) lettuce (3.0 µg g
-1
FW). It was difficult to make a direct comparison since the
values in USDA database were reported from two studies, and growing conditions, leaf color, and cultivar were
not provided.
Biosynthesis of carotenoids is regulated by light. Differences in carotenoid content in lettuce types was
suggested to be related to head structure. Crisphead lettuce forms a closed head that obstructs light penetration
into the head. In contrast, romaine lettuce has an open head structure which allows more light penetration,
resulting in higher amount of carotenoids accumulated (Mou, 2005; Mou & Ryder, 2004). In these studies
carotenoid contents were higher in green than red cultivars. In another study, the contents of carotenoids were
evaluated in two differently pigmented lettuce cultivars; ‘Blonde of Paris Batavia’ (green) and ‘Oak Leaf’
(Pérez-López, Miranda-Apodaca, Muñoz-Rueda, & Mena-Petite, 2015). The carotenoids were higher in red than
in green cultivar by 130%. In our study red leaf ‘Outredgeous’ contained the highest amount of total carotenoids
(P ≤ 0.05), and there was no difference between leaf colors within the same lettuce type. Of the major
carotenoids measured in our study, there was an interaction of type and color on antheraxanthin content. Red
romaine was higher (P ≤ 0.001) in antheraxanthin than in green cultivars.
Other carotenoids detected in our lettuce samples were neoxanthin and violaxanthin (~13-18%). There was a
significant effect of lettuce type, but not leaf color. Romaine was higher in neoxanthin (P ≤ 0.01) than in
crisphead lettuce. Violaxanthin was higher (P ≤ 0.05) in red romaine than in the green crisphead, but no
difference was found in cultivars differing leaf colors within the same type. Baslam et al. (2013) analyzed one
romaine and two crisphead type lettuces and reported significantly higher levels of both neoxanthin and
violaxanthin in romaine lettuce. Advantages of higher consumption of neoxanthin and violaxanthin in humans is
unknown. We detected only small quantities of zeaxanthin and α-carotene at 1-1.8% of the total carotenoid
content. Among the lettuce types evaluated in our study, total carotenoid content was highest in red ‘Outredgeous’
romaine lettuce.
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Table 4. Carotenoid content in green and red cultivars of romaine (L. sativa L. var longifolia) and crisphead (L.
sativa L. var capitata) lettuces
Type Color (Cultivar) α-Carotene β-Carotene Lutein
Antheraxanthin
Neoxanthin Violaxanthin Zeaxanthin Total
--------------------------------------------------------------µg g
-1
DW-------------------------------------------------------------------
Romaine Green
(Coastal Star)
16.5±2.9a
z
(1.4%)
y
280.6±7.8ab
(23.6%)
338.5±11.1ab
(28.5%)
132.5±5.1c
(11.1%)
215.2±6.6a
(18.1%)
184.2±6.5ab
(15.5%)
22.0±1.1a
(1.8%)
1189.4±38.0b
Red
(Outredgeous)
16.0±2.2a
(1.0%)
344.2±35.1a
(20.7%)
373.4±34.7a
(22.5%)
450.8±44.8a
(27.1%)
228.5±23.8a
(13.8%)
226.2±27.7a
(13.6%)
21.4±5.3a
(1.3%)
1660.4±167.3a
Crisphead Green
(Ithaca)
12.7±1.3a
(1.3%)
193.3±9.8c
(19.1%)
220.3±14.3c
(21.8%)
296.7±18.9b
(29.3%)
135.6±8.4b
(13.4%)
138.8±8.5b
(13.7%)
14.4±0.9a
(1.4%)
1011.7±60.6b
Red
(Red Iceberg)
12.2±0.6a
(1.0%)
232.0±18.0bc
(18. 7%)
268.9±21.2bc
(21.7%)
374.2±19.1ab
(30.2%)
160.6±12.6b
(13.0%)
177.7±12.9ab
(14.3%)
13.5±1.6a
(1.1%)
1239.1±84.4b
Significance
Type (T) * ** ** NS ** * * *
Color (C) NS NS NS *** NS NS NS *
T × C NS NS NS ** NS NS NS NS
Note.
z
Data are presented as mean ± standard error (n = 4). Mean separation within columns by Fisher’s LSD at
P ≤ 0.05;
y
Relative ratio of each carotenoid to the total carotenoid content;
NS
,
*
,
**
,
***
Nonsignificant or
significant at P ≤ 0.05, 0.01, or 0.001, respectively.
3.5 Total Phenolic and Anthocyanin
Total phenolic and anthocyanin contents were significantly higher in romaine than in crisphead lettuce in both
green and red cultivars (Figure 2, Table 1). Biosynthesis of phenolic compounds might be light dependent as
suggested by Tsormpatsidis et al. (2008). As discussed above, romaine lettuces have an open head structure that
allows greater light penetration compared to crisphead lettuces. Therefore, romaine lettuce could accumulate
higher levels of phenolic compounds including anthocyanins.
Total phenolic content was significantly higher in red compared to green cultivars of romaine and crisphead
(Figure 2, Table 4). Llorach, Martínez-Sánchez, Tomás-Barberán, Gil, and Ferreres (2008) reported higher total
phenolic content in red oak leaf and lollo rosso than in green crisphead (iceberg) and romaine lettuces. Liu et al.
(2007) also reported higher total phenolic content in red compared to green cultivars of romaine lettuces as well
as leaf type lettuces. We evaluated anthocyanin content since this subgroup of phenolic compound is the main
plant pigment responsible for the red color in lettuce (Ferreres, Gil, Castañer, & Tomás-Barberán, 1997). Total
anthocyanin content in red ‘Outredgeous’ romaine and crisphead ‘Red Iceberg’ was 3418.2 and 442.5 µg g
-1
DW,
respectively (Figure 2). No anthocyanin was detected in green cultivars of either type lettuce. Similarly,
anthocyanin in red oak leaf and lollo rosso lettuce was reported, but anthocyanin was not detected in green
cultivars of crisphead (iceberg) and romaine (Llorach et al., 2008). Based on the results, red pigmentation was
indicative of total anthocyanin and phenolic content. There was also an interaction between leaf color and lettuce
type (Table 4). Total phenolic and anthocyanin was significantly higher in red ‘Outredgeous’ romaine than
crisphead ‘Red Iceberg’ (Figure 2). Red lettuce is increasing in popularity due to public perception that red color
is associated with greater health benefits. Among lettuces evaluated in our study, choosing red leaf lettuce such
as ‘Outredgeous’ romaine lettuce may result in greater intake of anthocyanin and phenolic compounds which
have been associated with diverse health benefits (Kris-Etherton et al., 2002).
jas.ccsenet.org Journal of Agricultural Science Vol. 8, No. 11; 2016
8
Cultivar
Coastal Star Outredgeous Ithaca Red Iceberg
Total phenolic conte nt
(mg gallic aci d equi v. g-1 DW)
0
5
10
15
20
25
30
Cultivar
Coastal Star Outredgeous Ithaca Red Iceberg
Total anthocyanin c ontent
(µg cy ani din 3 -gluc os ide equi v. g-1 DW)
0
1000
2000
3000
4000
5000
ND ND
a
bb
b
a
bb
c
Green
Red
Green
Red
Romaine
Crisphead
Figure 2. Total anthocyanin and phenolic contents in green and red cultivars of romaine (L. sativa L. var
longifolia) and crisphead (L. sativa L. var capitata) lettuces
Note. Vertical bars are standard errors of the means with four replications (n = 4). Different letters indicate
significant difference at P ≤ 0.05 by Fisher’s LSD.
4. Conclusions
Although lettuce is considered low in nutritional value, the results of the current study showed that nutrient
content depended on lettuce type, as well as the morphological factor of leaf color. Generally, the crisphead
lettuces were lower in all nutrients analyzed compared to romaine lettuces. Romaine cultivars contained higher
amount of insoluble fiber and the essential fatty acids, ALA and LA, which comprise over 75% of the total fatty
acids regardless of leaf color. Red cultivars had higher phenolic content than green cultivars with the highest
amount in red ‘Outredgeous’ romaine lettuce. The results of this study provide comparative nutrient data of
several popularly consumed lettuce cultivars that were grown under the same growing condition. This
information will assist consumers to make food choices that provide higher nutritional value.
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