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Quinoa (Chenopodium quinoa, Willd.) as a source of dietary fiber and other functional components

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Four varieties of an Andean indigenous crop, quinoa (Chenopodium quinoa Willd.), were evaluated as a source of dietary fiber, phenolic compounds and antioxidant activity. The crops were processed by extrusion-cooking and the final products were analyzed to determine the dietary fiber, total polyphenols, radical scavenging activity, and in vitro digestibility of starch and protein. There were no significant differences in the contents of total dietary fiber between varieties of quinoa. In all cases, the contents of total and insoluble dietary fiber decreased during the extrusion process. At the same time, the content of soluble dietary fiber increased. The content of total phenolic compounds and the radical scavenging activity increased during the extrusion process in the case of all 4 varieties. There were significant differences between the varieties and the content of total polyphenols. The in vitro protein digestibility of quinoa varieties was between 76.3 and 80.5% and the in vitro starch digestibility was between 65.1 and 68.7%. Our study demonstrates that quinoa can be considered a good source of dietary fiber, polyphenols and other antioxidant compounds and that extrusion improves the nutritional value.
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Ciência e Tecnologia de Alimentos ISSN 0101-2061
Recebido para publicação em 2/6/2009
Aceito para publicação em 25/9/2009 (004229)
1
Universidad Nacional Agraria La Molina (Lima), Lima, Peru, E-mail: ritva@lamolina.edu.pe
*A quem a correspondência deve ser enviada
Quinoa (Chenopodium quinoa, Willd.) as a source of dietary ber
and other functional components
Quinoa (Chenopodium quinoa, Willd.) como fonte de bra alimentar e de outros componentes funcionais
Ritva Ann-Mari REPOCARRASCOVALENCIA
1
*, Lesli Astuhuaman SERNA
1
1 Introduction
Quinoa (Chenopodium quinoa Willd.) is a crop used by
pre-Columbian cultures in South America for centuries. ere
is a long history of safe use of the grain in South America.
Cultivated and collected Chenopodium species have been part
of the Tiahuanacotan and Incan cultures. Quinoa has fullled
various roles in these ancestral cultures, in addition to its role in
human and animal nutrition, quinoa had a sacred importance
(BONIFACIO, 2003). Archaeological studies have shown that
quinoa was already known in 5000 B.C. (TAPIAetal., 1979). For
the Incas, quinoa was a very important crop together with corn
and potato. Quinoa is currently grown for its grain in the South
American countries of Peru, Bolivia, Ecuador, Argentina, Chile
and Colombia. e plant is cold and drought tolerant and it can
be cultivated in high altitudes in the mountain areas. Quinoa
can be grown in a wide range of pH of the soil, including acidic
soils, and it can tolerate poor and rough environments. is crop
grows perfectly at high altitudes, where it is not possible to grow
maize, and it matures in 4 to 7 months, depending on the variety
or ecotype (CARMEN, 1984). e genetic variability of quinoa is
great, with cultivars of quinoa being adapted to growth from sea
level to an altitude of 4,000 m, from 40° S to 2° N latitude, and
from the cold highland climate to subtropical conditions. is
makes it possible to select, adapt, and breed cultivars for a wide
range of environmental conditions, providing basic nutrition
in demanding environmental conditions (JACOBSEN, 2003).
Quinoa is usually referred to as a pseudo-cereal since it is
not a member of the Gramineae family, but it produces seeds
that can be milled in to our and used as a cereal crop. It is an
annual dicotyledonous plant usually standing 0.5-2.0 m high
Resumo
Quatro variedades de quinoa (Chenopodium quinoa Willd.), cultura de origem andina, foram avaliados como fonte de bra dietética, de
compostos fenólicos e atividade antioxidante. As quinoas foram processadas por extrusão e os produtos nais foram analisados para determinar
a bra alimentar, o total de polifenóis, atividade de ligar os radicais livres e digestibilidade in vitro do amido e proteínas. Não houve diferença
signicativa no conteúdo de bra dietética total entre as variedades de quinoa. Em todos os casos, o teor de bra alimentar insolúvel e total
diminuiu durante o processo de extrusão. Ao mesmo tempo, o teor de bra alimentar solúvel teve um incremento. O teor de compostos
fenólicos totais e a atividade de ligar os radicais livres foram aumentados durante o processo de extrusão, no caso das quatro variedades.
Houve diferenças signicativas entre o conteúdo total de polifenóis por variedades. A digestibilidade proteica in vitro das variedades de quinoa
cou entre 76,3 e 80,5%, e a digestibilidade in vitro do amido situou-se entre 65,1 e 68,7%. Nosso estudo demonstra que a quinoa pode ser
considerada como uma boa fonte de bra dietética, polifenóis e outros compostos antioxidantes e que o processo de extrusão – cocção pode
melhorar o valor nutricional dos grãos.
Palavras-chave: quinoa; Chenopodium quinoa Willd.; bra dietética; componentes funcionais; processo de extrusão – cocção.
Abstract
Four varieties of an Andean indigenous crop, quinoa (Chenopodium quinoa Willd.), were evaluated as a source of dietary ber, phenolic
compounds and antioxidant activity. e crops were processed by extrusion-cooking and the nal products were analyzed to determine the
dietary ber, total polyphenols, radical scavenging activity, and in vitro digestibility of starch and protein. ere were no signicant dierences
in the contents of total dietary ber between varieties of quinoa. In all cases, the contents of total and insoluble dietary ber decreased during
the extrusion process. At the same time, the content of soluble dietary ber increased. e content of total phenolic compounds and the
radical scavenging activity increased during the extrusion process in the case of all 4 varieties. ere were signicant dierences between the
varieties and the content of total polyphenols. e in vitro protein digestibility of quinoa varieties was between 76.3 and 80.5% and the in
vitro starch digestibility was between 65.1 and 68.7%. Our study demonstrates that quinoa can be considered a good source of dietary ber,
polyphenols and other antioxidant compounds and that extrusion improves the nutritional value.
Keywords: quinoa; Chenopodium quinoa Willd.; dietary ber; bioactive compounds; extrusion.
225
Original
Ciênc. Tecnol. Aliment., Campinas, 31(1): 225-230, jan.-mar. 2011
Quinoa as a source of dietary fiber
GORINSTEINetal., 2007; NSIMBA; KIKUZAKI; KONISHI,
2008). ese investigations have shown that quinoa is a very
good source of antioxidants and it can be a substitute for
common cereals. However, we did not nd any reports on the
content of phenolic compounds and antioxidant activity of
processed quinoa. us, the aim of this study was to analyze the
content of dietary ber, phenolic compounds, and antioxidant
activity in 4 varieties of raw and extruded quinoa.
2 Materials and methods
2.1 Raw material
The following 4 varieties of quinoa (Chenopodium
quinoa, Willd.) were used in this study: ´La Molina 89´, of
the leguminous and cereal program of National Agrarian
University La Molina, Lima, Peru; ´Kcancolla´, ‘Blanca de Juli´
and ´Sajama´ from the Agronomical Experimentation Center
of Altiplano University, Puno, Peru.
2.2 Preparation of raw material
Quinoa was washed for 20 minutes with tap water with the
aim to eliminate bitter taste and toxic saponins. Washed grains
were dried at 45 °C for 12 hours. Dried seeds were packed in
polyethylene bags and stored at 4 °C until they used in analysis
and processing.
2.3 Extrusion
Washed and dried quinoa seeds were moistened to 12%
humidity for the extrusion process. e extrusion process
was carried out using a low-cost extruder simulating local
processing conditions (Jarcon del Peru, Huancayo, Peru).
e single-screw extruder was operated with the following
parameters: 389.4 rpm, 10-13 seconds resident time, 200 °C
work temperature, 2 orices on the die. No external heat was
transferred to the barrel on the screw during extrusion. e aim
was to produce results applicable to local conditions where this
technology is widely used.
2.4 Extraction of polyphenols and radical scavenging
analysis
All samples were ground through a Foss cyclotec mill
before extraction. Five grams of milled quinoa or extrudate
were mixed with 25mL methanol and homogenized using the
Ultra-Turrax homogenizer. e homogenates were allowed
to stand for 12-24 hours under refrigeration (4 °C) and then
centrifuged for 15 minutes. e supernatant was recovered and
stored until analysis.
2.5 Analysis
Proximate analysis. Water content, proteins (N × 6.25),
fat, crude ber and ash were determined according to AOAC
(ASSOCIATION..., 1995). The carbohydrates (CHO) were
calculated by dierence, using the Equation 1:
(
)
100 CHO fat protein crude fiber ash=−+ + +
(1)
with large panicles of 1.8-2.2 mm long seeds produced at the
end of the stem. e seed is usually pale yellow, but it may
vary from almost white through pink, orange or red to brown
and black. e embryo can hold 60% of the seed weight and it
forms a ring around the endosperm that loosens when the seed
is cooked (NATIONAL RESEARCH COUNCIL, 1989). Grain
yields vary from 1 to 3 t.ha
–1
, depending on the variety and the
level of cultivation technology (CARMEN, 1984).
Most of the varieties of quinoa contain saponins, bitter-
tasting triterpenoid glycosides, which are concentrated in the
seed coat and must be removed before consumption. e most
popular method for removing saponins involves washing the
grains with water in the ratio of 1:8 quinoa:water, although
there are several other traditional methods (ANTUNEZ DE
MAYOLO, 1981). Large-scale commercial methods haven been
developed in Peru and Bolivia.
Quinoa is one of the most nutritive grains used as human
food and it has been selected by FAO as one of the crops
destined to oer food security in this century (FOOD..., 1998).
Its protein content is remarkable and the composition of the
essential amino acids is excellent. e nutritional value of quinoa
protein is comparable to that of milk protein (KOZIOL, 1992;
RANHOTRAetal., 1993). e content of lysine, methionine
and cysteine in quinoa is higher than in common cereals and
legumes, making it complementary to these crops. Quinoa is
rich in oil, containing benecial fatty acids and a high content
of tocopherols (REPO-CARRASCO-VALÊNCIA; ESPINOZA;
JACOBSEN, 2003). Based on the high quality of the oil, and
on the fact that some varieties show oil concentrations of up to
9.5%, quinoa could be considered as a potentially valuable new
oil crop (KOZIOL, 1992).
Actually, quinoa is widely used in many South American
countries, especially in Peru, Bolivia, Ecuador, Chile, and
Argentina. In Peru, the population of Lima is aware of the
nutritive qualities of quinoa and other Andean crops. ey
consume quinoa once a day on average. In rural areas of
southern Peru, the population is accustomed to eating quinoa
every day in dierent preparations (AYALA, 2003).
Quinoa grains do not contain gluten and thus, they
cannot be used alone for bread- making. However, they can
be mixed with wheat our in the preparation of bread with
high nutritional value (MORITAetal., 2001). e fact that
quinoa contains no gluten, makes it an interesting ingredient
for the diets of persons who have celiac disease. Quinoa our
is commonly used in infant foods. Flakes, similar to oat akes,
have also been prepared from quinoa. Pued grains of quinoa
are produced commercially in Peru and Bolivia. e plant is
sometimes grown as a green vegetable and its leaves are eaten
fresh or cooked (NATIONAL RESEARCH COUNCIL, 1989).
e saponins obtained as a by-product in the processing of
quinoa can be utilized by the cosmetics and pharmaceutical
industries (TAPIAetal., 1979).
Cereals are commonly known as good sources of dietary
ber, phenolic compounds, and natural antioxidants. Some
studies on dietary ber, phenolic compounds, and antioxidant
activity of quinoa have been published (RUALES; NAIR, 1994;
Ciênc. Tecnol. Aliment., Campinas, 31(1): 225-230, jan.-mar. 2011
226
Repo-Carrasco-Valencia; Serna
Guzman-Maldonado and Paredes-Lopez (1998). According
to these authors, the protein content of quinoa is between
11.0and15.0%. ere were dierences between the 4 varieties
of quinoa in protein content, La Molina 89 having the highest
and Blanca de Juli the lowest protein contents. Extrusion aected
the protein content, decreasing it. La Molina 89 had the highest
crude fat content. e contents of moisture, ash, and crude ber
were reduced during the extrusion process in all varieties.
In Table 2, the contents of total (TDF), insoluble (IDF), and
soluble dietary ber (SDF) are presented for raw and extruded
quinoa varieties. ere were no signicant dierences in the
contents of TDF, IDF, and SDF between the varieties. In all cases,
the contents of total and insoluble dietary ber decreased during
the extrusion process, however, this decrease was signicant
only in the case of the Sajama variety. At the same time, the
content of soluble dietary ber increased during the extrusion
process. e increase of content of soluble dietary ber was
statistically signicant in the case of Blanca de Juli, Kcancolla,
Dietary fiber. The total, soluble and insoluble dietary
ber were determined by an enzymatic-gravimetric method
according to the Approved Method 32-21 (AMERICAN...,
1995) using the TDF-100 kit from Sigma Chemical Company
(St. Louis, MO, U.S.A.). Radical scavenging activity. Radical
scavenging activity was determined according to Reetal. (1999)
based on the decrease of absorbance at 734 nm produced by
reduction of ABTS (2,2´-azinobis-(3-ethylbenzothiazoline-6-
sulfonic acid)) by an antioxidant. Trolox was used as the
reference compound for calibration curve. e percentage
inhibition of absorbance at 734 nm was calculated using the
formula according to Katalinicetal. (2006), Equation 2:
( )
(0) ( )
(0)
% 100
C At
C
AA
inhibition
A




(2)
where A
C(0)
is the absorbance of the control at t = 0 minute and
A
A(t)
is the absorbance of the antioxidant at t = 16 minutes.
Total polyphenols. e content of total polyphenols was
analyzed according to the method of Swain and Hillis (1959).
e phenolic compounds were extracted with methanol and
the extract was allowed to react with the Folin- Ciocalteau
phenol reagent. e absorbance was measured at 725 nm.
Gallic acid equivalents (GAE) were determined from a standard
concentration curve.
Protein in vitro digestibility. Digestibility of proteins was
determined by an in vitro method according to Hsuetal. (1977).
e multi-enzymatic method is based on the decrease of pH
during 10 minutes. e percentage of digestibility was calculated
using the Equation 3:
210.464 18.103
YX
=−
(3)
where: X = pH of the protein suspension aer 10 minutes of
digestion and Y = percentage of protein hydrolysis.
Starch in vitro digestibility. e digestibility of starch was
determined by an in vitro method according to Holmetal.
(1985). Starch (500 mg) was mixed with phosphate buer
(pH6.9) and incubated with α-amylase at 37 °C for 1 hour. e
sugars released were determined by spectrophotometry.
Degree of gelatinization was based on the method of
Wooton; Weeden and Munk (1971).
2.6 Statistical analysis
Each analysis was done at least in duplicate and expressed as
means and standard deviation (SD). e data were analyzed by
analysis of variance and Tukey´s test (signicance of dierences
p < 0.05) was used to nd signicant dierences between the
samples and treatments.
3 Results and Discussion
The results of analysis of the proximate composition
of 4 quinoa varieties and their extrudates are presented in
Table 1. en, protein content of the grain of the 4 varieties
was between 14.0 and 15.5%. is coincides with values by
Table 1. Proximate composition of 4 quinoa varieties (% dry basis).
Component Blanca de
Juli
Kcancolla La Molina
89
Sajama
Raw
Moisture 11.39 10.78 12.03 12.62
Ash 3.38 3.52 5.46 3.04
Protein 13.96 15.17 15.47 14.53
Crude fat 5.51 5.77 6.85 4.69
Crude ber 2.00 3.07 3.38 1.92
Carbohydrates 75.15 72.47 68.84 75.82
Extruded
Moisture 7.46 8.98 7.31 7.58
Ash 2.61 2.61 2.45 2.55
Protein 13.41 14.19 15.45 14.08
Crude fat 3.48 3.75 4.21 4.13
Crude ber 1.64 2.60 2.13 1.62
Carbohydrates 78.86 76.85 75.76 77.62
All data are the means of 2 replicates. All contents g.100 g
–1
dry weight except moisture
g.100 g
–1
fresh weight.
Table 2. Total, insoluble, and soluble dietary ber content in 4 quinoa
varieties, raw and extruded, g.100 g
–1
dry basis.
Variety TDF IDF SDF
Raw
Blanca de Juli 13.72 ± 1.63
a,x
12.18 ± 1.65
a,x
1.54 ± 0.01
a,y
Kcancolla 14.11 ± 1.02
a,x
12.70 ± 1.15
a,x
1.41 ± 0.13
a,y
La Molina 89 15.99 ± 0.63
a,x
14.39 ± 0.81
a,x
1.60 ± 0.18
a,y
Sajama 13.56 ± 0.23
a,x
11.99 ± 0.28
a,x
1.58 ± 0.05
a,x
Extruded
Blanca de Juli 10.77 ± 0.32
b,x
8.64 ± 0.17
b,x
2.13 ± 0.15
a,x
Kcancolla 12.52 ± 0.64
b,x
10.13 ± 0.88
b,x
2.39 ± 0.23
a,x
La Molina 89 15.27 ± 0.49
a,x
12.54 ± 0.59
a,x
2.73 ± 0.10
a,x
Sajama 11.64 ± 0.30
b,y
9.38 ± 0.71
b,y
2.26 ± 0.41
a,x
TDF = total dietary ber, IDF = insoluble dietary ber, SDF = soluble dietary ber. All
data are the means ± SD of three replicates.
a-d
Varieties are compared. Means in the
same row followed by same letter are not signicantly dierent (p < 0.05).
x,y
Raw and
extruded quinoa are compared. Means in the same column followed by same letter are
not signicantly dierent (p < ±0.05).
Ciênc. Tecnol. Aliment., Campinas, 31(1): 225-230, jan.-mar. 2011
227
Quinoa as a source of dietary fiber
from 1.43 to 1.97 mg.GAE.g
–1
. Paskoetal. (2009) dened the
content of total polyphenols in quinoa to be 3.75 mg.GAE.g
–1
by
using a 2-step extraction process, rst with methanol and then
with acetone. As we used methanol only, some polyphenols may
not have been included in the extract.
Figure 1 presents the radical scavenging activities of raw and
extruded quinoa. ese increased during the extrusion process.
e nding is in agreement with the report of Dewanto, Wu and
Hai Liu (2002) who discovered that the antioxidant activity and
the content of total phenolics of sweet corn increased during
thermal processing. Increase of the total antioxidant activity
in processed grains could be explained by the increase of
soluble phenolic compounds released by thermal processing. In
cereals, the phenolic acids are in free, esteried and insoluble
bound forms. Dewanto, Wu and Hai Liu (2002) found that heat
treatment increased the free and conjugated ferulic acid contents
in sweet corn due to the release of bound ferulic acid across both
the heating time and heating temperature parameters.
Xu and Chang (2008) studied the effect of thermal
processing on total phenolics and specic phenolic compounds
in yellow and black soybeans. ey found that the content of
phenolics increased in yellow varieties and decreased in black
varieties during the heat treatment. In yellow soybean varieties,
thermal processing caused more free gallic acid to be released
leading to higher total phenolic content and antioxidant activity
compared to raw beans.
e values of degree of gelatinization (DG), as well as the in
vitro digestibility of starch and protein are presented in Table4.
DG of the 4 quinoa varieties was between 79.9and89.0%. ese
values are lower than those found by Ruales and Nair (1994)
for drum-dried (96%) and cooked (97%) quinoa samples, but
higher than for the autoclaved (27%) samples. Dogan and
Karwe (2003) investigated the physicochemical properties
of quinoa extrudates. ey found that the starch was only
partially gelatinized with a maximum of 84.4%, depending on
the extrusion conditions (feed moisture, screw speed and die
temperature). During extrusion-cooking, both temperature and
shear are responsible for starch gelatinization.
Ruales and Nair (1994) reported the in vitro digestibility
of starch in raw, autoclaved, cooked, and drum-dried quinoa.
eir values for raw, autoclaved and cooked quinoa were lower
than ours (22, 32 and 45%, respectively), but the value for drum-
dried quinoa was higher (73%). As the starch granules of quinoa
are surrounded by a protein matrix, they are not very easily
and La Molina 89 varieties. Gualbertoetal. (1997) also found a
decrease in the content of insoluble dietary ber and an increase
in the content of soluble ber during extrusion-cooking. is
could be due to shear stress caused by high screw speed and
also to high temperature. e exposure to shear stress and high
temperature causes chemical bond breakage creating smaller
particles, which are soluble. ere is a transformation of some
insoluble ber components into soluble ber during extrusion.
Rinaldi; NG and Bennink (2000) studied the eect of extrusion
on dietary ber of wheat extrudates enriched with wet okara
and his results coincide with ours. Extrusion of the formulations
resulted in decreased insoluble ber and increased soluble ber
contents of the products. Extrusion-cooking of white heat our
has also been found to cause a redistribution of insoluble to
soluble dietary ber (BJORCK; NYMAN; ASP, 1984).
e extrusion-cooking process was investigated by Lue,
Hsieh and Hu (1991) with the expectancy that mechanic
rupture of the glycosidic bonds would lead to an increase of
soluble ber. In some cases, an increase of insoluble ber was
observed (UNLU; FALLER, 1998). Espositoetal. (2005) studied
the eect of extrusion on dietary ber of durum wheat. e data
showed that the extrusion-cooking process did not have an eect
on the amount of soluble dietary ber, independently from the
ber typology of the dierent samples. is dierence in ber
solubilization during processing could be explained by the
variability in the raw material composition, but also by dierent
experimental conditions, for example, screw share forces and
pressure in extrusion. The high mechanical stress during
extrusion may cause breakdown of polysaccharide glycosidic
bonds releasing oligosaccharides and, therefore, end up with
an increase of soluble dietary ber (ESPOSITOetal., 2005).
Ruales and Nair (1994) determined the contents of dietary
ber in raw and processed quinoa samples. ey found 13.4%
of total dietary ber for raw quinoa. is value is comparable to
our values for Blanca de Juli and Sajama varieties. e content of
total dietary ber was decreased only in cooked quinoa, while
in autoclaved and drum-dried samples it remained the same.
Some soluble ber was lost during cooking, and in autoclaved
samples, it was lost probably due to depolymerization of ber
components.
Content of the total phenolic compounds and the radical
scavenging activity increased during the extrusion process in the
case of all 4 varieties (Table 3.). ere were signicant dierences
between the varieties and the contents of total polyphenols. e
contents of total polyphenols in the 4 quinoa varieties ranged
Table 3. Total polyphenols and radical scavenging activity of 4 varieties of quinoa, raw and extruded.
Variety Raw quinoa total
polyphenols mg
GAE/g d.b.*
Extruded quinoa total
phenolics mg gallic
acid/g d.b.*
Raw quinoa radical
scavenging activity microg
trolox/g sample**
Extruded quinoa radical
scavenging activity microg
trolox/g sample**
Blanca de Juli 1.42 ± 0.5
c,y
1.70 ± 0.1
c,x
2351.9
3960.8
Kcancolla 1.57 ± 0.3
b,y
1.82 ± 0.4
b,x
2389.9
4095.4
La Molina 89 1.97 ± 0.2
a,y
3.28 ± 0.3
a,x
3689.5
4165.6
Sajama 1.63 ± 0.1
b,x
1.66 ± 0.2
c,x
2440.3
4118.8
*Data are the means ± SD of 3 replicates. **Data are means of duplicate analyses.
a-d
Varieties are compared. Means in the same column followed by same letter are not signicantly
dierent (p < 0.05).
x,y
Raw and extruded quinoa are compared. Means in the same row followed by same letter are not signicantly dierent (p < 0.05). d.b. = dry basis.
Ciênc. Tecnol. Aliment., Campinas, 31(1): 225-230, jan.-mar. 2011
228
Repo-Carrasco-Valencia; Serna
should be conducted to characterize the phenolic compound
composition and the antioxidant content and activity, especially
of colored varieties of quinoa.
Acknowledgements
We thank Dr. Seppo Salminen and Dr. Heikki Kallio,
Department of Biochemistry and Food Chemistry, University of
Turku, Finland, for their critical reading and helpful comments
on the manuscript. Financing from CONCYTEC (Concejo
Nacional de Ciencia, Tecnologia e Innovación Tecnológica,
Peru) is gratefully acknowledged.
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4 Conclusions
Altogether, this study demonstrates that quinoa can be
considered a very nutritive cereal when compared to commonly
consumed cereals such as wheat, barley, and corn. It has a
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provide a new ingredient for specic foods for particular target
populations with potential health benets. us, further studies
Figure 1. Radical scavenging activity of raw and extruded quinoa
e results given here are means values of two separate experiments.
Table 4. Degree of gelatinization of starch, in vitro digestibility of starch
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Variety Degree of
gelatinization%
In vitro
digestibility
of starch%
In vitro
digestibility
of protein%
Blanca de Juli 79.85 68.53 80.54
Kcancolla 86.15 65.11 79.34
La Molina 89 86.74 68.42 76.80
Sajama 89.02 68.69 76.32
All data are the means of two replicates.
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230
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