Accepted Manuscript

Wheat br ead enr iched with gr een coffee – in vitro bioaccessibility and bio
availability of phenolics and antioxidant activity

Michał Świeca, Urszula Gawlik-Dziki, Dariusz Dziki, Barbara Baraniak

PII: S0308-8146(16)31829-5
DOI: http://dx.doi.org/10.1016/j.foodchem.2016.11.006
Reference: FOCH 20138

To appear in: Food Chemistry

Received Date: 21 June 2016

Revised Date: 7 October 2016
Accepted Date: 1 November 2016

Please cite this article as: Świeca, M., Gawlik-Dziki, U., Dziki, D., Baraniak, B., Wheat br ead enr iched with gr een
coffee – in vitro bioaccessibility and bioavailability of phenolics and antioxidant activity, Food Chemistry
(2016), doi: http://dx.doi.org/10.1016/j.foodchem.2016.11.006

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Wheat bread enriched with green coffee – in vitro bioaccessibility and bioavailability of

phenolics and antioxidant activity

Running title: Bread enriched with green coffee beans

Michał Świeca*a, Urszula Gawlik-Dzikia, Dariusz Dzikib, Barbara Baraniaka

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

* Corresponding author. Tel.:+ 48-81- 4623327 fax: +48-81-4623324

E-mail address: michal.swieca@up.lublin.pl;

ABSTRACT

The potential bioaccessibility and bioavailability of phenolics, caffeine and antioxidant

activity of wheat bread enriched with green coffee were studied. Supplementation enhanced

nutraceutical potential by improving phenolic content and lipid protecting capacity. The

simulated-digestion-released phenolics (mainly caffeic acid, syringic acid and vanillic acid)

from bread, also caused significant qualitative 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 contents of phenolics

were 1.6 and 3.33 times higher. Also, an approximately 2.3-fold increase in antioxidant

activity was found in bread containing 5% of the supplement. The compounds responsible for

antioxidant potential have high bioaccessibility but poor bioavailability. The qualitative

composition of the phenolic fraction has a key role in developing the antioxidant potential of

bread; however, caffeine and synergism between antioxidants are also important

considerations.

1
Keywords: bioaccessibility; bioavailability; bread; fortification; green coffee; in vitro,

phenolics.

1. Introduction

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 additional benefits (Allen, de

Benoist, Dary, & Hurrell, 2006). Fortification 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, Różyło, 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 activities, are widely used for food fortification (Arts and

Hollman, 2005; Wang, Melnyk, Tsao, & Marcone, 2011). So far, there is no substantial

evidence as to which method is the most appropriate for measuring the

bioaccessibility/bioavailability of these bioactive 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

2
bioaccessibility and bioavailability of functional supplements whilst evaluating the quality of

new products. 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 50µM

(e.g. like in coffee and coffee-based products) is toxic and by destroying the monolayer of

cells causes an unspecific transfer of phenolics, 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 absorption of 5-caffeoylquinic acid (5-CQA) has been

studied in a cell culture, using the human colon carcinoma cell line Caco-2. The absorption

rate for 5-CQA was found to be about 0.10% at physiological concentrations equivalent to gut

lumen concentrations (0.1~1 mM). Transepithelial transport experiments with chlorogenic

acid (CGA) using Caco-2 intestine epithelia cultured monolayer demonstrated bidirectional

permeation with no transport into the basolaterial side, regardless of pH gradient. The

permeation rate was concentration-dependent and not saturable, thus indicating passive

diffusion. In addition, the transport was inversely correlated 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 et al.,

3
2006). An important role has also been proven for the interactions of phenolics with other

active components 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, & Czyż, 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,

Żyżelewicz, & Oracz, 2014; Gawlik-Dziki, Świeca, Dziki, Kowalska, Pecio, Durak, &

Sęczyk, 2014; Liang and 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 bioaccessibility 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

4
 α–amylase, pancreatin, pepsin, bile extract, linoleic acid, Tween-20 and haemoglobin

were purchased from Sigma–Aldrich Company (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 laboratory

hammer mill (POLYMIX-Micro-Hammermill MFC, Kinematica. 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 acceptance (data previously published (Dziki et al.,

2015)). Besides this, 6 g of instant yeast and 12 g of salt were used for dough preparation. 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)

5
 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 KH2PO4, 8 g NaCl and 100 mg of mucin in 1 liter of distilled water. The solution was

adjusted to pH 6.75 and α-amylase (E.C. 3.2.1.1.) was added to obtain 200 U/ml of enzyme

activity. For gastric digestion, 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 gastrointestinal digestion as

follows: 1 g of powdered sample (wheat and green coffee flours as well as enriched bread)

was homogenized in a Stomacher laboratory blender for 1 min to simulate mastication 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 37ºC. 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 mixture 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 ºC and in the dark. Thereafter, the samples were centrifuged and the supernatants were

used for further analysis.

2.4.3. Absorption in vitro – potentially bioavailability (AE)

6
 Fluids obtained after in vitro digestion were transferred to dialysis sacks (D9777–

100FT, Sigma-Aldrich), placed in an Erlenmeyer flask containing 50 ml of PBS buffer and

incubated in a rotary shaker (2 times per 2 h, 37 °C). The PBS buffer, together with the

compounds that passed through the membrane (dialysate, GDA), was treated as an equivalent

of the raw material absorbed in the intestine 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 detector

(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 min−1 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 detection 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 phenolics 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 Groupy, Vulcain, Caris-Veyrat, & 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)

7
containing 0.05 % Tween 20 and 4 mM linoleic acid and then equilibrated at 37 oC for 3 min.

The peroxidation of linoleic acid in the above-mentioned reaction mixture was initiated by

adding 20 µl of 0.035% bovine haemoglobin (in water) followed by incubation in a shaking

water bath at 37 oC for 10 min. The reaction was stopped by adding 5 ml 0.6% HCL (in

ethanol). Hydroxyperoxide 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 haemoglobin 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 [%]= [1-(As-A0)/(A100-A0)] × 100 (1)

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 understanding of the

relationships between biologically active compounds in the light of their bioaccessibility,

bioavailability, and bioefficiency (Gawlik-Dziki et al., 2015):

– the phenolics bioaccessibility index (PAC), which is an indication of the bioaccessibility of

phenolic compounds:

PAC= CGE/CBE (2)

– the phenolics bioavailability index (PAV), which is an indication of the bioavailability of

phenolic compounds:

PAV= CAE/CGE (3)

8
where CBE is the phenolic content in raw extract (BE), CGE is the phenolic 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 indication of the bioaccessibility

of antioxidative compounds:

BAC= AGE/ABE (4)

– the antioxidant bioavailability index (BAV), which is an indication of the bioavailability of

antioxidative compounds:

BAV= AAE/AGE (5)

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

considered 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 concerning 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 et al. (2011), who demonstrated that free phenolics (regardless of wheat variety)

9
account for only 10% to 40% of total wheat phenolics. Green coffee contained high amounts

of chlorogenic 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 compared 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 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 extraction 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, & Crozier (2014), the lowered bioavailability of phenolics 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 bioavailable phenolics predispose green

coffee beans to be a functional 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

10
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, 3 and 4, respectively. The control

bread (BC) contained all of the phenolics previously found in wheat flour; however, their

contents were about 2 times higher than in flour (Table 2). These phenomena may be partially

explained by the fact that during dough fermentation some phenolics are released from flour,

which has previously been described by Chandrasekara & 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 contained significant amounts of chlorogenic acids derived from the

functional ingredient (Table 2). The in vitro digestion effectively released both phenolics and

11
caffeine from the studied breads (Table 3). The phenolics bioaccessibility index (PAC) being

significantly 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 supplement (Swieca et al., 2013; Swieca et al., 2014).

Effectiveness of fortification 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. Additionally, 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, supplementation 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 compared to the potentially bioaccessible fraction. As

mentioned above, some quantitative changes were found, however, there were no qualitative

12
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 responsible for the lipid protecting activity were highly bioaccessible 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 antioxidant 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 compounds

size (Dupas et al., 2006). It is also well documented that phenolics are able to interact with

the food matrix and form indigestible 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 qualitative 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

13
extracts. The buffer and absorbed extracts were characterized by a similar content of

phenolics; however, 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 confirms

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 correlations, which may indicate that

also interactions (e.g. synergism, antagonism) between them are important. Such an

observation was previously described by Williamson (2001) in different plant formulations.

4. Conclusion

The addition of green coffee flour into wheat bread significantly improved the

phenolic content and their ability to protect lipids against oxidation. The in vitro digestion

induced the release of phenolics from bread, causing significant qualitative changes. Most

importantly, the introduced phenolics were potentially bioaccessible and bioavailable in vitro,

however the influence of the food matrix and its interaction with CGAs also played an

important role in the bioactivity of the functional product. According to the results, it may be

concluded that the qualitative composition of the phenolic fraction plays a key role in creating

antioxidant potential; however caffeine and potential synergism between low-molecular

weight antioxidants are important as well. To sum up, 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.

Acknowledgements

14
 This scientific study was financed by the Polish National Science Centre (grant

2013/09/B/NZ9/01801). We are grateful to Romuald Zalewski who provided the raw material

for research.

Conflict of interests

The authors declare that there is no conflict of interest regarding the publication of this

paper.

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Figure caption

Fig. 1. Ability of buffer extractable, potentially bioaccessible and bioavailable fractions of

control and enriched bread to inhibit lipid peroxidation

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 different letters are

significantly different (p< 0.05).

90
CB m
80
B1
70 B2
Inhibition of lipids peroxidation

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

50
B5
40 i

30
h h
20
fg
de f
10 ab d
a a b b c
0
Buffer extractable Potentially bioaccessible Potentially bioavailable
compounds compounds compounds

21
Table 1. Potentially bioaccessible and bioavailable phenolics and caffeine in green coffee beans and wheat flour

Green coffee Wheat flour

Compounds Extract
Extract after Extracts Buffer Extracts
Buffer extract after
digestion absorbed extract absorbed
[µg/ g DM] digestion
in vitro in vitro 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
Nd 134±29.9a 121.4±7.2a Nd Nd Nd
acid

Caffeic
Nd 9803±758c 3687±240b 5.8±4.4a 19.4±13.3a Nd
acid

Protocatechuic
580±172 Nd Nd Nd Nd Nd
acid

Syringic
Nd 183±17b 86.5±2.4a 87.9±6.4a 377.7±30c 77.5±11a
acid

Ferulic
Nd 2628±554c 225.4±96b Nd 15.9±13.3a 6.21±0.98a
acid

p-coumaric
Nd 123±6.8b 71.6±5.8a Nd Nd Nd
acid

Vanillic
Nd Nd Nd Nd 128.7±45b 46.2±4.2a
acid

3-caffeoylquinic
35574±743c 1380±63b 446±75.5a Nd Nd Nd
acid

5-caffeoylquinic
4239±796c 287±30b 188±15a Nd Nd Nd
acid

4-caffeoylquinic
2435±616c 282±62b 193±5.3a Nd Nd Nd
acid

3-feruloylquinic
1082±294b 227±42.3a 195±46a Nd Nd Nd
acid

5-p-coumaroylquinic
1894±948b 188±61.4a 167±120a Nd Nd Nd
acid

22
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 66794 16803 7919 160.9 562.8 381.8

Caffeine 31460±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

23
Table 2. Phenolics and caffeine content in the control and bread enriched with green coffee - buffer extracts

Bread
Compounds
CB B1 B2 B3 B4 B5
[µg/ g DM]
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
a bc c cd
Caffeine Nd 71±5.7 133±32.5 163±0.5 175±56.8 225±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

24
Table 3. Phenolics and caffeine content in the control and bread enriched with green coffee – extracts obtained
after digestion in vitro.

Compounds Bread
[µg/ g DM] CB B1 B2 B3 B4 B5
a a a a
Gallic acid Nd 5.3±1.8 5.6±2.0 6.3±0.8 6.5±0.7 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
b c c
Caffeine Nd 304±9.4a 714±10.9 1072±24.3 1186±49.9 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

25
Table 4. Phenolics and caffeine content in the control and bread enriched with green coffee – extracts obtained
after absorption in vitro.

Compounds Bread
[µg/ 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±7.5cd 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±2.2cd 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

26
Table 5. Relationships between biologically active compounds in the light of their bioaccessibility,
bioavailability and bioefficiency

Phenolics Phenolics Antioxidant Antioxidant

Bread sample bioavailability bioaccessibility bioavailability bioaccessibility
index (PAV) index (PAC) index (BAV) 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%)

27
1. Phenolics and antioxidant activity of bread with green coffee were studied
2. Caffeic and ferulic acids are released from chlorogenic acid after digestion in vitro
3. Antioxidant from the enriched bread are potentially bioaccessible and bioavailable
4. Addition of green coffee improves the ability to protect lipids against oxidation

28