Food Chemistry 221 (2017) 1451–1457

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Wheat bread enriched with green coffee – In vitro bioaccessibility and

bioavailability of phenolics and antioxidant activity
Michał Świeca a,⇑, Urszula Gawlik-Dziki a, Dariusz Dziki b, Barbara Baraniak 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, Doświadczalna 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 potential bioaccessibility and bioavailability of phenolics, caffeine and antioxidant activity of wheat
Received 21 June 2016 bread enriched with green coffee were studied. Supplementation enhanced nutraceutical potential by
Received in revised form 7 October 2016 improving phenolic content and lipid protecting capacity. The simulated-digestion-released phenolics
Accepted 1 November 2016
(mainly caffeic acid, syringic acid and vanillic acid) from bread, also caused significant qualitative
Available online 2 November 2016
changes (chlorogenic acids were cleaved and significant amounts of caffeic acid and ferulic acid were
determined). Compared to the control, for the bread with 1% and 5% of the functional component the con-
Keywords:
tents of phenolics were 1.6 and 3.33 times higher. Also, an approximately 2.3-fold increase in antioxidant
Bioaccessibility
Bioavailability
activity was found in bread containing 5% of the supplement. The compounds responsible for antioxidant
Bread potential have high bioaccessibility but poor bioavailability. The qualitative composition of the phenolic
Fortification fraction has a key role in developing the antioxidant potential of bread; however, caffeine and synergism
Green coffee between antioxidants are also important considerations.
In vitro Ó 2016 Elsevier Ltd. All rights reserved.
Phenolics

1. Introduction suring the bioaccessibility/bioavailability of these bioactive

Fortification (enrichment) of food is one of the most popular
strategies aimed at achieving products that are characterized by
an increased content of health-promoting components. It includes
the addition of one or more functional components to particular
foods, thereby preventing their deficiency and/or providing addi-
tional benefits (Allen, de Benoist, Dary, & Hurrell, 2006). Fortifica-
tion of foods, especially of widely consumed products (e.g. bakery
products, pasta, juices), makes this strategy more effective and
allows it to reach a larger number of consumers (Dziki, Rózyło, _
Gawlik-Dziki, & Świeca, 2014; Takahama, Tanaka, & Hirota,
2011). Additionally, targeted as well as market-driven fortification
enables new food products to be designed, that usually meet the
criteria of the so-called ‘‘functional food” (Fletcher, Bell, &
Lambert, 2004; Siró, Kápolna, Kápolna, & Lugasi, 2008).
Phenolics, a group of plant secondary metabolites with well-
documented antioxidant, anti-inflammatory and anticancer activi-
ties, are widely used for food fortification (Arts & Hollman, 2005;
Wang, Melnyk, Tsao, & Marcone, 2011). So far, there is no substan-
tial evidence as to which method is the most appropriate for mea-
⇑ Corresponding author.
E-mail addresses: michal.swieca@up.lublin.pl (M. Świeca), urszula.gawlik@up.
lublin.pl (U. Gawlik-Dziki), dariusz.dziki@up.lublin.pl (D. Dziki), barbara.baraniak@
up.lublin.pl (B. Baraniak).
ingredients; however these two factors are strongly determined
by phenolics’ chemical structure, hydrophobicity as well as the
current status of an organism, including microbiota action
(Laparra & Sanz, 2010).
It is a well-known fact that the effectiveness of fortification and
the final effect observed after consumption of phenolic-rich foods
is a result of many factors (Li, Tsao, & Deng, 2012). A high activity
in vitro is not always translated into comparable activity in vivo
(González-Sarrías et al., 2015; Siviero et al., 2015), thus, it is very
important to determine the bioaccessibility and bioavailability of
functional supplements whilst evaluating the quality of new prod-
ucts. As in vivo studies are very expensive and usually difficult to
perform, some in vitro strategies based on simulated digestion
and absorption have been developed and successfully introduced
(Hur, Lim, Decker, & McClements, 2011; Minekus et al., 2014). In
general, in vitro methods are somewhat limited due to the omitted
role of the colon in digestion and absorption (Etcheverry, Grusak, &
Fleige, 2012). It has been proven that Caco-2 systems are useful to
study the bioaccessibility of phenolics (Barrington et al., 2009).
Unfortunately, they may only be applied to samples characterized
by a relatively low content of phenolics, as there is some evidence
that a high concentration of phenolics - up to 50 lM (e.g. like in
coffee and coffee-based products) is toxic and by destroying the
monolayer of cells causes an unspecific transfer of phenolics,

http://dx.doi.org/10.1016/j.foodchem.2016.11.006
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
1452 M. Świeca et al. / Food Chemistry 221 (2017) 1451–1457
which overestimates their bioaccessibility (Calvello et al., 2016).
The absorption system used in this study (based on the passive
transport) allows estimation of the minimal bioavailability and
minimizes the risk of overestimation. So far, the intestinal absorp-
tion of 5-caffeoylquinic acid (5-CQA) has been studied in a cell cul-
ture, using the human colon carcinoma cell line Caco-2. The
absorption rate for 5-CQA was found to be about 0.10% at physio-
logical concentrations equivalent to gut lumen concentrations
(0.1
1 mM). Transepithelial transport experiments with chloro-
genic acid (CGA) using Caco-2 intestine epithelia cultured mono-
layer demonstrated bidirectional permeation with no transport
into the basolateral side, regardless of pH gradient. The permeation
rate was concentration-dependent and not saturable, thus indicat-
ing passive diffusion. In addition, the transport was inversely cor-
related with transepithelial electrical resistance, indicating limited
passive diffusion when intestinal junctions are tight (Liang & Kitts,
2015). On the other hand, according to absorption studies in the
Caco-2 system and in vivo experiments in a rat model, in which
CQA was studied together with a realistic food matrix, it has been
reported that CQA is poorly absorbed in its native form (Dupas,
Baglieri, Ordonaud, & Tomø, 2006). An important role has also been
proven for the interactions of phenolics with other active compo-
nents and/or food matrices. Such relationships may significantly
diminish the bioactivity of phenolics in the upper parts of the
digestive tract and may also lower the digestibility of nutrients
(Budryn et al., 2016; Dupas et al., 2006; Swieca, Gawlik-Dziki,
Dziki, Baraniak, & Czyz,_ 2013).
Green coffee is a rich source of chlorogenic acids and caffeine
(Budryn, Zaczyńska, & Rachwał-Rosiak, 2015; Dziki et al., 2015;
Mehari et al., 2015). Chlorogenic acids, due to a high antiradical
and reducing potential as well as the ability to modify the activity
of pro-oxidative enzymes, exhibit in vitro many biological activities
including antimutagenic, anticarcinogenic, anti-inflammatory and
antioxidant properties (Budryn, Nebesny, Zy _ zelewicz,
_ & Oracz,
2014; Dziki et al., 2014; Liang & Kitts, 2015). In the last years, green
coffee has also been considered as a functional ingredient useful in
regulating the metabolism aiming at reducing body weight.
According to literature data, this effect is ambiguous (Onakpoya,
Terry, & Ernst, 2011). It has been proven that in some cases green
coffee bean phytochemicals show a tendency to reduce visceral fat
and body weight (Thom, 2007); however, another study provides
opposite findings (Li Kwok Cheong et al., 2014).
The aim of this study was to evaluate the in vitro bioaccessibil-
ity and absorption of phenolics and caffeine. The antioxidant
potential of green coffee bean as well as wheat bread enriched with
this functional component, was also explored.
2. Materials and methods
2.1. Chemicals
a-amylase, pancreatin, pepsin, bile extract, linoleic acid, Tween-
20 and haemoglobin were purchased from Sigma–Aldrich Com-
pany (Poznan, Poland). All others chemicals were of analytical
grade.
2.2. Green coffee flour preparation
The samples of green coffee (Coffea arabica) beans from Kenya
(GCB) were purchased from Caffeine Co. Marki, Poland. They were
prepared by adding water to adjust moisture content to 10 g/100 g
(w.b.) and storing for 48 h. The beans were ground using the labo-
ratory hammer mill (POLYMIX-Micro-Hammermill MFC, Kinemat-
ica. AG, Littau/Lucerne, Switzerland) equipped with round holes of
3.0 mm (Dziki et al., 2015).
2.3. Bread making
Wholemeal wheat flour (type 2000; average 1.95% ash content,
humidity 14%) used in the formula of control bread (CB) (600 g)
was purchased in a local market from Lublin, Poland. The flour
was replaced with GCB at 1%, 2%, 3%, 4% and 5% levels (B1-B5,
respectively). The percentage of green coffee flour addition was
chosen on the basis of previous test concerning consumer accep-
tance (data previously published (Dziki et al., 2015)). Besides this,
6 g of instant yeast and 12 g of salt were used for dough prepara-
tion. The general quantity of water necessary for the preparation
of the dough was established through the marking of water
absorption properties of flour to a consistency of 350 Brabender
units. The batches of dough were mixed in a spiral mixer for
6 min. After fermentation (optimal time was about 2 h), the pieces
of dough (300 g) were put into an oven at a temperature of 230 °C.
The baking time was 30 min. After baking, the bread was left to
stand for 24 h at room temperature, lyophilized and ground
(Dziki et al., 2015).
2.4. Extract preparation
2.4.1. Buffer extracts (BE)
The samples of wheat and green coffee flours as well as
enriched bread (1 g of dry weight (DW)) were extracted for 1 h
with 20 ml of PBS buffer (phosphate buffered saline, pH 7.4). The
extracts were separated by decantation and the residues were
extracted again with 20 ml of PBS buffer. Extracts were combined
and stored in the dark at 20 °C.
2.4.2. Digestion in vitro – potentially bioaccessibility (GE)
In vitro digestion was performed as described previously by
Minekus et al. (2014) with some modifications. Artificial saliva
solution was prepared by dissolving 2.38 g Na2HPO4, 0.19 g KH2-
PO4, 8 g NaCl and 100 mg of mucin in 1 L of distilled water. The
solution was adjusted to pH 6.75 and a-amylase (E.C. 3.2.1.1.)
was added to obtain 200 U/ml of enzyme activity. For gastric diges-
tion, 300 U/ml of pepsin (from porcine stomach mucosa, pepsin, EC
3.4.23.1) was prepared in 0.03 mol/l NaCl, pH 1.2. Simulated
intestinal juice was prepared by dissolving 0.05 g of pancreatin
(activity equivalent 4  USP) and 0.3 g of bile extract in 35 ml
0.1 mol/l NaHCO3. The samples were subjected to simulated gas-
trointestinal digestion as follows: 1 g of powdered sample (wheat
and green coffee flours as well as enriched bread) was homoge-
nized in a Stomacher laboratory blender for 1 min to simulate mas-
tication in the presence of 15 ml of simulated salivary fluid. The
samples were then shaken for 10 min. at 37 °C. The samples were
adjusted to pH 3 using 5 mol/l HCl, and then 15 ml of simulated
gastric fluid was added. The samples were shaken for 60 min at
37 °C. After digestion with the gastric fluid, the samples were
adjusted to pH 6 with 0.1 mol/l of NaHCO3 and then 15 ml of a mix-
ture of bile extract and pancreatin was added. The extracts were
adjusted to pH 7 with 1 mol/l NaOH and finally 5 ml of
120 mmol/l NaCl and 5 ml of 5 mmol/l KCl were added to each
sample. Once prepared, the samples were submitted for in vitro
digestion for 120 min., at 37 °C and in the dark. Thereafter, the
samples were centrifuged and the supernatants were used for fur-
ther analysis.
2.4.3. Absorption in vitro – potentially bioavailability (AE)
Fluids obtained after in vitro digestion were transferred to dial-
ysis sacks (D9777–100FT, Sigma-Aldrich), placed in an Erlenmeyer
flask containing 50 ml of PBS buffer and incubated in a rotary sha-
ker (2 times per 2 h, 37 °C). The PBS buffer, together with the com-
pounds that passed through the membrane (dialysate, GDA), was
treated as an equivalent of the raw material absorbed in the intes-
 M. Świeca et al. / Food Chemistry 221 (2017) 1451–1457
tine after digestion (at least 75% efficiency) (Gawlik-Dziki et al.,
2014).
2.5. Phenolics and caffeine content
Samples were analyzed with a Varian ProStar high-performance
liquid chromatography (HPLC) system separation module (Varian,
Palo Alto, CA, USA) equipped with a Varian ChromSpher C18
reverse phase column (250 mm  4.6 mm) and ProStar DAD detec-
tor (Świeca & Baraniak, 2014). The column thermostat was set at
40 °C. The mobile phase consisted of 4.5% acetic acid (solvent A)
and 50% acetonitrile (solvent B), and a flow rate of 0.8 ml min1
was used. At the end of the gradient, the column was washed with
50% acetonitrile and equilibrated to the initial conditions for
10 min. The gradient elution was used as follows: 0 min, 92% A;
30 min, 70% A; 45 min, 60% A; 80 min, 60% A; 82 min, 0% A;
85 min, 0% A; 86 min, 92% A; and 90 min, 92% A. Phenolics detec-
tion was carried out at 270 and 370 nm (250 nm for caffeine).
Spectrum analysis and a comparison of their retention times with
those of the standard compounds enabled identification of the phe-
nolics in a sample. Quantitative determinations were carried out
by means of the external standard calculation, using calibration
curves of the standards. Phenolics and caffeine were expressed in
micrograms per gram of dry mass (DW).
2.6. Inhibition of linoleic acid peroxidation (LPO)
The inhibition of the haemoglobin-catalyzed peroxidation of
linoleic acid was determined according to Goupy, Vulcain, Caris-
Veyrat, and Dangles (2007). Ten microliters of the extract obtained
after digestion in vitro was mixed with 0.37 ml of 5 mM phosphate
buffer (pH 7) containing 0.05% Tween 20 and 4 mM linoleic acid
and then equilibrated at 37 °C for 3 min. The peroxidation of lino-
leic acid in the above-mentioned reaction mixture was initiated by
adding 20 ll of 0.035% bovine haemoglobin (in water) followed by
incubation in a shaking water bath at 37 °C for 10 min. The reac-
tion was stopped by adding 5 ml 0.6% HCl (in ethanol). Hydrox-
yperoxide formation was assayed according to a ferric
thiocyanate method that consists of mixing first with 0.02 mol/l
FeCl2 (0.1 ml) and then with 30% ammonium thiocyanate
(0.1 ml). Absorbance (As) at 480 nm was measured (Lambda 40
UV–vis spectrophotometer, Perkin-Elmer Inc. Waltham, USA). The
absorbance of blank (Ao) was obtained without the addition of hae-
moglobin to the above reaction mixture; the absorbance of control
(A100) was obtained with no sample addition to the above mixture.
Thus, the antioxidative activity of the sample was calculated
according to the following equation:
LPO ½% ¼ ½1  ðAs  A0 Þ=ðA100  A0 Þ  100
The activity was expressed as Trolox equivalent in mg per g of
dry mass (DW).
2.7. Theoretical calculations
The following factors were determined to permit better under-
standing of the relationships between biologically active com-
pounds in the light of their bioaccessibility, bioavailability, and
bioefficiency (Gawlik-Dziki et al., 2015):
The phenolics bioaccessibility index (PAC), which is an indica-
tion of the bioaccessibility of phenolic compounds:
PAC ¼ CGE =CBE
The phenolics bioavailability index (PAV), which is an indication
of the bioavailability of phenolic compounds:
PAC ¼ CAE =CGE
1453
where CBE is the phenolic content in raw extract (BE), CGE is the phe-
nolic content in extracts after simulated gastrointestinal digestion
(GE), and CAE is the phenolic content in extracts after simulated
intestinal absorption (AE),
The antioxidant bioaccessibility index (BAC), which is an indica-
tion of the bioaccessibility of antioxidative compounds:
BAC ¼ AGE =ABE ð4Þ
The antioxidant bioavailability index (BAV), which is an indica-
tion of the bioavailability of antioxidative compounds:
BAV ¼ AAE =AGE ð5Þ
where, ABE is the activity of raw extract (BE), AGE is the activity of
extract after simulated gastrointestinal digestion (GE) and AAE is
the activity of extract after simulated intestinal absorption (AE).
2.8. Statistical analysis
All experimental results were expressed as mean ± S.D. of three
independent experiments (n = 18). One-way analysis of variance
(ANOVA) and Turkey’s post hoc test were used to compare groups
(STATISTICA 6, StatSoft, Inc., Tulsa, USA). Differences were consid-
ered significant at p < 0.05.
3. Results and discussion
The phenolic composition of wheat flour and green coffee bean
is well characterized; however, there are only a few studies con-
cerning the bioavailability and bioaccessibility of phenolics from
these sources. As shown in Table 1, wheat flour was characterized
by a high content of bound phenolics that were effectively released
during in vitro digestion. Such an observation confirms the results
of previous studies conducted by Budryn et al. (2015) and Hung,
Hatcher, and Barker (2011), who demonstrated that free phenolics
(regardless of wheat variety) account for only 10–40% of total
wheat phenolics. Green coffee contained high amounts of chloro-
genic acids, mainly 3-caffeoylquinic acid, which are bioavailable
and rapidly metabolizable in the human body (Farah, Monteiro,
Donangelo, & Lafay, 2008). In the cultured gastric epithelial model,
multiple chlorogenic acid isomers showed intact transfer across
the gastric barrier at an acidic apical pH, with dicaffeoylquinic
acids showing a relatively higher permeability coefficient com-
pared to CQA. Experiments conducted in a rat model showed that
CGAs are not hydrolyzed in the stomach but absorbed in an intact
form. This could explain the early detection of CGA in plasma
within 30 min of coffee consumption (Liang & Kitts, 2015). Also,
according to previous studies (Dziki et al., 2015) concerning green
ð1Þ
coffee from different locations (Brazil, Columbia, Ethiopia and
Kenya), these compounds are easily bioaccessible in vitro. The
results obtained after digestion in this study were comparable with
those obtained for the chemical extraction by the cited researchers.
The relatively low bioaccessibility and bioavailability determined
in these studies may be due to the fact that the results obtained
after in vitro digestion were compared to those achieved for buffer
extraction - chlorogenic acid is excellently isolated using an extrac-
tion system based on water.
Dupas et al. (2006) showed that about 10% and 12% of CGA were
bound to gastric or intestinal enzymes during in vitro digestion,
respectively. Additionally, according to a study by Stalmach,
Williamson, and Crozier (2014), the lowered bioavailability of phe-
ð2Þ nolics from green coffee may also be associated with a higher
ingested dose. Considering the above and by comparing these
two factors, it may be pointed out that the approximately 30 and
20 times higher contents of potentially bioaccessible and bioavail-
ð3Þ able phenolics predispose green coffee beans to be a functional
1454 M. Świeca et al. / Food Chemistry 221 (2017) 1451–1457

Table 1
Potentially bioaccessible and bioavailable phenolics and caffeine in green coffee beans and wheat flour.

Compounds Green coffee Wheat flour

[lg/g DM]
Buffer extract Extract after digestion Extracts absorbed Buffer Extract after digestion Extracts absorbed
in vitro in vitro extract in vitro in vitro
(+)-catechin 481 ± 12.5c Nd 75.2 ± 2.9b 49.9 ± 14.7a 84.1 ± 6.9b 75.2 ± 2.9b
Gallic acid Nd 134 ± 29.9a 121.4 ± 7.2a Nd Nd Nd
Caffeic acid Nd 9803 ± 758c 3687 ± 240b 5.8 ± 4.4a 19.4 ± 13.3a Nd
Protocatechuic acid 580 ± 172 Nd Nd Nd Nd Nd
Syringic acid Nd 183 ± 17b 86.5 ± 2.4a 87.9 ± 6.4a 377.7 ± 30c 77.5 ± 11a
Ferulic acid Nd 2628 ± 554c 225.4 ± 96b Nd 15.9 ± 13.3a 6.21 ± 0.98a
p-coumaric acid Nd 123 ± 6.8b 71.6 ± 5.8a Nd Nd Nd
Vanillic acid Nd Nd Nd Nd 128.7 ± 45b 46.2 ± 4.2a
3-caffeoylquinic acid 35,574 ± 743c 1380 ± 63b 446 ± 75.5a Nd Nd Nd
5-caffeoylquinic acid 4239 ± 796c 287 ± 30b 188 ± 15a Nd Nd Nd
4-caffeoylquinic acid 2435 ± 616c 282 ± 62b 193 ± 5.3a Nd Nd Nd
3-feruloylquinic acid 1082 ± 294b 227 ± 42.3a 195 ± 46a Nd Nd Nd
5-p-coumaroylquinic 1894 ± 948b 188 ± 61.4a 167 ± 120a Nd Nd Nd
acid
5-feruloylquinic acid 968 ± 327 Nd Nd Nd Nd Nd
3.5-dicaffeoylquinic 6855 ± 721c 241 ± 77b 84.1 ± 19.8a Nd Nd Nd
acid
4.5-dicaffeoylquinic 5682 ± 345 Nd Nd Nd Nd Nd
acid
Sum of phenolics 66,794 16,803 7919 160.9 562.8 381.8
Caffeine 31,460 ± 5589b 9683 ± 692a 9231 ± 576a – – –

Results were expressed as mean ± SD (n = 9). The values designated by the different letters (a,b,c) are significantly different (p < 0.05). Nd – not detected.

ingredient for food enrichment. However, the influence of the type
of food matrix, which affected CGA digestion and bioavailability,
remains unclear and represents an interesting area for more
research on factors that influence the bioaccessibility of CGAs
and other important dietary polyphenols. There is some evidence
that CGAs exhibit high affinity to proteins and Maillard reaction
products, which may lower their bioaccessibility (Dupas et al.,
2006). Strong evidence has shown that the majority of CGA is not
absorbed in the proximal part of the gastrointestinal tract, unless
it is transformed into caffeic and ferulic acids before being
absorbed (Liang & Kitts, 2015). Another aspect is that even when
absorption occurs in the small intestine, substantial quantities pass
to the large intestine where the parent compounds and their
catabolites can impact both colonic health and colonic microbiota.
Some of these compounds may play a key role in the protective
effects of phenolic rich foods (Crozier, Del Rio, & Clifford, 2010).
It this study, green coffee beans were used for the enrichment of
wheat bread. For better estimation of the fortification, systems
based on in vitro digestion and absorption were used to mirror
the amount of potentially bioaccessible and bioavailable bioactive
compounds. The contents of buffer extractable (BE), potentially
bioaccessible (extracts after in vitro digestion, DE) and potentially
bioavailable (extracts after in vitro absorption, AE) phenolics and
caffeine in the studied bread are presented in Table 2–4, respec-
tively. The control bread (BC) contained all of the phenolics previ-
ously found in wheat flour; however, their contents were about 2
times higher than in flour (Table 2). These phenomena may be par-
tially explained by the fact that during dough fermentation some
phenolics are released from flour, which has previously been
described by Chandrasekara and Shahidi (2012). On analysis of
the buffer extract, it was found that the addition of green coffee
flour into the bread formula significantly increased the phenolic
content - up to 4.17–times for the bread with 5% of supplement.
Also, the caffeine content significantly increased (about 3.3-fold
for the 5% bread). Syringic acids and (+)-catechin dominated in
the phenolic profile of the control bread. The enriched bread also

Table 2
Phenolics and caffeine content in the control and bread enriched with green coffee - buffer extracts.

Compounds Bread
[lg/g DM]
CB B1 B2 B3 B4 B5
Gallic acid 8.7 ± 5.0b 8.2 ± 1.0b 5.0 ± 0.2a 5.0 ± 0.4a 5.1 ± 2.0a 5.1 ± 2.0a
(+)-catechin 92.4 ± 31a 72.8 ± 24a 94.0 ± 11a 103.3 ± 15a 89.1 ± 29a 96.2 ± 13a
Caffeic acid 3.4 ± 0.8a 81.0 ± 3.2b 157.8 ± 12c 192.3 ± 21d 278.4 ± 28e 397.7 ± 94f
Ferulic acid 13.8 ± 2.1a 15.9 ± 0.8a 13.8 ± 1.6a 24.8 ± 2.4a 12.2 ± 3.9a 15.1 ± 2.0a
Syringic acid 107 ± 7.2c 86.2 ± 1.4b 83.3 ± 8.9b 87.6 ± 6.6b 43.8 ± 8.6a 47.3 ± 10.0a
3-caffeoylquinic acid Nd 40.6 ± 4.5a 99.8 ± 13b 135.5 ± 16c 187.1 ± 50cd 229.1 ± 59d
5-caffeoylquinic acid Nd 26.3 ± 3.1a 31.7 ± 10.9ab 37.3 ± 3.3b 42.3 ± 6.0b 45.7 ± 11.9b
4-caffeoylquinic acid Nd 2.0 ± 0.6a 57.7 ± 6.6c 45.1 ± 3.6b 37.9 ± 9.5b 64.0 ± 13.6d
3-feruloylquinic acid Nd 4.1 ± 2.3a 12.6 ± 2.1bc 9.0 ± 2.1b 17.1 ± 3.5c 9.0 ± 4.7bc
5-p-coumaroylquinic acid. Nd 1.4 ± 1.7a 1.7 ± 1.0a 1.4 ± 1.6a 1.3 ± 0.5a 1.6 ± 0.3a
5-feruloylquinic acid Nd 0.5 ± 0.15a 0.4 ± 0.15a 2.6 ± 0.9b 3.1 ± 0.9b 2.0 ± 0.4b
3.5-dicaffeoylquinic acid Nd 0.3 ± 0.03a 0.9 ± 0.08c 0.5 ± 0.05b 1.4 ± 0.4a 2.7 ± 0.5e
4.5-dicaffeoylquinic acid Nd 6.3 ± 1.8a 21.6 ± 2.9bc 18.9 ± 1.8b 15.3 ± 3.6b 26.2 ± 3.6c
Sum of phenolics 225.8 445.4 619.5 569.4 734.0 941.6
Caffeine Nd 71 ± 5.7a 133 ± 32.5bc 163 ± 0.5c 175 ± 56.8cd 225 ± 56.8d

CB – control bread; B1–B5 – breads supplemented with green coffee beans (1–5%).
Results were expressed as mean ± SD (n = 9). The values designated by the different letters (a,b,c,d,e,f) in the rows of the table are significantly different (p < 0.05). Nd – not
detected.
 M. Świeca et al. / Food Chemistry 221 (2017) 1451–1457 1455

Table 3
Phenolics and caffeine content in the control and bread enriched with green coffee – extracts obtained after digestion in vitro.

Compounds Bread
[lg/g DM]
CB B1 B2 B3 B4 B5
Gallic acid Nd 5.3 ± 1.8a 5.6 ± 2.0a 6.3 ± 0.8a 6.5 ± 0.7a 8.7 ± 3.1a
(+)-catechin 105.1 ± 8.4c 59.7 ± 10.8a 67.3 ± 6.9ab 77.8 ± 8.0ab 63.5 ± 10.8ab 77.3 ± 3.6b
Caffeic acid 25.8 ± 1.3a 220.5 ± 26.2b 351.4 ± 20.9c 532.2 ± 95.8d 733.4 ± 111.7de 844.6 ± 122.6e
Ferulic acid 12.3 ± 2.6a 11.3 ± 1.1a 14.3 ± 1.2ab 14.9 ± 1.7b 21.5 ± 1.7c 25.9 ± 2.5c
Syringic acid 287 ± 15.9a 296 ± 45.9ab 288 ± 44.4ab 3091 ± 12.0ab 324 ± 15.4b 327 ± 11.5b
p-coumaric acid Nd 1.5 ± 0.6a 4.1 ± 1.2b 4.4 ± 1.0b 4.4 ± 1.0b 7.4 ± 1.3c
Vanillic acid 46.9 ± 3.3b 48.6 ± 3.5b 46.4 ± 3.2b 39.6 ± 5.2a 41.2 ± 1.58a 38.6 ± 2.6a
3-caffeoylquinic acid Nd 38.6 ± 22.3a 90.1 ± 17.4b 123.9 ± 30.4bc 128.7 ± 49.0c 146.4 ± 9.7c
5-caffeoylquinic acid Nd 46.2 ± 7.6a 65.5 ± 9.5b 68.7 ± 3.9b 69.3 ± 2.3b 120.0 ± 12.1c
4-caffeoylquinic acid Nd 36.2 ± 2.5a 69.2 ± 4.1b 64.9 ± 2.1b 72.4 ± 7.8b 93.7 ± 10.4c
3-feruloylquinic acid Nd 11.4 ± 2.7a 13.3 ± 3.6a 11.8 ± 1.9a 18.1 ± 1.2b 19.3 ± 2.3b
Sum of phenolics 477.3 763.6 977.5 1212.2 1464.9 1694.3
Caffeine Nd 304 ± 9.4a 714 ± 10.9b 1072 ± 24.3c 1186 ± 49.9c 1296 ± 111d

CB – control bread; B1–B5 – breads supplemented with green coffee beans (1–5%).
Results were expressed as mean ± SD (n = 9). The values designated by the different letters (a,b,c,d,e) in the rows of the table are significantly different (p < 0.05). Nd -not
detected.

Table 4
Phenolics and caffeine content in the control and bread enriched with green coffee – extracts obtained after absorption in vitro.

Compounds Bread
[lg/g DM]
CB B1 B2 B3 B4 B5
Gallic acid 7.3 ± 1.1ab 5.9 ± 1.1a 5.4 ± 1.3a 7.0 ± 2.6ab 8.4 ± 1.5b 9.0 ± 2.0b
(+)-catechin 43.2 ± 12.16a 58.5 ± 13.6ab 69.9 ± 12.7bc 73.7 ± 2.9bc 76.8 ± 7.5 cd 88.1 ± 3.0d
Caffeic acid Nd 82.3 ± 34.6a 157.3 ± 26.0b 191.4 ± 26.0bc 202.4 ± 23.6bc 238.3 ± 56.3c
Ferulic acid 5.4 ± 0.7a 5.1 ± 1.8ab 8.6 ± 3.4abc 6.9 ± 3.3abc 8.0 ± 1.5bc 9.4 ± 1.3c
Syringic acid 77.5 ± 11.7a 71.2 ± 9.2a 75.2 ± 13.0a 77.5 ± 20.0a 66.9 ± 6.6a 58.7 ± 10.1a
Vanillic acid 36.9 ± 3.3bc 38.6 ± 3.5c 35.4 ± 3.2bc 29.6 ± 2.7ab 38.0 ± 3.4c 27.5 ± 2.5a
3-caffeoylquinic acid Nd 26.6 ± 2.8a 52.4 ± 6.8b 82.2 ± 11.2c 110.1 ± 13.0d 131.3 ± 10.0e
5-caffeoylquinic acid Nd 3.9 ± 0.0a 12.6 ± 3.8b 18.8 ± 0.9c 21.4 ± 2.2 cd 29.1 ± 6.9d
4-caffeoylquinic acid Nd 10.6 ± 2.6a 12.1 ± 4.3ab 15.9 ± 2.6b 25.7 ± 5.3c 27.8 ± 3.0c
3-feruloylquinic acid Nd 3.4 ± 0.1a 4.6 ± 0.1b 6.3 ± 0.2c 7.4 ± 0.2d 9.2 ± 0.3e
Sum of phenolics 197.1 333.3 444.6 535.8 583.6 633.6
Caffeine Nd 66.6 ± 3.5a 296.5 ± 14.4b 366.0 ± 7.5c 561.9 ± 24.0d 836.3 ± 13.9e

CB – control bread; B1–B5 – breads supplemented with green coffee beans (1%–5%).
Results were expressed as mean ± SD (n = 9). The values designated by the different letters (a,b,c,d,e) in the rows of the table are significantly different (p < 0.05). Nd – not
detected.

contained significant amounts of chlorogenic acids derived from
the functional ingredient (Table 2). The in vitro digestion effectively
released both phenolics and caffeine from the studied breads
(Table 3). The phenolics bioaccessibility index (PAC) being signifi-
cantly higher than 1, indicates that phenolics from the studied
bread were highly bioaccessible in vitro. Most importantly, the
PAC values determined for the enriched bread were lower than
those recorded for the control (2.11) (Table 5), which may indicate
the occurrence of phenolic – bread matrix interactions previously
described for wheat bread enriched with a phenolic-rich supple-
ment (Swieca et al., 2013; Swieca et al., 2014). Effectiveness of for-
tification was obvious – compared to the control, for the bread
with 1% and 5% of the functional component phenolic contents
were 1.6 and 3.33 times higher, respectively. Conditions similar
to those occurring in the human digestive tract caused significant
qualitative changes in the phenolics. Caffeic acid, syringic acid
and vanillic acid were released from the wheat matrix. Addition-
ally, ester linkages of chlorogenic acids in the enriched bread were
cleaved – those samples were characterized by significant contents
of caffeic and ferulic acids. Compared to the buffer extract, caffeine
contents were significantly higher – about 4.3-fold for the bread
with 5% of supplement (Table 3). The values of the phenolics
bioavailability index (PAV) confirmed that potentially bioactive
compounds from the studied breads were poorly bioavailable
in vitro. The highest bioavailability in vitro was found for
the bread with 1%–3% (PAV = 0.44), whereas the lowest value
was determined for those with 5% of supplement (PAV = 0.37)
(Table 5). The diminished potential bioavailability of phenolics
may be partially explained by the use of model systems (exhibiting
by definition only 82.5% of passive transport). Despite this, supple-
mentation of bread increased contents of both phenolics as well as
caffeine (Table 4). Comparing these samples (AE), it was found that
3-caffeoylquinic acid was very effectively absorbed; however, the
sum of phenolics after absorption was about 2.7 times lower com-
pared to the potentially bioaccessible fraction. As mentioned
above, some quantitative changes were found, however, there
were no qualitative changes in the phenolics (generally phenolics
present in the extract obtained after in vitro digestion were also
found after the simulated absorption).
As the antioxidant potential of food is mainly created by low-
molecular weight antioxidants, thus the effect of incorporation of
green coffee phenolics as well as caffeine into wheat bread on
the ability to protect lipids against induced oxidation was also
studied (Fig. 1). The enriched bread exhibited a significantly higher
antioxidant activity (compared to the control) probably due to a
significantly increased content of caffeine and phenolics acid –
antioxidants with well–documented antioxidant capacity (Dziki
et al., 2015; Farah et al., 2008; Li Kwok Cheong et al., 2014).
Between the studied fractions, the highest activity was found for
the extracts obtained after in vitro digestion (DE). The activity of
bread with 5% of supplement was about 2.3-fold higher than that
of the control (Fig. 1). Most importantly, the bioactive compounds
1456 M. Świeca et al. / Food Chemistry 221 (2017) 1451–1457

Table 5
Relationships between biologically active compounds in the light of their bioaccessibility, bioavailability and bioefficiency.

Bread sample Phenolics Phenolics bioaccessibility Antioxidant bioavailability Antioxidant

bioavailability index (PAC) index (BAV) bioaccessibility
index (PAV) index (BAC)
CB 0.41 2.11 0.13 17.69
B1 0.44 1.71 0.18 15.19
B2 0.45 1.58 0.21 21.42
B3 0.44 1.83 0.18 17.84
B4 0.40 2.00 0.28 19.20
B5 0.37 1.80 0.19 21.71

CB – control bread; B1–B5 – breads supplemented with green coffee beans (1–5%).

90 relations, which may indicate that also interactions (e.g. synergism,

CB m antagonism) between them are important. Such an observation
80
B1 was previously described by Williamson (2001) in different plant
70 B2 formulations.
Inhibition of lipids peroxidation

l
B3 kl
60 jk
B4 ij 4. Conclusion
[mg TE/ g]

50
B5
i
40 The addition of green coffee flour into wheat bread significantly
30
improved the phenolic content and their ability to protect lipids
h h against oxidation. The in vitro digestion induced the release of phe-
20
fg
nolics from bread, causing significant qualitative changes. Most
de f
10 ab d importantly, the introduced phenolics were potentially bioaccessi-
b b c
a a ble and bioavailable in vitro, however the influence of the food
0 matrix and its interaction with CGAs also played an important role
Buffer extractable Potentially bioaccessible Potentially bioavailable
compounds compounds compounds in the bioactivity of the functional product. According to the
results, it may be concluded that the qualitative composition of
Fig. 1. Ability of buffer extractable, potentially bioaccessible and bioavailable the phenolic fraction plays a key role in creating antioxidant poten-
fractions of control and enriched bread to inhibit lipid peroxidation CB- control
tial; however caffeine and potential synergism between low-
bread; B1-B5 – breads supplemented with green coffee beans (1%–5%) Results were
expressed as mean ± SD (n = 9). The values designated by different letters are molecular weight antioxidants are important as well. To sum up,
significantly different (p < 0.05). the fortification of wheat bread with green coffee is an effective
tool that allows obtaining functional food with a significantly
enhanced nutraceutical potential to be obtained.

responsible for the lipid protecting activity were highly bioaccessi-

ble in vitro – the values of the antioxidant bioaccessibility index
(BAC) ranged from 15.2 to 21.7 (the effect of supplementation
was not clearly visible) (Table 5). Unfortunately, according to the
antioxidant bioavailability index (BAV) it was shown that antioxi-
dant compounds were very poorly bioavailable in vitro. It may be
suggested that antioxidants realized during digestion in vitro are
not able to permeate the membrane due to polarity and/or com-
pounds size (Dupas et al., 2006). It is also well documented that
phenolics are able to interact with the food matrix and form indi-
gestible complexes that cannot be absorbed (Jakobek, 2015).
The highest bioactivity is not always directly translated into
higher values of factors describing potential bioavailability and
bioaccessibility. Comparing the level of activity with the content
of bioactive compounds it may be stated that the most important
role in creating the antioxidant potential is ascribed to the qualita-
tive composition of phenolics; however, the role of caffeine as well
as interactions of bioactive components seem to be important as
well. The studied activity was significantly higher in the absorbed
extracts compared to the buffer extracts. The buffer and absorbed
extracts were characterized by a similar content of phenolics; how-
ever, their qualitative composition varied. In turn, the potentially
bioaccessible and bioavailable fractions had the same phenolics
but the extracts after in vitro digestion exhibited much higher
activity – they had much higher amounts of phenolics. This con-
firms earlier findings reported by Baeza et al. (2014) concerning
the protective effect of green coffee hydroxycinnamic acids against
oxidative stress in a human HepG2 cells model. Moreover, the
chemical composition (phenolics as well as caffeine) only partially
explains differences between the extracts. There are no linear cor-
Conflict of interests
The authors declare that there is no conflict of interest regard-
ing the publication of this paper.
Acknowledgement
This scientific study was financed by the National Science
Centre, Poland (grant 2013/09/B/NZ9/01801). We are grateful to
Romuald Zalewski who provided the raw material for research.
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