LWT - Food Science and Technology 62 (2015) 1226e1234

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LWT - Food Science and Technology

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Bread enriched with Chenopodium quinoa leaves powder e The

procedures for assessing the fortification efficiency


Urszula Gawlik-Dziki a, *, Dariusz Dziki b, Michał Swieca a
, Łukasz Se˛ czyk a,

zyło
Renata Ro _ c
, Urszula Szymanowska a
a
Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland
b
Department of Thermal Technology, University of Life Sciences, Do
swiadczalna Str. 44, 20-280, Lublin, Poland
c
Department of Equipment Operation and Maintenance in Food Industry, University of Life Sciences, Do
swiadczalna Str. 44, 20-280 Lublin, Poland

a r t i c l e i n f o a b s t r a c t

Article history: The objectives of the study were to investigate the effect of fortification with ground Chenopodium quinoa
Received 22 February 2014 leaves (QL) on the sensory value and nutraceutical potential of breads and to compare an in vitro
Received in revised form physiological and chemical procedure for determination their quality. By QL addition, breads were
31 December 2014
mainly enriched with rutin and gallic acid. The highest content of rutin was found in buffer extracts (from
Accepted 8 February 2015
Available online 18 February 2015
73.55 mg/g DM to 209.89 mg/g DM depending from QL addition), whereas the highest gallic acid content
was observed after simulated digestion (from 1527.81 mg/g DM (control bread) to 2214.60 (bread with
5 g/100 g QL addition)). Antioxidant potential of enriched breads was significantly higher than activity of
Keywords:
Antioxidant activity
control. Breads were especially fortificated with bioaccessible lipids preventers and reductive
Bioaccessibility in vitro compounds. Essential differences between both used procedures were illustrated by extractability and
Chenopodium quinoa fortification factors. Chemical extraction overestimates the ability to chelate metal ions, while
Enriched bread underestimates the ability to lipids prevention and reducing power of potentially bioaccessible and
Functional food mastication-extractable compounds. The sensory evaluation, antioxidant tests, extractability and
fortification factors showed that the replacements of wheat flour with up to 3 g/100 g QL powder in
breads gives satisfactory results. Additionally, we propose the universal protocol for preliminary
evaluation of efficiency of fortificated food.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction Thus, today consumers prefer to eat healthier foods in order to

The unbalanced diet usually goes hand in hand with the way of
life leading to the prevalence of oxidative stress. Under these
conditions, reactive oxygen species (ROS) can oxidize lipids,
proteins and nucleic acids, ultimately leading to cell death or


transformation (Cai, Luo Sun, & Corke, 2004; Gawlik-Dziki, Swieca


& Sugier, 2012; Swieca, _ 2013).
Gawlik-Dziki, Dziki, Baraniak, & Czyz,
Abbreviations: QL, quinoa leaves; QL1-QL5, bread breads with 1, 2, 3, 4 and 5 g/
100 g quinoa leaves addition, respectively; CE, chemical extracts; BE, raw (buffer)
extracts; GE, extracts after simulated digestion; AE, dialysate, extracts after simu-
lated absorption; TPC, total phenols content; GAE, gallic acid equivalent; EF,
extractability factor; BAC, the antioxidant bioaccessibility index; BAV, the antioxi-
dant bioavailability index; BEF, the antioxidant bioefficiency index; FF, the fortifi-
cation factors; AA, antiradical activity; RED, reducing power; CHEL, chelating
power; LPO, degree of inhibition of linoleic acid peroxidation.
* Corresponding author. Tel.: þ48 81 4623327; fax: þ48 81 4623324.
E-mail address: urszula.gawlik@up.lublin.pl (U. Gawlik-Dziki).
prevent non-communicable diseases. In the response to the market
needs researchers and industry are involved in optimizing tech-
nologies to improve the nutritional and nutraceutical quality of


food (Balestra, Cocci, Pinnavaia, & Romani, 2011; Swieca, Baraniak,
& Gawlik-Dziki, 2013). At the epidemiological level, plant poly-
phenols have been suggested to reduce the risk of cardiovascular
diseases, neurodegenerative disorders, cancer and diabetes (Arts &
Hollman, 2005). Fortification with phenolics-rich ingredients is the
most commonly used for improving the antioxidant potential of
food and the resulting in followed improvement of the consumer
antioxidant status (Chlopicka, Pasko, Gorinstein, Jedryas, &
Zagrodzki, 2012; Fan, Zhang, Yu, & Ma, 2006; Gawlik-Dziki, Dziki,


Baraniak, & Lin, 2009; Gawlik-Dziki, Swieca, Dziki, et al., 2013; Lim,
Park, Ghafoor, Hwang, & Park, 2011). However, results of random-
ized trials looking at the possible preventive effect of dietary anti-
oxidant supplementation with one or more selected antioxidants
have been contradictory (Serrano, Goni, & Saura-Calixto, 2007).

http://dx.doi.org/10.1016/j.lwt.2015.02.007
0023-6438/© 2015 Elsevier Ltd. All rights reserved.
 U. Gawlik-Dziki et al. / LWT - Food Science and Technology 62 (2015) 1226e1234
The best vehicle for functional supplements, due to the wide-
spread consumption, are considered cereals food products (e.g.
bread), which in developed communities provide more than 50% of
the total energy intake (Akhtar, Anjum & Anjum, 2011). Somehow
wheat breads possess some antioxidant potential, its fortification is
reasonable due to deficit of antioxidants in the common diet. Since
now, there are some successful trials concerning improvement of
nutraceutical potential of breads by fortification (Chlopicka et al.,
2012; Fan et al., 2006; Gawlik-Dziki et al., 2009; Gawlik-Dziki,


Swieca, Dziki, et al., 2013; Lim et al., 2011).
In the number of countries (especially in Europe) there has been
growing interest initiating introduction and research work on
quinoa (Chenopodium quinoa Willd). In Asian, North and South
American countries, young quinoa leaves are ingested as vegetable,
however, like all members of the Amaranthaceae-Chenopodiaceae
plant family (e.g. spinach, chard), quinoa leaves does contain oxa-
lates. On the other hand, the risks associated with quinoa leaves
consumption are minimal, provided that the leaves are not eaten to
excess (Siener, Ho € now, Seidler, Voss, & Hesse, 2006).
Our previous observations concerning the chemical composi-
tion and bioactivity in vitro of quinoa leaves (QL) fill this gap and
open new perspectives for their introduction into the normal diet,
at least as supplements. The high bioavailability of bioactive QL
compounds, parallelled by their in vitro effects on prostate cancer
cells, suggests that they can help in the chemoprevention of cancer
and other diseases related to oxidative stress, such as atheroscle-

were used for dough preparation. The general quantity of water
rosis (Gawlik-Dziki, Swieca, Sułkowski, et al., 2013).
As far as selection of “vehicle” and supporting material poses no
major problems, from the consumer point of view the most sig-
nificant things, often overlooked in the design of fortified foods, are
the real value of the received nutraceutical foods, determined by
bioaccessibility and bioavailability of biologically active com-
pounds. The most of studies concerning bioactivity of foods deal
only with the antioxidant capacity extracted by chemical solvents
(normal antioxidant capacity assays are limited by the extraction
technique employed). But some antioxidants may remain in the
residues from organic extraction of foods. Therefore, the biological
properties of antioxidants may depend on their release from the
food matrix during the digestion process (bioaccessibility) and may
differ quantitatively and qualitatively from those produced by the
chemical extraction employed in most studies (Serrano et al.,
2007). In the light of this, the first criterion of determining bio-
accessibility is extractability of bioactives during in vivo conditions.
In vitro digestion models are widely used for studying structural
changes, digestibility, and release of food components (Oomen
et al., 2002).
Thus, the aim of this study was to test two hypotheses: 1) the
amount and bioactivity of chemically-extracted polyphenols and
other active compounds principally differ from these obtained in
gastrointestinal system action; 2) QL consist a valuable source for
bread fortification from both (sensory and nutraceutical properties)
points of view.
2. Materials and methods
2.1. Plant material
C. quinoa Willd, (Caryophyllales: Amaranthaceae) ‘Faro’ seeds
were cropped in Peru in 2011 and imported by Bio Planet S.A
(product specification QNR/20417). Plant material was harvested in
experimental farm of University of Life Sciences in Lublin, Poland
(voucher specimen was deposited in Department of Biochemistry
and Food Chemistry, University of Life Sciences, Lublin, No#CQ/BB/
0112). The soil was characterized by mean content of humus, very
low phosphorus, potassium and magnesium content, and was acid
1227
in reaction. During vegetation plants were weeded by hand three
times, and the inter-rows were cultivated. Leaves (QL) were har-
vested after 90 days of growth and dried at 50  C for 12 h in labour
oven. Dried material was powdered using laboratory mill (POLY-
MIX-Micro-Hammermill MFC, Kinematica. AG, Littau/Lucerne,
Switzerland) and sieved to 0.25 mm by using a laboratory screen
Thyr 2 (Saskia, Essen, Germany).
2.2. Chemicals
Ferrozine (3-(2-pyridyl)-5,6-bis-(4-phenyl-sulfonic acid)-1,2,4-
triazine), ABTS (2,20 -azino-bis (3-ethylbenzothiazoline-6-sul-
phonic acid)), a-amylase, pancreatin, pepsin, bile extract, Folin-
Ciocalteau reagent, linoleic acid and ammonium thiocyanate were
purchased from SigmaeAldrich company (Poznan, Poland). All
others chemicals were of analytical grade.
2.3. Bread making and sample preparation
The flour used in the formula of control bread (C) was wheat
bread flour (600 g), type 750 (average 0.75 g/100 g ash content,
humidity 14%). The flour was replaced with QL at 1e5 g/100 g levels
(QL1eQL5, respectively). The percentage of QL addition had been
chosen on the basis of the previous test on the sensory and anti-
oxidant activity. Beside of this 6 g of instant yeast and 12 g of salt
necessary for the preparation of the dough was established through
the marking of water absorption properties in the flour of consis-
tency of 350 Brabender units. The batches of dough, were mixed in
a spiral mixer for 6 min. After fermentation, the pieces of dough

(300 g) were put into the oven heat up to a temperature of 230 C.
The baking time was 30 min. The bread after baking was left to


stand for 24 h at room temperature (Gawlik-Dziki, Swieca, Dziki,
et al., 2013). The breads were sliced (slices about 1.5 cm thick).
The crust was removed aseptically and kept frozen (at 20  C) until
analysis. After thawing, the slices were dried and then manually
crumbed, grounded in a mill and screened through 0.5 mm sieve to
obtain bread powder.
2.4. Sensory evaluation
Sensory evaluation was carried out on bread samples with the
different percentages of QL according to Lim et al., 2011. Subse-
quently, the samples were sliced (slices about 1.5 cm thick), coded
with a number and served to untrained consumers. Attributes
recognized by the sensory panel as describing the sensory prop-
erties of the bread products were: crumb colour, aroma, texture and
taste.
The panel consisted of 35 consumers (22e50 years old; 20
women and 15 men), who evaluated the bread's overall accept-
ability. This hedonic test was used to determine the degree of
overall liking for the different types of bread based on degree of
liking or disliking according to a nine-point hedonic scale (1: dislike
extremely, 5: neither like nor dislike, 9: like extremely). Plain water
was used for mouth rinsing before and after each sample testing.
2.5. Extracts preparation
2.5.1. Buffer extracts (BE)
Powdered samples of breads (1 g) were extracted for 1 h with
20 mL of PBS buffer (phosphate buffered saline, pH 7.4). The ex-
tracts were separated by decantation and the residues were
extracted again with 20 mL of PBS buffer. Extracts were combined,
and stored in darkness at 20  C.
1228 U. Gawlik-Dziki et al. / LWT - Food Science and Technology 62 (2015) 1226e1234

2.5.2. In vitro digestion and absorption 2.7. Antioxidant activities

In vitro digestion and absorption were performed according to
Gawlik-Dziki et al.(2009). The bread samples (1 g) were homoge- Antiradical activity (AA) was determined using an improved
nized in a stomacher laboratory blender for 1 min to simulate ABTS decolorization assay (Re et al., 1999). Chelating power (CHEL)
mastication with the presence of 15 mL of simulated salivary fluid was determined by the method of Guo, Lee, Chiang, Lin, and Chang
(prepared by dissolving 2.38 g Na2HPO4, 0.19 g KH2PO4, and 8 g (2001). The degree of inhibition of linoleic acid peroxidation (LPO)
NaCl, 100 mg of mucin in 1 L of distilled water. The solution was was performed according to Kuo, Yeh and Pan (1999). All activities
adjusted to pH ¼ 6.75 and a-amylase (E.C. 3.2.1.1.) was added to were expressed as EC50 e extract concentration provided 50% of
obtain 200 U per mL of enzyme activity. For the gastric digestion activity based on dose-dependent mode of action. Reducing power
300 U/mL of pepsin (from porcine stomach mucosa, pepsin A, EC (RP) was determined using the method described by Oyaizu (1986).
3.4.23.1) in 0.03 mol/L NaCl, pH ¼ 1.2 was prepared. Further, EC50 value (mg/mL) is the effective concentration at which the
simulated intestinal juice was prepared by dissolving 0.05 g of absorbance was 0.5 for reducing power and was obtained by
pancreatin (activity equivalent 4  USP) and 0.3 g of bile extract in interpolation from linear regression analysis.
35 mL 0.1 mol/L NaHCO3), and subsequently, the samples were

shaken for 10 min at 37 C. The samples were adjusted to pH ¼ 1.2
using 5 mol/L HCl, and subsequently, 15 mL of simulated gastric 2.8. Theoretical approach

fluid was added. The samples were shaken for 120 min at 37 C.
After digestion with the gastric fluid, the samples were adjusted to The following factors were determined for better understanding
pH ¼ 6 with 0.1 mol/L of NaHCO3 and then 15 mL of a mixture of the relationships between biologically active compounds in the
bile extract and pancreatin was added. The extracts were adjusted light of their bioaccessibility, bioavailability, and bioefficiency:
to pH ¼ 7 with 1 mol/L NaOH and finally 5 mL of 120 mmol/L NaCl
and 5 mL of mmol/LKCl were added to each sample. The prepared - the extractability factor (EF), which indicating the efficiency of

samples were submitted for in vitro digestion for 60 min, at 37 C in extraction (compared to chemical extraction):
the darkness. After that samples were centrifuged and superna-
tants (extracts after simulated digestion, GE) were used for further For phenolic compounds:
analysis.
In vitro absorption. Considering that antioxidants absorption EFBE ¼ CBE =CCE
takes place mainly at the intestinal digestion stage, the resulting
mixture (fluids obtained after in vitro digestion) was transferred to EFGE ¼ CGE =CCE
the dialysis sacks (D9777-100FT, Sidma-Aldrich, Poznan, Poland),
placed in an Erlenmeyer flask containing 50 mL of PBS buffer and
 EFAE ¼ CAE =CCE
incubated in a rotary shaker (2 times per 2 h, 37 C). The PBS buffer
together with the compounds that passed through the membrane where, CCE is phenolics content in chemical extract, CBE phenolics
(dialysate, AE) was treated as an equivalent of the raw material content in of raw (buffer) extract, CGE is phenolics content in extract
absorbed in the intestine after digestion. after simulated gastrointestinal digestion, CAE is phenolics content
in extract after simulated absorption.
2.5.3. Methanolic extracts (chemical extracts; CE) For antioxidants:
Powdered samples (1 g) were extracted for 1 h with 25 mL of
methanol: water mixture (4:2, v/v). The extracts were separated by EFBE ¼ ACE =ABE
decantation and the residues were extracted again with 25 mL of
methanol: water mixture (1:1, v/v). Extracts were combined and
EFGE ¼ ACE =AGE
stored in darkness at 20  C.

2.6. Determination of phenolics EFA ¼ ACE =AAE

Total phenols content (TPC) were estimated according to the

- the antioxidant bioaccessibility index (BAC), which is an indi-
FolineCiocalteau method (Singleton & Rossi, 1965). The amount of
cation of the bioaccessibility of antioxidative compounds:
total phenolics was expressed as gallic acid equivalents (GAE).
Qualitative-quantitative analysis of main potentially
mastication-extractable, bioaccessible and bioavailable phenolic
compounds was performed according to Swieca,
Gawlik-Dziki, BAC ¼ ABE =AGE
Kowalczyk, and Złotek (2012). Samples were analysed with a Var-
ian ProStar HPLC System separation module (Varian, Palo Alto, CA,
USA) equipped with a Varian ChromSpher C18 reverse phase col-
umn (25 mm  4.6 mm) column and a ProStar DAD detector. The - the antioxidant bioavailability index (BAV) which is an indica-

column thermostat was set at 40 C. The mobilephase consisted of tion of the bioavailability of antioxidative compounds:
4.5:95.5 (v/v) acetic acid: water (solvent A) and 1:1 (v/v) acetoni-
trile: water (solvent B) and a flow rate of 0.8 mL/min. At the end of
the gradient, the column was washed with 1:1 (v/v) acetonitrile:
water and equilibrated to the initial condition for 10 min. Gradient BAV ¼ AGE =AAE
elution was used as follows: 0 min 92% A, 30 min 70% A, 45 min 60%,
80 min 61% A, 82 min 0% A, 85 min 0% A, 86 min 92% A, 90 min 92%
A. Detection was carried out at 270 and 370 nm. Quantitative de-
terminations were carried out with the external standard calcula- - the antioxidant bioefficiency index (BEF), which is an indication
tion, using calibration curves of the standard. of the bioactivity of bioavailable antioxidant compounds:
 U. Gawlik-Dziki et al. / LWT - Food Science and Technology 62 (2015) 1226e1234 1229

3.2. Phenolic compounds analysis

BEF ¼ ABE =AAE
where, ABE is activity (EC50) of raw (buffer) extract, AGE is activity
(EC50) of extract after simulated gastrointestinal digestion (GE), AAE
is activity (EC50)of extract after simulated intestinal absorption.
- the fortification factors (FF), which are indication of effective-
ness of fortification:
FF ¼ ACB =AFB
where, ACB activity of control bread (expressed as EC50), AFB activity
of fortificated bread (expressed as EC50)
2.9. Statistical analysis
All experimental results are displayed as ± S.D. of three parallel
experiments (n ¼ 9) and data were evaluated by one-way analysis
of variance (one-way Anova). The statistical differences between
the groups were estimated using the Tukey test. The Pearson cor-
relation analysis was also carried out on this data. Statistical tests
were evaluated using Statistica 6.0 software (StatSoft, Inc., Tulsa,
USA). All the statistical tests were carried out at a significance level
of a ¼ 0.05.
3. Results
3.1. Sensory analysis
The results of hedonic tests on different types of bread are given
in Table 1. The colour of crumb of the enriched bread was much
darker (green) than the control bread what had a slight but a sig-
nificant and negative influence on bread acceptability. The taste,
aroma and overall acceptability of bread at substitution levels of
1e2 g/100 g did not differ significantly in respect to control breads.
Higher levels of QL addition, especially 5 g/100 g caused an un-
pleasant aroma and taste. Quinoa leaves addition up to 3 g/100 g
had no statistically significant influence on bread texture. Higher
levels of QL addition had negative influence of this parameter. The
sensory characteristics linking results indicated that a partial
replacement of wheat flour in bread with up to 3 g/100 g QL powder
gives satisfactory overall consumer acceptability. However, breads
containing 4 g/100 g and 5 g/100 g of QL were rated comparatively
lower, which might be due to excessive amounts of ground leaves
which negatively affected the aroma, taste, and texture of breads.
Supplementation of wheat breads with QL caused an increase of
phenolic compounds content. The amount of chemically extract-
able (CE) phenolics was strongly linked with percentage of QL
addition; the lowest TPC was determined for control bread whereas
the highest for bread with 5 g/100 g QL addition (144.35 and
321.01 mg GAE/g DW, respectively). These results which may
indicate lipophilic character of QL phenolics. Contents of buffer-
extractable (BE) phenolic in all breads was significantly lower
than those determined in CE and averaged from 56.63 mg GAE/g
DW to 135.28 mg GAE/g DW (C and QL5 breads, respectively).
Simulated gastrointestinal digestion caused a release of phenolics
from all tested breads. Importantly, taking into account the TPC in
the extracts after simulated absorption, the released phenolics
were highly bioavailable in vitro. In the cases of DE and AE, the
highest TPCs were obtained for QL4, whereas the lowest for control
breads. Analysis of extractability factors (EFs) clearly showed that
simulated gastrointestinal tract consists an effective extractor of
phenolic compounds from all samples e about two times more
effective than chemical extraction. These results are most impor-
tant (and unexpected) in the light of EF values for BE indicating a
low extractability in the buffer system (Table 2).
In the breads supplemented with QL nine phenolic acids (gallic,
protocatechuic, p-hydroxybenzoic, vanillic, caffeic, syryngic, p-
coumaric, ferulic and synapinic) and rutin were identified (Table 3).
All of these compounds were potentially mastication-extractable,
however only rutin, gallic, protocatechuic, and ferulic acids were
potentially bioaccessible and bioavailable. QL addition especially
enriched breads with rutin and gallic acid, while the main source of
protocatechuic, caffeic, p-hydroxybenzoic and syryngic acids was
wheat flour.
Taking into account bioaccessibility it can be stated that simu-
lated digestion released gallic and protocatechuic acids, while
bioaccessibility of ferulic acid and rutin was limited. Unfortunately,
p-hydroxybenzoic, vanilic, syryngic, p-coumaric and synapinic
acids were not detected in fluids obtained after simulated digestion
and absorption, which may indicate their limited bioaccessibility
and bioavailability in model system.
3.3. Antioxidants potential study
3.3.1. Activity of chemical-extractable phytochemicals
As it can be seen from Fig. 1 chemical extracts possessed
multifaceted antioxidant properties. All studied antioxidant activ-
ities (LPO, CHEL, AA and RED) may be ordered as follow
QL5 > QL4 > QL3 > QL2 > QL1 > C (Fig. 1) and were significantly,
positively correlated with QL addition (r ¼ 0.94, 0.94, 0.99 and 0.93
for LPO, CHEL, AA and RED, respectively). Thus, it may be
concluded, that QL consist valuable source of antioxidants espe-
cially lipids preventers and metal chelators.

Table 1
3.3.2. Activity of bioaccessible and bioavailable in vitro antioxidants
Sensory evaluation of bread prepared by the substitution of wheat flour with ground Results obtained after chemical extraction were satisfactory
quinoa leaves. (Fig. 1), but because chemical extraction does not mirror the con-
Attribute Substitution level (g/100 g)
ditions in the human gastrointestinal tract, we decided to evaluate
the bioaccessibility and bioavailability in vitro using human
0 1 2 3 4 5
gastrointestinal tract model.
Crumb colour 8.5a 7.8b 7.9b 7.3c 7.0d 6.8d Taking into account antiradical activity it may be stated that QL
Aroma 8.2a 8.1a 7.8b 6.9c 5.3d 4.2e
contains buffer-extractable radical scavengers. Antiradical activity
Texture 7.9a 7.8a 7.8a 7.6ab 7.0b 6.4c
Taste 8.8a 8.5ab 8.1b 7.5c 6.5d 4.8e of BE was correlated with percent of QL addition (r ¼ 0.97).
Overall 8.4a 8.1ab 7.9b 7.6c 6.5d 5.6e Digestion in vitro released antiradical compounds from all tested
Nine-point hedonic scale with 1, 5 and 9 representing extremely dislike, neither like
samples; the highest activity was observed in the case of QL3, QL4
nor dislike, and extremely like, respectively. Means with different letter superscript and QL5, however bioactive compounds from these breads were
within a same row are significantly different (a < 0.05). poorly bioavailable (BAV values 0.77, 0.69 and 0.95, respectively)
1230 U. Gawlik-Dziki et al. / LWT - Food Science and Technology 62 (2015) 1226e1234

Table 2
Comparison of phenolic contents and extractability factors (n ¼ 9).

Sample Total phenolics content [mg GAE/g DW] Extractability factor (EF)

CE BE DE AE BE DE AE
aA bA cA dA
C 144.35 ± 6.12 56.63 ± 2.33 296.87 ± 9.11 199.17 ± 5.66 0.39 2.06 1.38
QL1 170.34 ± 7.21aB 59.87 ± 2.75bA 339.31 ± 11.52cB 202.83 ± 4.37dA 0.35 1.99 1.19
QL2 196.68 ± 7.35aC 63.58 ± 3.21bB 357.33 ± 10.22cC 226.56 ± 5.22dB 0.32 1.82 1.15
QL3 245.15 ± 8.17aD 91.64 ± 4.51bC 360.55 ± 9.69cC 260.44 ± 5.44aC 0.37 1.47 1.06
QL4 259.55 ± 11.25aD 108.71 ± 5.33bD 413.21 ± 15.24cD 307.75 ± 6.14dD 0.42 1.38 1.19
QL5 321.01 ± 13.02aE 135.28 ± 7.88bE 396.86 ± 13.43cE 298.53 ± 7.88dE 0.42 1.29 0.93

C e control bread, QL1, QL2, QL3, QL4, QL5 e breads with 1, 2, 3, 4 and 5 g/100 g Chenopodium quinoa leaves addition, respectively, CE e chemical extract, BE e buffer extract,
DE e extract after simulated digestion, AE e extract after simulated absorption. Means with different small letter within the same rows and capital letter within the same
columns (within each activity) are significantly different (a < 0.05).

(Table 4). It is worth nothing that lipids preventers were already
effectively extracted by simulated mastication and subsequent
digestion in vitro caused their further release from food matrix. It
should be also noted that the lowest supplementation level caused
a strong elevation of activity. Unfortunately, compounds able to
prevent lipids against oxidation were poorly bioavailable in model
system (BAV values did not excess 1) (Table 4). Fortification breads
with QL significantly influenced on chelating activity of all studied
extracts. After digestion in vitro, in respect to BE, significant in-
crease of activity was observed, especially in the case of QL1 and
QL2 breads (72.5% and 65.1%, respectively). Also, a large part of
bioactive compounds passed through dialysis membrane, which
may suggest their hydrophilic character (Table 4). In spite of the fact
that digestion in vitro released potential metal chelators (BAC
values ranged from 1.35 to 3.64) their bioavailability was signifi-
cantly lower (BAV values for enriched breads ranged from 0.66 to
1.06). In respect to control breads enriched breads were also
characterized by much higher reducing ability. Activity of extracts
containing potentially mastication-extractable compounds (BE)
was significantly, positively correlated with percent of QL addition
(r ¼ 0.92), although already 1 g/100 g of QL addition increased
tested activity by about 50%. Reductive compounds were highly
bioaccessible in vitro (BAC for QL1 e 10.24) but similarly to lipid
preventers and metal chelators were poorly bioavailable (BAV for
QL1 e 0.97).
3.4. Extractability of antioxidative compounds
Extractability factors describing the efficiency of the different
extraction system used in the study are presented in Table 5. Based
on EF values it may be supposed that antiradical and lipid pre-
venting compounds from control breads possess rather lipophilic
character, and QL supplemented them with hydrophilic active
compounds, a reverse situation was observed for CHEL, while
reductive compounds were more effectively extracted from all
samples using PBS buffer. As expected, LPO from control breads
were most effectively extracted using MeOH:H2O (1:1, v/v)
(FF ¼ 0.38), however QL addition enriched breads with buffer-
extractable phytochemicals exhibiting this activity (FF values
about 1). Simulated digestion released multifaceted antioxidant
compounds from all samples much more effectively than chemical
extraction MeOH:H2O (1:1, v/v). The most important differences
were visible in the cases of reductive (EF values from 7.24 to 23.86)
and antiradical (EF values from 3.85 to 5.24) compounds, the lowest
for LPO (EF values from 1.15 to 3.16) and CHEL (1.73e3.75). The
same relationship was observed for AE. Regardless of used
methods, extractability of all antioxidants decreased with QL
addition higher than 3 g/100 g. Obtained data clearly illustrate
essential differences between both used procedures (chemical
extraction and simulated gastrointestinal tract action) and point
the need for standardization and harmonization of procedures for
assessing the activity of newly designed food products.
3.5. Effectiveness of fortification
A well-designed enriching component should have high effi-
ciency, thus to evaluate the effectiveness of enrichment with
bioactive ingredient the fortification factor (FF) was proposed. As
shown in Fig. 2 significant differences between FF values for CE, BE,
DE and AE were observed. Taking into account CE it can be
concluded that values of FF in all tested methods strongly depen-
dent of QL percent (linear relationships were observed). Similar
relationships were observed for BE, however FF values (in except
CHEL) were significantly higher than those determined for CE. It
may suggested that these active compounds can be extractable
already during mastication. Taking into account AA and RED the
highest FF values were determined for QL4. On the other hand QL
most effectively fortificated wheat breads with (potentially bio-
accessible) reductive compounds, while the least for metal chela-
tors. On the basis of the curves of DE it may be resumed that the
supplementation of breads with more than 3e4 g/100 g was not
translated into subsequent and proportional increase of all activ-
ities. Interesting results were obtained for AE. Fig. 2 bed clearly
showed, that the maximal effect was achieved for QL2 taking into
account LPO, CHEL and RED. Only in the case of antiradical com-
pounds rather linear relationship was observed.
In general, hydrophilic compounds, probably passively absor-
bed, derived from QL enhanced antioxidant activity of breads about
2e3 times (except chelating power). It is interesting, that in all
cases, except AA, the highest FF values were determined from QL2
bread.
4. Discussion
Reactive oxygen species (ROS) are suspected in “civilization
diseases”, and accelerated ageing due their abilities to react with
various cellular components leading to oxidative degradation


(Gawlik-Dziki, Swieca, Dziki, Tomiło & Baraniak, 2012). In this
reason the total antioxidant capacity of some foods has increasingly
become a sales argument for professionals in the food industry
(Michiels, Kevers, Pincemail, Defraigne, & Dommes, 2012). One of
the ways to improve of nutraceutical potential of food is their
fortification with components of documented activity.
The criterion of choice of supplement should be first of all their
activity, not without significance is also the availability and cost. In
our study choice of QL was dictated by their documented multi-


faceted antioxidant an anticancer activities (Gawlik-Dziki, Swieca,


Dziki, et al, 2012; Gawlik-Dziki, Swieca, Sułkowski, et al., 2013).
Additionally, QL are the waste material the use of which can bring
environmental and economic benefits. Our results clearly show
Table 3
Qualitative-quantitative determination of potentially mastication-extractable, bioaccessible and bioavailable phenolics from bread (n ¼ 9).

Sample Compound [mg/g DM]

Gallic acid Protocateuchuic acid p-hydroxybenzoic acid Vanilic acid Caffeic acid Syryngic acid p-coumaric acid Ferulic acid Synapinic acid Rutin

BE C 58.26 ± 4.11a 25.68 ± 2.33ab 2.43 ± 0.21a 2.13 ± 0.32a 2.89 ± 0.19a 0.99 ± 0.05a 3.25 ± 0.09a 16.81 ± 1.11ef 15.89 ± 2.01a Nd
QL1 69.38 ± 3.45ab 28.75 ± 1.23bc 3.69 ± 0.06b 3.84 ± 0.51b 2.93 ± 0.22a 1.49 ± 0.22bc 6.49 ± 0.70b 20.79 ± 0.76g 20.37 ± 0.24d 73.55 ± 3.11f
QL2 77.29 ± 4.51bc 24.90 ± 0.11a 3.58 ± 0.17bc 4.44 ± 0.09b 2.81 ± 0.55a 1.52 ± 0.08b 9.05 ± 0.48c 24.27 ± 2.43hj 18.03 ± 0.32bc 80.20 ± 3.02g
QL3 87.12 ± 2.53c 31.23 ± 0.65c 4.04 ± 0.40cd 5.49 ± 0.13c 4.83 ± 0.41b 2.18 ± 0.37cd 12.59 ± 0.85d 27.91 ± 1.14i 22.11 ± 0.49e 145.41 ± 5.03i
QL4 97.82 ± 5.81d 25.00 ± 3.02a 5.65 ± 0.38de 6.28 ± 0.43d 5.14 ± 0.90bc 2.15 ± 0.19d 16.96 ± 3.48de 28.96 ± 2.97jk 17.41 ± 3.63abcd 146.61 ± 28.37ij
QL5 121.31 ± 7.35e 24.98 ± 0.64a 6.09 ± 0.05e 10.08 ± 1.39e 6.66 ± 0.45c 2.74 ± 0.02e 20.45 ± 0.19e 31.96 ± 2.77k 19.67 ± 0.63c 209.89 ± 13.72k
DE C 1527.81 ± 52.38k 98.96 ± 5.55ef Nd Nd Nd Nd Nd 8.35 ± 2.16c Nd Nd
QL1 1682.78 ± 29.22l 98.92 ± 2.61ef Nd Nd Nd Nd Nd 13.54 ± 1.26d Nd 31.61 ± 1.06b
QL2 1703.62 ± 15.76l 93.70 ± 2.97ef Nd Nd Nd Nd Nd 17.70 ± 1.20ef Nd 61.16 ± 1.36e
QL3 1947.59 ± 41.17m 111.44 ± 6.39g Nd Nd Nd Nd Nd 20.51 ± 1.04gh Nd 111.80 ± 15.35h

U. Gawlik-Dziki et al. / LWT - Food Science and Technology 62 (2015) 1226e1234

QL4 2088.49 ± 51.12m 111.56 ± 12.22fg Nd Nd Nd Nd Nd 19.22 ± 2.19fg Nd 151.27 ± 33.31ij
QL5 2214.60 ± 45.59n 122.09 ± 5.31g Nd Nd Nd Nd Nd 20.74 ± 1.84gh Nd 163.07 ± 18.34j
AE C 855.80 ± 10.38f 78.63 ± 2.58de Nd Nd Nd Nd Nd 2.59 ± 0.05a Nd Nd
QL1 933.11 ± 20.76g 75.72 ± 1.78d Nd Nd Nd Nd Nd 4.36 ± 0.14b Nd 1.72 ± 0.00a
QL2 1260.28 ± 4.03h 75.13 ± 11.71df Nd Nd Nd Nd Nd 7.29 ± 0.20c Nd 41.88 ± 2.99c
QL3 1375.21 ± 65.34hi 82.84 ± 3.62e Nd Nd Nd Nd Nd 7.24 ± 0.84c Nd 51.67 ± 4.65d
QL4 1496.55 ± 45.15ij 86.68 ± 10.30de Nd Nd Nd Nd Nd 8.15 ± 0.78c Nd 64.77 ± 4.54ef
QL5 1521.48 ± 33.13j 95.15 ± 10.15f Nd Nd Nd Nd Nd 9.97 ± 1.99c Nd 121.71 ± 21.87hij

Nd e not detected, C e control bread, QL1, QL2, QL3, QL4, QL5 e breads with 1, 2, 3, 4 and 5 g/100 g Chenopodium quinoa leaves addition, respectively, BE e buffer extract, DE e extract after simulated digestion, AE e extract after
simulated absorption. Means with different small letter within the same columns are significantly different (a < 0.05).
breads with 1, 2, 3, 4 and 5 g/100 g quinoa leaves addition, respectively.
chelating power, D e reducing power. C e control bread, QL1, QL2, QL3, QL4, QL5 e
wheat breads. A e antiradical activity, B e inhibition of lipid peroxidation, C e
Fig. 1. Antioxidant activity of chemical extracts obtained from control and enriched

1231
1232 U. Gawlik-Dziki et al. / LWT - Food Science and Technology 62 (2015) 1226e1234

Table 4
Comparison of antioxidant activities of bread, bioaccessibility (BAC), bioavailability (BAV) and bioefficiency (BEF) factors (n ¼ 9).

Activity Sample

C QL1 QL2 QL3 QL4 QL5

aA aA bA cA dA
Antiradical activity BE 448.61 ± 12.87 442.80 ± 13.56 257.54 ± 9.58 152.41 ± 6.78 112.68 ± 4.32 63.45 ± 2.69eA
GE 68.79 ± 3.25aB 63.34 ± 3.33bB 41.29 ± 2.45cB 29.38 ± 2.47dB 25.57 ± 4.44eB 26.44 ± 4.98eB
AE 55.17 ± 5.14aC 57.79 ± 4.15aC 38.79 ± 6.45bC 37.98 ± 6.28bC 36.87 ± 6.33bC 27.73 ± 4.55cB
BAC 6.52 6.99 6.24 5.19 4.41 2.40
BAV 1.25 1.10 1.06 0.77 0.69 0.95
BEF 8.13 7.66 6.64 4.01 3.06 2.29
Inhibition of linoleic acid peroxidation BE 75.16 ± 5.61aA 19.77 ± 2.11bA 19.86 ± 1.99bA 15.04 ± 2.01cA 14.26 ± 3.13cA 11.88 ± 2.25dA
GE 19.90 ± 1.45aB 6.53 ± 0.49bB 7.25 ± 0.37bB 7.27 ± 0.31bB 7.98 ± 0.28bB 10.50 ± 0.85cA
AE 17.92 ± 0.82aC 9.89 ± 0.28bC 7.41 ± 0.31cB 10.18 ± 0.41bC 15.30 ± 0.45dC 18.95 ± 0.95aB
BAC 3.78 3.03 2.74 2.07 1.79 1.13
BAV 1.11 0.66 0.98 0.71 0.52 0.55
BEF 4.19 2.00 2.68 1.48 0.93 0.63
Chelating power BE 15.32 ± 0.77aA 24.65 ± 1.13bA 21.92 ± 1.09cA 15.10 ± 0.87dA 13.89 ± 0.88eA 12.19 ± 0.79fA
GE 10.24 ± 0.75aB 6.78 ± 0.22bB 7.64 ± 0.29bB 7.15 ± 0.31bB 7.50 ± 0.30bB 9.06 ± 0.42cB
AE 7.94 ± 0.29aC 10.06 ± 0.36bC 7.22 ± 0.31aB 9.00 ± 0.39bC 11.40 ± 0.44cC 9.65 ± 0.45bB
BAC 1.50 3.64 2.87 2.11 1.85 1.35
BAV 1.29 0.67 1.06 0.79 0.66 0.94
BEF 1.93 2.45 3.04 1.68 1.22 1.26
Reducing power BE 270.72 ± 11.32aA 145.03 ± 11.21bA 110.26 ± 7.52cA 63.17 ± 3.52dA 50.35 ± 2.25eA 34.70 ± 1.15fA
GE 75.89 ± 3.38aB 14.16 ± 2.11bB 13.76 ± 1.18bB 11.39 ± 1.22cB 10.34 ± 0.33cB 11.15 ± 0.26cB
AE 34.41 ± 1.18aC 14.54 ± 0.32bB 11.09 ± 0.28cC 14.12 ± 0.35bC 17.21 ± 0.23dC 14.92 ± 0.32bC
BAC 3.57 10.24 8.01 5.54 4.85 3.11
BAV 2.21 0.97 1.24 0.81 0.60 0.75
BEF 7.87 9.97 9.94 4.47 2.93 2.33

C e control bread, QL1, QL2, QL3, QL4, QL5e breads with 1, 2, 3, 4 and 5 g/100 g Chenopodium quinoa leaves addition, respectively, CE e chemical extract, BE e buffer extract, DE
e extract after simulated digestion, AE e extract after simulated absorption. Means with different small letter within a same row and capital letter within a same column
(within each activity) are significantly different (a < 0.05).

that QL enriched breads with bioaccessible and bioavailable rutin. As our research shows, wheat bread is a good source of bio-
Most importantly, rutin from QL seems to be resistant to conditions accessible and bioavailable in vitro compounds with multidirec-
that occur during baking. Similar results were founded by Ma, Li, tional antioxidant activity (Table 3). In the light of the presented
Yuan, and Yang (2008). Beside of this, QL enriched breads with facts enrichment of wheat breads may seem unnecessary, but
phenolic acids. Bread contains bioaccessible phenolic antioxidants, should be take into account that these activities are relatively low,
especially ferulic acid and alkylresorcinols (Mateo Anson, Hemery, and even at a reduced bioavailability activity of enriched breads
Bast, & Haenen, 2012). Additionally, thermally processed foods may are significantly higher than activity of control (Table 4). Our
contain various levels of Maillard reaction products that have been previous study proved that the main phenolics of QL were ferulic,

zyło, Gawlik-Dziki, &
reported to have antioxidant activity (Dziki, Ro sinapinic and gallic acids, kaempferol, rutin and isorhamnetin and


Swieca, 2014). Interestingly, the content of protocateuchic and there was a strong relationship between their content, antioxidant
synapinic acid in QL4 and QL5 is lower than in QL3 sample, but this activities and anticancer potential (Gawlik-Dziki, Swieca,

inconsistency is observed only in the case of BE. According to Sułkowski, et al., 2013). In the light of these data, use of QL for
previous studies this fact may be partially explained by the ability breads fortification seems to be justified. As being presented in
to interact with other components of the food matrix (e.g. protein Table 2. QL addition significantly enriched breads with phenolic


and/or starch) (Swieca, Sȩczyk, Gawlik-Dziki, & Dziki, 2014). compounds.
Taking into account EF values, it can be concluded, that simu-
lated digestive tract was more effective extractor of phenolic
compounds than usually used chemical solvent. In the light of this
Table 5
using only chemical extraction, content of potentially mastication
Comparison of extractability factors.
extractable phenolics could be overestimated, while content of
Antioxidant assay Factor Sample potentially bioaccessible and bioavailable phenolics e under-
C QL1 QL2 QL3 QL4 QL5 estimated (Table 2).
Antiradical EFBE 0.65 0.61 0.84 1.02 1.14 1.61 The antioxidant properties of food matrices are due to the
activity EFDE 4.21 4.18 5.24 5.02 5.02 3.85 presence of a complex mixture of compounds of varying polarity.
EFAE 5.25 4.58 5.58 3.48 3.48 3.68 Thus, we decided to use four methods (based on differ mechanism
Inhibition of linoleic EFBE 0.38 1.04 0.87 1.06 0.98 1.01 of action) for determine antioxidant activity of designed products.
acid peroxidation EFDE 1.42 3.16 2.38 2.20 1.75 1.15
EFAE 1.58 2.09 2.32 1.57 0.92 0.64
In most studies only “chemical” extraction was taken into account.
Chelating power EFBE 2.35 1.03 1.04 1.36 1.28 1.29 In the study concerning the influence of extraction conditions on
EFDE 3.52 3.75 2.98 2.87 2.36 1.73 antioxidant capacity Michiels et al. (2012) proposed standardiza-
EFAE 4.54 2.52 3.15 2.28 1.56 1.63 tion of sample preparation and extraction to allow comparing the
Reducing power EFBE 2.03 2.33 1.92 2.55 2.34 2.69
antioxidant capacities and phenolic contents of different food
EFDE 7.24 23.86 15.40 14.12 11.38 8.38
EFAE 15.97 23.24 19.11 11.39 6.84 6.26 matrices. Generally, this procedure (similarly to other chemical
extraction) is by all means correct and is useful for fast evaluation
C e control bread, QL1, QL2, QL3, QL4, QL5e breads with 1, 2, 3, 4 and 5 g/100 g
Chenopodium quinoa leaves addition, respectively, CE e chemical extract, BE e
of antioxidant potential, however it does not reflect the conditions
buffer extract, DE e extract after simulated digestion, AE e extract after simulated occurring in the gastrointestinal tract/human organism while this
absorption. issue is crucial to assess the biological activity of new products.
 U. Gawlik-Dziki et al. / LWT - Food Science and Technology 62 (2015) 1226e1234
Fig. 2. Comparison of fortification factors. A e antiradical activity, B e inhibition of
lipid peroxidation, C e chelating power, D e reducing power. CE e chemical extract, BE
e buffer extract, DE e extract after simulated digestion, AE e extract after simulated
absorption. QL1, QL2, QL3, QL4, QL5 e breads with 1, 2, 3, 4 and 5 g/100 g quinoa leaves
addition, respectively.
1233
Based on data obtained for CE (Fig. 1) it may be concluded, that QL
consist valuable material for bread fortification and may be rec-
ommended for food technologists. In the light of previous state-
ments in this study the bioaccessibility and bioavailability were
determined on the basis of model of human gastrointestinal tract
(GI). In should be underlined that use the simulated GI eliminates
differences arising during chemical extraction (type of solvent,
pH, temperature, number of steps, volume of solvent, solvent
saturation, solvent selectivity due to composition) and tempera-
ture. Essential differences between both used procedures (CE and
GI) were illustrated by EF and FF values (Table 5, Fig. 2). Our ob-
servations were in accordance with those provided by Perez-
Jimenez and Saura-Calixto (2005), Saura-Calixto et al., 2007.
Generally, relationship between antioxidant activities level and
percent of QL addition were not proportional. It should be noted
that additives with health-promoting properties required specific
studies in view of the possibility of various interactions e both
synergistic (between antioxidants) and negative e creating and
indigestible inactive complexes with proteins or starch (Gawlik-


Dziki, 2012; Swieca, Gawlik-Dziki, et al., 2013). The bioactivity of
bioavailable antioxidant compounds defined by BEF justified sup-
plementation of breads up to 3 g/100 g of QL. On the other hand it
should be kept on mind that a lower bioavailability of antioxidant
from breads with higher percent of QL is balanced by their signif-
icantly higher activity.
Our results point the need for standardization and harmoniza-
tion of procedures for assessing the activity of newly designed food
products. We propose the following conditions:
1. Choice of vehicle and fortificant.
2. Sensory analysis and preliminary development of new products
recipe.
3. Preliminary estimation of active compounds content and anti-
oxidant activity based on chemical extraction.
4. Estimation of in vitro bioaccessibility of active compounds and
their antioxidant activity.
5. Description on new product recipe and evaluation in vivo.
In conclusion, QL constitutes a valuable supplement for devel-
opment of bread with enhanced functional properties. In the light
of the present data 3 g/100 g supplementation was optimal for
improving the antioxidant potential of bread without compro-
mising its sensory quality. The antioxidant activities of potentially
bioaccessible bioactives yielded higher then these determined for
chemical procedure. For this reason an universal way for determi-
nation nutraceutical potential in vitro was proposed.
Acknowledgements
This scientific study was financed by the Polish Ministry of
Scientific Research and Higher Education (grant NN312233738).
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