Improving Carotenoids and Amino-Acids in Cassava

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Recent Patents on Food, Nutrition & Agriculture, 2009, 1, 32-38

Improving Carotenoids and Amino-Acids in Cassava
Nagib M.A. Nassar†,*, Osmindo P. Junior†, Marcelo V. Sousa† and Rodomiro Ortiz‡
†Departamento de Genetica e Morfologia, Universidade de Brasilia, Brasilia, Brazil, ‡Centro Internacional de
Mejoramiento de Maíz y Trigo (CIMMYT), Km 45 Carretera México-Veracruz, Col. El Batan, Texcoco, Edo. de México,
C.P. 56130, México

Received: August 19, 2008; Accepted: September 15, 2008; Revised: October 1, 2008
Abstract: More than 800 million people in tropics and sub tropics use cassava as food. However, its roots are poor in
protein content (0.7-2%). Amino acids such as lysine and methionine are also low, and some research reports indicate the
absence of methionine in cassava edible roots. By inter-specific hybridization it was possible to increase true protein in
cassava roots measured by amino acid contents. The amino acid profiles of a common cassava cultivar and an inter-
specific hybrid, namely ICB 300, were determined using the computerized amino acid analyzer Hitachi L-8500. The inter-
specific hybrid has 10-fold lysine and 3-fold methionine than common cassava cultivar: lysine content was 0.010 g per
100 g in the common cassava cultivar while it reached 0.098 in the inter-specific hybrid. Methionine in the common
cassava cultivar was 0.014 g per 100 g whereas it reached 0.041 g per 100 g in the inter-specific hybrid. Total amino acid
content in the common cassava cultivar was 0.254 g per 100 g viz. a viz. 1.664 g per 100 g in the inter-specific hybrid.
The genetic variability of the profile and quantity of amino acids indicate the feasibility of selecting inter-specific hybrids
that are rich in both crude protein and amino acids. Carotenoid content could be improved in cassava edible roots by
selecting cultivars rich in carotenoids. In Brazil, the center of cassava origin, cassava landraces have acquired through
their domestication a large diversity in relation to many economic traits such as high content of carotenoids and excellent
palatability among other characters. One of these clones, which has been grown by indigenous farmers in Brazil and
available at the University of Brasília genebank, showed a high level of lycopene content (5 mg/kg viz. a viz. zero in
common cultivars, and 12-20 mg/kg in tomato-a lycopene-rich vegetable). The cassava landrace UnB 400 had a high
content of -carotene (up to 4 mg/kg). This article also discusses relevant patents to the main subject of this research.
Keywords: Biofortification, genetic resources enhancement, lycopene, wild Manihot species.
Dedicated to Joachim Voss, a pioneer of Cassava Research Development, and a Visionary Administrator on his
retirement from a distinguished service with IDRC and CGIAR.
INTRODUCTION
hydrocyanic acid. The remaining of the total nitrogen (38-
40%) remains unidentified.

Cassava is among the most important crops in the tropics
and a staple food for more than 800 million people. Cassava

The majority of cassava clones grown and consumed in
is also the principal food for about 60 million people living
northeast Brazil are known to be free of carotenoids, which
in northeast Brazil. Cassava roots provide more than 60% of
leads to many health problems for inhabitants of this region.
the daily energy intake for the population of northeast Brazil
One of the interesting aspects is to screen indigenous clones
and many countries in Africa.
for cultivars rich in carotenoids. This concept is based on the
fact that crop landraces have accumulated, in their center of
Cassava protein is comparable to rice protein in
diversity, desirable mutations that were selected by
digestibility. The biological value (Block and Michell
indigenous farmers during their history of cultivation. The
equivalent) of the total protein is 48%. The crude protein
nutritive importance of carotenoids is attributed to its
content of roots appears to be relatively stable and constant
conversion to vitamin A, as in the case of -carotene, and to
with maturity of the plant. Cassava roots, however, are a
its antioxidant property and ability to quench singlet oxygen
poor source of protein, despite that the quality of this protein
as in the case of lycopene. Lycopene interacts with free
is fairly good, as is the proportion of amino acids as well [1,
radicals eliminating their deleterious effect. Some landraces
2]. Methionine and lysine are limiting amino-acids in
maintained at the genebank of the Univ. of Brasilia may be a
cassava edible roots [3]. Furthermore, the protein of pro-
source of carotenoids as shown by their red root flesh and
cessed cassava includes the highest percentage of glutamic
yellow color after cooking [5].
acid and the lowest of methionine (1%) [4]. If cultivars could
be developed with a higher quantity of these amino acids, it

Efforts have been made in the past to increase the protein
would enhance the value of cassava as a food and/or feed.
content of cassava roots by interspecific hybridization with a
Only about 60% of the total nitrogen derives from amino
wild species, namely M. saxicola and M. melanobasis. Over
acids, and about 1% of it is in the form of nitrates and
a period of 10 years beginning in 1932 and ending with the

Japanese occupation of Java in 1942, Bolhuis [6] carried out
a program of cassava breeding for increased protein content
*Address correspondence to this author at the Departamento de Genetica e
in roots. Crosses with M. saxicola yielded a few seedlings
Morfologia, Universidade de Brasilia, Brasilia, Brazil; Tel: 556133493253;
Fax: 556133493562; E-mail: [email protected]
with as much as 2% protein in the root fresh. In the clones,

he propagated from these seedlings protein content fell back

1876-1429/09 $100.00+.00
© 2009 Bentham Science Publishers Ltd.

Potential of Patents in Cassava Carotenoids and Amino-Acids
Recent Patents on Food, Nutrition & Agriculture, 2009, Vol. 1, No. 1 33
to typical levels. Moreover, Jennings [7] reported that the
respectively. The cis isomer of lycopene was quantified
roots of the F1 progeny of M. esculenta x M. melanobasis
using the calibration curve of lycopene. The vitamin A
possessed approximately twice as much protein as their
values were calculated according to the conversion factor
cassava parent. Such progenies were, however, lost and not
given by NAS-NRC, in which 6 mg of trans- -carotene
grown anywhere, probably because of poor root yield.This
correspond to 1 mg of retinol equivalent (RE), and the
article reports the assessment of lycopene content and
activities are related as follows: 100% for trans- -carotene,
amino-acid profiles of cassava landraces and a inter-specific
50% and for trans- -carotene and cis- -carotene.
hybrid, respectively.
Evaluation of Amino-acid Profiles
MATERIAL AND METHODS
Sample
extraction.
Samples (500 mg) of cassava root
Evaluation of Carotenoids
powder were extracted with 1 mL 10 mM HCl for 4 h, at
25°C, under agitation at 1,200 rpm in Thermomixer
The landraces UnB 400 - with yellow color after
(Eppendorf, Hamburg, Germany). The suspensions were
cooking, and UnB 500 - with red root flesh, which are
then centrifuged for 4 min at 6,000 rpm in a bench
indigenous cassava clones grown in the Amazon and
centrifuge. The supernatant (800 L), called acid extract, and
maintained in the University of Brasília’s living Manihot
the remaining powder were dried down in a SpeedVac
species collection were analyzed spectrophotometrically for
vaccum centrifuge (Savant, NY, USA). The dried powder
lycopene content along with the cassava cultivar Pohlii and
was extracted in the same way with 1 mL 10 M NH
ICB 300 –an inter-specific hybrid between cassava UNB 01
4OH
producing an alkaline extract. The dried acid extract was
and Manihot oligantha. Root powders were also taken from
resuspended with 750 L 10 mM HCl, washed with an extra
inter-specific hybrids ICB 300 and Diploide and ICB 300-
750 L of the same dilute acid, and added to 750 L of the
derived offspring (Progeny 4, Progeny 9 and Progeny 10) for
alkaline extract. The total extract was exhaustively dialyzed
amino-acid profiling.
against MilliQ water and vacuum-dried in a SpeedVac
Lycopene Analysis
centrifuge.
Extraction. Ten grams of mature roots was extracted

Amino acid analysis. Aliquots of 150 g of each extract
three times with acetone (5 ml per gram). The filtered
were dissolved in 75 L 100 mM HCl. Acid hydrolysis of
acetone extract was added in separation funnel containing
the samples was performed in 6 M HCl under vacuum for 24
petroleum ether, distilled water, ethilic ether (100/100/0.3:
h at 109°C. After acid hydrolysis, the hydrolyzed samples
v/v/v) The aqueous fraction was discarded and the organic
were solubilized in 75 L 100 mM HCl, and 50 L was
fraction was submitted to saponification. Saponification was
injected into an amino acid analyzer (Hitachi L8500, Tokyo,
preferred since it removes accompanying lipids and
Japan). The analyses for determination of amino acid
chlorophylls. In this study, the optimal conditions for mild
compositions were performed in triplicate. The total protein
saponification were achieved with 10% methanolic
contents of the samples were calculated by summing up the
potassium hydroxide solution (100 ml) overnight at room
amounts of the amino acids. Amino acid compositions from
temperature. After saponification, the aqueous fraction was
Manihot proteins were determined by analyzing sample
discarded and the organic fraction was dried with anhydrous
extracts which were dialyzed with water to remove free
sodium sulfate. The organic fraction was evaporated to
amino acids, salts, monosaccharides, and other small
dryness at 30°C, re-suspended in 1000 l ethyl acetate and
molecules. Tryptophan could not be analyzed since it is
methanol (v/v; 50/50) and submitted to HPLC separation.
degraded upon acid hydrolysis. By summing the amounts of
the analyzed amino acids, it was possible to determine the
Equipment. Carotenoid analyses were performed with a
protein content for each sample.
Shimadzu LC-10A HPLC equipped with a photodiode array
detector SPD MXA-10 and a Rheodyne injection valve with
RESULTS AND DISCUSSION
a 20- l loop. The separation was carried out in a C18 Vydac
Selection of Carotenoid-Rich Cassava Landraces
218TP54 column 250 x 4.6 mm i.d. (5- m particle size) with
100% MeOH as mobile phase at a flow rate of 1 ml/min at a

UNB 400 has a gray stem, which is 1.5 m high, has large
temperature of 15°C. The chromatograms were processed at
raised scars and 2 or 3 branches. Leaves have 5 to 7 lobes,
wavelengths of maximum absorption (450 nm). The
and the leaf lobe is obvater, with the margins slightly
identification of carotenoids was achieved by retention time
sinuous, whereas the medium lobe is 14 to 16 cm in length.
(tR) comparisons with those of the standard compounds and
The leaf has a green petiole, and the young foliage is
using the wavelength of maximal absorption ( max) and the
reddish. The inflorescence is a 5- to 8-cm glabrous panicle.
spectrum profile between 300 to 600 nm compared with data
Bracts and bracteoles are inconspicuous and caduceus.
available in the literature [8].
Flowers are monoecious showing pistillate flowers with
basal opening, whereas the staminate apical opening occurs 3
Quantification.
The calibration curves for lycopene were
weeks later. Fruits are green and winged and the roots are
purchased from Sigma Inc., and purified from tomato, while
conical, with a rough, pink-brownish surface. The root flesh
for trans- -carotene (purchased from Sigma Inc.), and for -
is creamy but turns yellow after cooking (Fig. 1a). UNB 500
carotene (purified from alfalfa) were constructed with a
has a gray stem, which is 1.5 m high with large raised scars
minimum of the concentration levels thrice. All curves
and 2 or3 branches. Its leaves are 7-lobed, and the leaf lobe
showed a good relation of area and concentration achieving a
is linear, with the margins slightly sinuous, whereas the
coefficient of determination (R2) of 0.96, 1 and 0.98, for
medium lobe shows 10 to 12 cm in length. The leaf has a
lycopene, trans- -carotene and trans- -carotene,
green petiole, and the young foliage is reddish. The

34 Recent Patents on Food, Nutrition & Agriculture, 2009, Vol. 1, No. 1
Ortiz et al.
inflorescence is a 3- to 6-cm glabrous panicle. Bracts and
a source for this micro-nutrient among indigent populations
bracteoles are inconspicuous and caduceus. Flowers are
who depend on the daily consumption of cassava as an
monoecious showing pistillate flowers with basal opening,
essential food. The root of this clone is creamy colored but
whereas the staminate apical opening occurs 3 weeks later.
turns yellow when cooked. Its palatability is excellent and
Fruits are green and winged and the roots are conical, with a
almost free of fibers.
rough, pink-brownish surface. The root flesh is slightly red

The retention time in HPLC system, and the similarity of
and turns dark red after cooking Fig. (1b).
the spectrogram profile 300-600nm in photo-diode array of

The chromatogram profiles of lycopene isolated from
0.98 Fig. (3) confirms the presence of lycopene in this
tomato and the cassava clone extract are shown in Fig. (2a &
cassava clone.
b), respectively. Lycopene was shown to be the major

This research provides a means of better understanding
carotenoid, although -carotene and cis-lycopene were also
cassava domestication and further breeding by indigenous
found. The identification and characterization of the peaks
Amazon farmers. The clone UNB-400 is grown in the
are given in Table 1. Other carotenoids could not be detected
Amazon, and from there it was brought to the State of São
in the cassava clone, which showed a concentration of 5 mg
Paulo, where it was further grown by a few farmers. This
lycopene per gram of wet root weight. Fig. (3a & b) shows
clone could have originated from a gene mutation that breaks
the spectrogram profile (range 300-600nm) of peak referent
the sequence of -carotene formation, and then adopted by
to lycopene isolated from tomato and of cassava clone roots,
Amazon farmers who used it probably for rituals or cultural
respectively. Carotenoids can be encapsulated through novel
ceremonies. This clone forms few roots compared to other
proprietary methods such as the one included in the patent by
improved cultivars. However, increasing its root yield
Barenholz et al. [9].
appears feasible by crossing with another clone possessing
Table 1. Quantification (ug/g of Tissue) of Lutein, trans- -
high combining ability for root yield. A patent
Carotene and cis- -Carotene of some Manihot
(US200874322873) on a carotenoid ketolase mutant was
Cultivar Organs
submitted by Stead et al. [10].

Lycopene occurs in tomato, guava, watermelon, and pink
Lutein
trans- -carotene
cis- -carotene
grapefruit and its consumption appears to be associated with
reduced degenerative diseases. Formulations of lycopene and
Roots

whey protein are used for the treatment of atherosclerotic
Pohlii
-
0.16
0.09
disorder [11]. Other potential human health benefits include
a possible role in the fight against digestive tract, breast,
UnB 400
236.83
1.24
0.96
lung, stomach and prostate cancers [8, 12-15]. King et al.
indicated the carotenoid and analogs are medically suitable
ICB 300
-
0.19
0.12
for the treatment of prostate cancer [16]. Epidemiological
Leaves
studies have shown that high intake of vegetables containing
lycopene is inversely associated with the incidence of certain
Pohlii
782.15 13.85
2.37
types of cancer. For example, regular intake of tomato
UnB 400
3081.69
24.12
3.28
products, with high lycopen content [17], has been inversely
associated with the risk of cancer of the digestive tract.
ICB 300
9108.98
18.02
1.88
Lycopene is a precursor of -carotene, whose synthesis

includes an enzymatic cycle in the chain-end [18]. The high
lycopene level found in this cassava clone may indicate a

The retention time in HPLC system and the similarity of
disruption in the biosynthesis of -carotene. The lycopene
the spectral profile 300-600 nm in photo-diode array of 0.98
accumulation in this cultivar may therefore be the result of a
confirm the presence of lycopene in this cassava clone.
deficiency in -carotene synthesis due to a mutation. UnB
Trans- -carotene reached 27.40 g/g in UnB 400, making it
400 can be considered a good source for -carotene in







Fig. (1). Cooked roots of clone UnB 400 (A) and UnB 500 (B).

Potential of Patents in Cassava Carotenoids and Amino-Acids
Recent Patents on Food, Nutrition & Agriculture, 2009, Vol. 1, No. 1 35











Fig. (2). Chromatogram profile of (A) lycopene and (B) cassava clone, showing peaks of trans- -carotene, lycopene and cis lycopene. HPLC
analysis conditions: RP column C18 Vydac 218TP54 column 250x4.6 mm, mobile phase 100% MeOH, flow 1.0 ml/min.











Fig. (3). Spectrogram profile (range 300-600nm) of peak referent to lycopene (A) isolated from tomato and (B) of cassava clone roots,
showing a similarity of 0.98. RP column C18 Vydac 218TP54 column 250 x 4.6 mm, mobile phase 100% MeOH, flow 1.0 ml/min.
regions where cassava is the day-to-day principal food. -
simple margins and green reddish petioles. Young foliage at
carotene-like lycopene is an antioxidant, besides being a
apices is green. Inflorescence is a 10 to 20 cm panicle
precursor of vitamin A, which is important in protecting
glabrous, with bracts and bracteoles inconspicuous and
against eye and skin diseases [19, 20]. Chomczynski has
caducous. Flowers are monoecious, pistillate, 2 to 3 based,
elaborated further on the significance of antioxidant dietary
open 3 weeks earlier than staminate ones. Perianth has 5
supplement for maintaining healthy skin [21].
separate tepals, and the yellow pistillate ovary has a ovary
sublended by a non-lobed yellow disk, 3 carpellate and
AMINO ACID PROFILES IN CASSAVA INTER-
glabrous. Fruit is winged and smooth surfaced. Seeds are
SPECIFIC HYBRID AND DERIVED OFFSPRING
caranculate elongate and brown.

ICB 300 derives from the wild species M. oligantha,

Among the six samples analyzed in this study Table 2,
which contains 4% crude protein [22]. This inter-specific
Fig. (4), the sample of the interspecific hybrid ICB 300
hybrid shows a shrub with 2 to3 m height, has conical roots
showed the highest amount of protein (1.654 g/100 g sample
of 40 to 60 cm length with a 3 to 4 cm neck, and they are
powder), followed by Diploide (1.454 g/100 g) and Progeny
abundant (5-10 kg per plant). Root surface color is dark
9 (0.922 g/100 g). Progeny 10, Progeny No. 4 and UnB 01
brown, whereas the root flesh is white. This hybrid possesses
showed poor protein content (0.350 g/100 g). The levels of
a brown story, and a 1-1.2 m height story. Scars on stem are
essential amino acids were also higher in inter-specific
moderately raised. Stems are 3-branched. Leaves have 3
hybrid ICB 300 (His, Leu, Lys, Met, Phe, and Val) and
lobes, occasionally 1 or 4 lobes. Leaf lobe is obovate, with
Diploide (Ile and Thr), with low or undetectable amounts in

36 Recent Patents on Food, Nutrition & Agriculture, 2009, Vol. 1, No. 1
Ortiz et al.
Table 2.
Amino Acid (AA) Profile (g per 100g Sample Mass) in Peeled Roots of Cassava Cultivar UnB, its Inter-Specific Hybrid
with Manihot oligantha ICB 300 (Sample 3 and Diplóide), and ICB 300-Derived Offspring (Progeny 4, Progeny 9 and Progeny 10)
AA
UnB 01
ICB 300 Sample 3
Progeny 10
Progeny 4
Progeny 9
ICB 300 Diplóide
Ala
0.020 0.093 0.017 0.019 0.040 0.098
Arg
0.037 0.261 0.061 0.082 0.320 0.108
Asp
0.016 0.146 0.023 0.033 0.052 0.137
Cys
0.027 0.029 0.026 0.025 0.026 0.025
Glu
0.039 0.222 0.044 0.065 0.151 0.221
Gly
0.012 0.078 0.012 0.015 0.037 0.075
His
0.000 0.038 0.010 0.010 0.027 0.036
Ile
0.008 0.068 0.010 0.010 0.018 0.069
Leu
0.016 0.131 0.013 0.000 0.041 0.127
Lys
0.010 0.098 0.020 0.019 0.034 0.079
Met
0.014 0.041 0.004 0.000 0.019 0.037
Phe
0.016 0.129 0.058 0.000 0.065 0.120
Pro
0.000 0.054 0.000 0.000 0.000 0.066
Ser
0.012 0.088 0.013 0.018 0.033 0.078
Thr
0.008 0.061 0.007 0.013 0.022 0.066
Tyr
0.000 0.000 0.000 0.000 0.000 0.000
Val
0.019 0.115 0.027 0.025 0.039 0.112
Total
0.254 1.654 0.344 0.336 0.922 1.454
the other materials. This results shows that inter-specific
the percentage of nitrogen by 3-fold. Hence, it is important
hybridizations provides materials that could be more
to determine protein content as amino acids jointly with its
interesting sources for breeding nutritious cassava for human
assessment as total nitrogen to ensure right screening of
consumption. Furthermore, Progeny 9 showed an equal
Manihot germplasm [26].
amount of protein as its inter-specific hybrid parent (ICB
CURRENT & FUTURE DEVELOPMENTS
300), i.e., doubling that of common cassava, thereby
indicating high heritability of this trait and the possibility of

Cassava cultivars UNB 120, UNB 122, UNB 123 and
selecting for high-protein cassava.
ICB 300 - all with average tuberous root yields above 15 kg

The results of amino acids profiles of cassava roots agree
plant 1 - are available to farmers in Brazil through cuttings
with those from the available literature. For example, Bailey
from the Univ. de Brasilia. They show either yellow or
[23] indicated a deficiency in sulfur-containing amino acids
orange flesh due to their carotenoid or lycopene content,
(methionine, cystine and cysteine), whereas Osuntokun et al,
respectively. Furthermore, the Government of Brazil plans to
[24] pointed out that both cysteine and cystine are involved
enact a law that will obligate mixing wheat flour with about
in cyanide detoxification. Cyanide is produced when the
20% of cassava flour for making bread. In this regard,
cyanogenic glucoside linamarin, present in cassava, is
cassava cultivars rich in essential amino acids and caro-
hydrolyzed by linamarinase or by acid. Cyanide is mainly
tenoids will be important for enhancing the nutritional
detoxicated by conversion to thiocyanate, in the process of
quality of this new bread with cassava and wheat flours.
which it reacts with cysteine and cystine. Excessive
CONFLICT OF INTEREST
detoxification may be responsible for the low concentration
of sulfur-containing amino acids [24]. In this regard, cassava

The first author acknowledge funding provided by the
with high-nitrogenous content could be bitter cultivars with
National Council for Scientific Development-CNPq, Brasilia
high-glucoside content [25]. Humidity could also influence
for undertaking research to enhance nutritional quality of
protein assessment as total nitrogen in cassava edible roots.
cassava.
Excessive drying of the root powder may increase drastically


Potential of Patents in Cassava Carotenoids and Amino-Acids
Recent Patents on Food, Nutrition & Agriculture, 2009, Vol. 1, No. 1 37





















Fig. (4). Morphology of interspecific hybrid ICB 300.
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