The influence of protein–flavonoid interactions on protein digestibility in vitro and the antioxidant quality of breads enriched with onion skin

Food Chemistry 141 (2013) 451–458

Contents lists available at SciVerse ScienceDirect

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

The influence of protein–flavonoid interactions on protein digestibility in vitro and the antioxidant quality of breads enriched with onion skin Michał S´wieca a,⇑, Urszula Gawlik-Dziki a, Dariusz Dziki b, Barbara Baraniak a, Jarosław Czyz_ c a

Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin, Poland Department of Thermal Technology, University of Life Sciences, Dos´wiadczalna Str. 44, 20-280 Lublin, Poland c Department of Cell Biology, Jagiellonian University, Gronostajowa Str.7, 30-387 Cracow, Poland b

a r t i c l e

i n f o

Article history: Received 10 December 2012 Received in revised form 11 March 2013 Accepted 13 March 2013 Available online 21 March 2013 Keywords: Breads Fortification Onion skin Protein–phenolics interaction Antioxidant activity Bioaccessibility

a b s t r a c t Different types of breads enriched with onion skin were studied. The objectives were twofold: to show and examine protein–phenolic interactions and to discuss results concerning phenolic content, antioxidant activity and protein digestibility in the light of in vitro bioaccessibility. Phenolic contents and antiradical abilities were linked with the level of onion skin supplement however, the amounts determined were significantly lower than expected. Fortification influenced protein digestibility (a reduction from 78.4% for control breads to 55% for breads with a 4% supplement). Electrophoretic and chromatographic studies showed the presence of indigestible protein–flavonoid complexes – with molecular weights about 25 kDa and 14.5 kDa; however, the reduction of free amino group levels and the increase in chromatogram areas suggest that flavonoids also bind to other bread proteins. The interaction of phenolics with proteins affects antioxidant efficacy and protein digestibility; thus, they have multiple effects on food quality and pro-health properties. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Breadmaking is a complex process in which dough, typically composed of flour, water, yeast, sugar, salt and oil, is mixed, kneaded, proofed and baked. Nowadays, consumers prefer to eat healthier foods in order to prevent non-communicable diseases. For this reason, the industry and researchers are involved in optimizing bread-making technology to improve the quality, taste, functionality and bioavailability of bakery goods. Among the ingredients that could be included in bread formulation are herbs, spices and other functional components, which may significantly improve its nutraceutical potential (Balestra, Cocci, Pinnavaia, & Romani, 2011; Gawlik-Dziki, Dziki, Baraniak, & Lin, 2009; Glei, Kirmse, Habermann, Persin, & Pool-Zobel, 2006). Onion skin (OS) has been shown to be an excellent source of quercetin and its derivatives (Gawlik-Dziki, S´wieca, et al., 2012; Wiczkowski et al., 2008). Quercetin is a strong in vitro antioxidant and, additionally, it possesses anti-inflammatory, antimicrobial and anticancer properties (Gawlik-Dziki, S´wieca, et al. 2012; Gawlik-Dziki, S´wieca, Sugier, & Cichocka, 2011). During bread preparation an increase in temperature can promote the formation of protein cross-links, causing the setting of the loaf during baking. Additionally, interactions between

⇑ Corresponding author. Tel.: +48 81 4623327; fax: +48 81 4623324. E-mail address: [email protected] (M. S´wieca). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.03.048

gelatinized starch granules and the gluten network occur in crumb, causing a loss of kinetic energy and subsequently an increase in firmness. Supplementation of bread dough with functional components, such as phenolics, dietary fiber etc., significantly change the relationships between bread components affecting the bioaccessibility and bioavailability of nutrients and bioactive ingredients (Sivam, Sun-Waterhouse, Quek, & Perera, 2010). The amount of bioaccessible food polyphenols, and thus antioxidants, may differ quantitatively and qualitatively from polyphenols included in food databases (Gawlik-Dziki, S´wieca, et al., 2012; Gaw_ lik-Dziki, Jezyna, et al., 2012). Some trials aimed at an explanation of this fact were undertaken by Arts et al. (2002) and Sivam, SunWaterhouse, Perera, and Waterhouse (2011). The cited researchers showed that phenolics could precipitate proteins through different mechanisms, such as hydrophobic and ionic interactions, and hydrogen and covalent bindings. Another consequence of the interaction might be a decrease in free phenolic levels, which may reduce the antioxidant status of food. Studies concerning the effect of these interactions on antioxidant capacity showed that a part of the activity is masked. The masking depends on both the protein and the phenolics used (Arts, Haenen, Voss, & Bast, 2001; Arts et al., 2002; Han & Koh, 2011a; Peng et al., 2010; Sivam et al., 2010). Additionally, due to lowering bioaccessibility, nutritional potential of fortified food is closely linked with interactions between phenolics with salivary proline-rich proteins and/or other digestive enzymes and food protein (Charlton et al., 2002; Kroll,

452

M. S´wieca et al. / Food Chemistry 141 (2013) 451–458

Rawel, Rohn, & Czajka, 2001). Phenolics are able to precipitate proteins; thus, their bioavailability may be significantly reduced. In the light of these facts, it may be speculated that the nutraceutical and nutritional potential of enriched breads is limited by some interaction, which influences bioaccessibility and bioavailability. Food enrichment is justified only when the bioactive components are bioaccessible and bioavailable. In our previous studies flavonoids from onion skin were highly bioavailable and bioaccessible; however, the bioactivities of enriched breads were significantly lower than expected (Gawlik-Dziki, S´wieca, et al., 2012). In the light of these results the existence of slightly in vitro bioavailable quercetin–protein complexes was suggested. The objectives of this study were twofold: (a) to show and examine potential protein–phenolic interactions, (b) to discuss results concerning phenolic levels, antioxidant activity and protein digestibility of enriched breads in the light of in vitro bioaccessibility. 2. Materials and methods 2.1. Dry onion skin-based supplement (OS) preparation The food supplement was prepared as follows: dry onion (Allium cepa, var. Wolska) skin was washed twice with deionised water and dried in an oven at 50 °C. Once dried, the material was powdered using a laboratory mill and then sieved (60 mesh). 2.2. Chemicals ABTS (2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)), a-amylase, pancreatin, pepsin, bile extract, were purchased from Sigma–Aldrich company (Poznan, Poland). All others chemicals were of analytical grade. 2.3. Bread making The flour used in the formula of control bread (BC) was wheat bread flour (600 g), type 750 (average 0.75% ash content, humidity 14%). The flour was replaced with OS at 1–4% levels (B1–B4%, respectively). The percentage of OS addition was chosen on the basis of a previous test on antioxidant activity (data not published). 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 in flour of a consistency 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 an oven heated up to 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. 2.4. Extracts preparation After baking, bread samples were allowed to cool down to room temperature for 24 h. Subsequently, the samples were sliced (slices about 1.5 cm thick). The crust was removed aseptically and crumb was kept frozen (at 20 °C) until analysis. After thawing, the slices were dried and then manually crumbed, ground in a mill and screened through a 0.5 mm sieve to obtain bread powder. 2.4.1. Buffer extracts (BE) Powdered samples of bread or dry onion skin (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 darkness at 20 °C.

2.4.2. Gastrointestinally digested (GE) In vitro digestion was performed as described previously Gawlik-Dziki, S´wieca, et al. (2012). Simulated saliva solution was prepared by dissolving 2.38 g Na2HPO4, 0.19 g KH2PO4, and 8 g NaCl, 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 digestion 300 U/mL of pepsin (from porcine stomach mucosa, pepsin A, EC 3.4.23.1) was prepared in 0.03 mol/L NaCl, pH = 1.2. Further, 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 onion skin and bread samples were subjected to simulated gastrointestinal digestion as follows: 1 g of powdered sample was homogenized in a Stomacher laboratory blender for 1 min to simulate mastication in the presence of 15 mL of simulated salivary fluid; and subsequently, the samples were shaken for 10 min at 37 °C. The samples were adjusted to pH = 1.2 using 5 mol/L HCl; and subsequently, 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 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 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 darkness. Thereafter, samples were centrifuged and supernatants were used for further analysis. 2.5. Phenolics content 2.5.1. Determination of total phenolic content (TPC) The amount of total phenolics was determined using Folin–Ciocalteau reagent (Singleton & Rossi, 1965). To 0.5 mL of the sample, 0.5 mL H2O, 2 mL Folin–Ciocalteau reagent (1:5 H2O) were added, and after 3 min, 10 mL of 10% Na2CO3 and the contents were mixed and allowed to stand for 30 min. Absorbance at 725 nm was measured in a UV–Vis spectrophotometer. The amount of total phenolics was calculated as a gallic acid equivalent (GAE) in mg/g of dry weight (DW). Expected total phenolic content (TPCe) was calculated as follow:



TPCe ¼ TPCBC þ

 TPCOS xN ; 100%

ð1Þ

where: TPCBC – activity of control bread; TPCos – activity of onion skin; N – percent of onion skin supplement. 2.5.2. Determination of flavonoids content (TFC) Total flavonoid content was determined according to the method described by Lamaison and Carnet (1990). One milliliter of extract was mixed with 1 mL of 2% AlCl3  6H2O solution and incubated at room temperature for 10 min. Thereafter, absorbance at 430 nm was measured. Total flavonoid content was calculated as a quercetin equivalent (QE) in mg/g of dry weight (DW). Expected total flavonoids content (TFCe) was calculated as follow:

TFCe ¼ TFCBC þ

  TFCOS xN ; 100%

ð2Þ

where: TFCBC – activity of control bread; TFCos – activity of onion skin; N – percent of onion skin supplement. 2.6. Free radical scavenging assay (ABTS assay) The experiments were performed using an improved ABTS decolorization assay (Re et al., 1999). ABTS+ was generated by

M. S´wieca et al. / Food Chemistry 141 (2013) 451–458

453

the oxidation of ABTS with potassium persulfate. The ABTS radical cation (ABTS+) was produced by reacting 7 mmol/L stock solution of ABTS with 2.45 mmol/L potassium persulphate (final concentration). The ABTS+ solution was diluted (with distilled water) to an absorbance of 0.7 ± 0.05 at 734 nm. Then, 40 lL of samples were added to 1.8 mL of ABTS+ solution and the absorbance was measured at the end time of 5 min. The ability of the extracts to quench the ABTS free radical was determined using the following equation:

The column thermostat was set at 30 °C. The amount of 20 lL of each sample solution was loaded on the column, and protein and peptides were eluted using a 20 mM PBS buffer pH 7.4. The flow rate was 1 mL min1. Ultraviolet detection was performed at a wavelength of 280 nm. The molecular mass of complexes was calculated with an external standard (Peptide Molecular Weight Marker, Sigma–Aldrich; SigmaMarker™ Low Range, Sigma–Aldrich) using comparison of retention times.

scavenging %¼ ½ðAC  ASÞ=AC  100

where: AABC – activity of control bread; AAOS – activity of onion skin; N – percent of onion skin supplement.

2.8.4. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) Samples were analyzed by SDS–PAGE in 12% (w/v) acrylamide gels, as described by Laemmli (1970). Samples were mixed with loading buffer (ratio 2:1, v:v) containing 2% SDS (w/v) and 0.1 M b-mercaptoethanol and boiled for 2 min, aliquots (40 ll) of protein samples were loaded into each lane. The polypeptides in gels were fixed with 10% (w/v) trichloroacetic acid and stained with Coomassie Brillant Blue G-250. Molecular mass of complexes was carried out with SigmaMarker™ Low Range, Sigma–Aldrich. Proteins electrophoresis was analyzed with Polydoc, Molecular Imaging System, Vilber Lourmat supplied with software PhotoCapt.

2.7. Protein content and in vitro protein digestibility

2.9. Statistic analysis

The proteins content was determined with the Bradford method (Bradford, 1976), using bovine serum albumin as the standard protein. The in vitro protein digestibility was evaluated on the basis of total soluble protein content and the content of protein determined after digestion in vitro.

All experimental results were mean ± S.D. of three parallel measurements. One-way analysis of variance post hoc, Tukey’s test was used to compare groups. a values < 0.05 were regarded as a significant.

where, AC – absorbance of control, AS –absorbance of sample. Antioxidant activities were determined as percent of radical scavenged by 1 mg of dry bread (%/g DW). Expected antioxidant activity (AAe) was calculated as follow:

AAe ¼ AABC þ

  AAOS xN ; 100%

  Pr PD½% ¼ 100%   100% ; Pt

ð3Þ

ð4Þ

where: PD – in vitro digestibility of protein; Pt – total protein content; Pr – content of proteins after in vitro digestion. 2.8. Analysis of proteins–phenolics interactions 2.8.1. Buffer and gastrointestinally digested extracts Soluble protein samples (4 mL), from each system tested (BE and GE), were mixed with 4 mL of cold acetone, incubated at 20 °C for 2 h, and pelleted by centrifugation at 14.000g for 20 min. The pellet was resuspended in PBS buffer (1 mL), pH 7.4, and analyzed. 2.8.2. Determination of free amino groups Changes in the content of free amino groups were analyzed using trinitrobenzenesulfonic acid (TNBS) (Habeeb, 1966). 2.8.3. Liquid chromatography 2.8.3.1. Size exclusion chromatography. Size exclusion chromatography of samples was carried out using Sephadex G-15 as a gel filtration medium. The volume of 0.5 mL of sample was loaded onto a Sephadex G-15 column (0.8  40 cm) that had been equilibrated and eluted with PBS buffer pH 7.4. The flow rate was 0.5 mL min1 and 40 (0.5 mL) fractions were collected. Protein content was monitored by changes of absorbance at 280 nm. Flavonoid content was monitored by the method described by Lamaison and Carnet (1990) (2.6.2.). 2.8.3.2. High-performance liquid chromatography. The samples were characterized by SEC-HPLC using a Varian ProStar HPLC System separation module (Varian, Palo Alto, USA) equipped with a column (COSMOSIL 5Diol- 20-II Packed Column 7.5 mm ID  300 mm) and a ProStar DAD detector.

3. Results In the buffer extracts, phenolics were strictly dependent on the percentage of OS supplement, the highest amounts of total phenolics and flavonoids were determined for breads with 4% OS addition; 6.47 and 1.27 mg/g DW of bread, respectively. The determined levels of phenolic compounds were significantly lower than those expected. This tendency was also observed for extracts obtained after digestion in vitro (Table 1). As the bioaccessible phenolic contents strongly affect on the antioxidant potential of food, the free radical scavenging abilities of breads were determined. In both extracts, the ability to quench ABTS radicals was dependent on the phenolic levels and onion skin supplement. Similar to the results concerning phenolic level, antioxidant potentials of breads were also lower than those expected. In the case of breads fortified with 2–4% of OS about 4.3%, 13.3%, and 21.2% of the activity was masked (Table 1). Supplementation of breads with OS strongly influenced protein digestibility. For the control breads it was 78.4% and even a 1% addition reduced it by about 7%. The lowest protein digestibility (55%) was determined for breads supplemented with 4% of OS (Table 2). Fig. 1A presents the absorbance profiles of the eluates for the buffer extracts of control and enriched breads. As expected, the control breads were characterized by peaks corresponding to buffer extractable wheat proteins (61–34.9 kDa, 29–22 kDa; 18 kDa and 6 kDa). Additionally, separation of the buffer extracts of supplemented breads led to peaks corresponding to those of wheat protein and OS components (Fig. 1A). As indicated by the peak areas, the larger amounts of protein determined for enriched breads could result from phenolic–protein interaction; phenolic compounds that interacted with the protein would remain in the protein matrix. In comparison to the control bread, an increase in chromatogram areas was observed; for 4% breads the increase was 28.1% (Fig. 1B). It should also be noted that supplementation of breads resulted in the disappearance of a protein peak with a molecular mass of about 52 kDa that was present in control breads (Fig. 1A). In spite of the fact that the peak corresponding with a

M. S´wieca et al. / Food Chemistry 141 (2013) 451–458

454

Table 1 Phenolics content and antioxidant activity control and enriched breads. Breads

Total phenolics [mg GAE/g DW]

Total flavonoids [mg QE/g DW]

Radical scavenging activity [%/100 lg DW]

Experimental

Experimental

Expected

Experimental

Expected

– 0.60 0.79 1.01 1.20 1.40

10.25 ± 0.092 0.30 ± 0.012a 0.39 ± 0.016b 0.44 ± 0.017bc 0.51 ± 0.020c 0.65 ± 0.026d

– 0.30 0.40 0.51 0.61 0.71

1.59 2.61 3.62 4.63 5.65

50.25 ± 2.96 2.91 ± 0.116e 3.41 ± 0.136ef 3.76 ± 0.150fg 3.90 ± 0.156g 4.06 ± 0.162g

2.91 3.41 3.92 4.42 4.92

Buffer extracts (BE) OS 85.62 ± 4.36 BC 3.99 ± 0.22a B1% 4.74 ± 0.05b B2% 5.14 ± 0.21b B3% 5.95 ± 0.03c B4% 6.47 ± 0.36c Gastrointestinally digested extracts (GE) OS 158.35 ± 4.65 BC 16.98 ± 0.15 d B1% 18.42 ± 0.07 e B2% 19.35 ± 0.20 f B3% 19.59 ± 0.10 f B4% 20.07 ± 0.73 g

Expected – 3.99 4.84 5.69 6.55 7.39

19.87 ± 1.21 0.60 ± 0.06a 0.78 ± 0.18ab 0.83 ± 0.08b 1.14 ± 0.02c 1.27 ± 0.01d 101.45 ± 1.58 1.59 ± 0.07e 2.58 ± 0.08f 3.30 ± 0.09g 4.42 ± 0.09h 4.70 ± 0.19h

16.98 18.56 20.15 21.73 23.31

Means followed by different small letters, in the columns, are significantly different at a < 0.05. OS – onion skin, BC – control breads, B1–B4% – breads enriched with 1–4% of onion skin, respectively.

Table 2 Protein digestibility and content of control and enriched breads. Breads

BC B1% B2% B3% B4%

Proteins [mg/g DW] Gastrointestinally digested extracts (GE)

Buffer extracts (BE)

2.88 ± 0.13a 3.94 ± 0.19b 5.01 ± 0.43c 6.40 ± 0.21d 8.53 ± 0.37e

13.30 ± 0.68a 14.57 ± 0.91ab 16.16 ± 0.65bcd 17.33 ± 1.02cd 18.95 ± 1.54d

Protein digestibility [%]

78.35 ± 2.85d 72.96 ± 2.03c 69.11 ± 1.98c 63.39 ± 2.57b 55.00 ± 3.89a

Means followed by different small letters, in the columns, are significantly different at a < 0.05. BC – control breads, B1–B4% – breads enriched with 1–4% of onion skin, respectively.

protein with molecular mass of about 18 kDa (retention time 13 min.) is present on both breads and onion skin chromatograms, it is clearly visible that its area is the result of wheat protein–onion flavonoid interaction. The area of these peaks determined for breads enriched with 1–4% of supplement were 2.1, 4.0, 5.4 and 6.8 times bigger than those obtained for control bread fractions and higher by about 10%, 38%, 41% and 40% than those expected (the sum of BC and appropriate onion skin percentage). As hydrogen bonding and covalent linkages are involved in the phenolic– protein interactions, free amino group levels were measured (Fig. 1C). A small reduction in their amounts, correlated with the percentage of OS supplement, was observed; however, the changes were not statistically significant. Fig. 2 presents the absorbance profiles of the eluates obtained after digestion in vitro of extracts of control and enriched breads. Samples were separated by size-exclusion chromatography and protein and flavonoid levels were determined. Chromatographic profiles of the studied samples obtained on a Sephadex G15 clearly show that flavonoid introduced with OS forms indigestible complexes with bread proteins (Fig. 2). The level of bound phenolics is linked with the percentage addition of OS. In bread with 1% supplement flavonoids are bound mainly to high molecular weight proteins (fractions 12–15; about 41–21 kDa) (Fig. 2B). In breads with a higher OS content there are visible complexes with molecular weights of about 25 kDa and 14.5 kDa (fractions 25–29) (Fig. 2C and D). Additionally, in breads with 4% OS supplement, the protein profile was wide and ‘‘blurred’’, and flavonoids were also determined in fractions containing low molecular weight proteins and peptides (Fig. 2E).

These results were confirmed by further electrophoretic and chromatographic studies. SDS–PAGE of the samples confirmed that the OS flavonoids form indigestible complexes with molecular mass 25 and 14.4 kDa (Fig. 3). It should be noted that the bands were not present in control breads and their intensity was correlated with OS addition (Fig. 3A). Theses complexes were also visible on the chromatograms of enriched breads (Fig. 3A); however, the reduction of free amino group levels and the increase of chromatogram areas suggest that flavonoids also bind to other bread proteins (Fig. 3C). The larger protein peaks for supplemented breads could result from onion flavonoids–wheat protein interaction (Fig. 3A). As indicated by the peak areas, the amounts of the proteins eluted for 7.5–10 min. (molecular masses ranged from about 60–35 kDa) were much higher than those of the control breads. In breads enriched with 1–4% supplement the amounts of proteins in these fractions were higher by about 43%, 38%, 20%, and 21% then those expected (the sum of BC and corresponding onion skin percentage). This same situation was observed for peaks with a maximum absorption at 11.6 min. (about 22–25 kDa) where the detected peaks areas were about 50–60% larger than those predicted. Finally, the chromatogram areas obtained for breads with 1–4% supplement additions were 1.2, 3.6, 4.3, 4.5 times greater than those obtained for the control bread (Fig. 3B). Additionally, analysis of the contents of free amino groups responsible for covalent binding of phenolics showed that their amounts were lower in enriched breads than those determined for control samples (Fig. 3C). 4. Discussion In many countries, breads are the staple food product, thus attempts at enriching them with materials rich in bioactive ingredients seem well targeted (Gawlik-Dziki et al., 2009; Han & Koh, 2011a; Han & Koh, 2011b; Peng et al., 2010). OS contains significantly high level of flavonoids, especially quercetin aglycone, with well-documented anti-inflammatory, antioxidant and anticancer abilities (Gawlik-Dziki, S´wieca, et al. 2012; Wiczkowski et al., 2008. In our previous study the antioxidant potential of bread with OS was significantly) higher than the activity noted in the control. In particular, OS addition significantly fortificated bread with bioaccessible lipid oxidation inhibitors and compounds with reducing and chelating abilities (Gawlik-Dziki et al., 2011). So far, some studies have been focused on evaluating the relationship between thermal processing of food and changes in the antioxidant capacities of phenolic additives. Thermal processing

M. S´wieca et al. / Food Chemistry 141 (2013) 451–458

455

29-22 kDa

A

18 kDa

61-34.9 kDa

6 kDa

a 4,50E+06

c

4,00E+06

b

3,50E+06 a

f

g

C

h

0,18 a

0,16

b

a

a

a a

0,14 a Free -NH2 groups

3,00E+06

Peaks area

e

2,50E+06 2,00E+06 1,50E+06

0,12 [mmol/ g DW]

B

bc d

0,1 0,08 0,06

1,00E+06

0,04

5,00E+05

0,02 0

0,00E+00 BC

B1%

B2%

B3%

B4%

BC

B1%

B2%

B3%

B4%

Fig. 1. Chromatographic analysis of protein–phenolic interactions in buffer extracts. A – size-exclusion chromatography (elution profiles) – molecular mass markers (kDa): a, 102; b, 42; c, 35; d, 22; e, 18; f, 6.5; g, 3; h, 1.5. B – size-exclusion chromatography (peaks area), C – free amino group contents BC – control breads, B1–B4% – breads enriched with 1–4% of onion skin, respectively Means followed by different small letters are significantly different at a < 0.05.

is generally considered to be destructive to nutrients, because most bioactive compounds become unstable when exposed to heat. On the other hand, the important role of Maillard reaction products as antioxidants is emphasized. Some researchers suggest that phenolics are unstable to heat and this is a reason why the detected activity is lower than predicted (Peng et al., 2010); however, in our study bread crumbs were studied (internal bread temperature did not exceed 80 °C during baking). Additionally, Han and Koh (2011a) have clearly shown that phonolics responsible for the antioxidant potential of enriched breads are already strongly bound to bread components at the stage of forming the bread mix and mixing the dough. They observed a reduction of phenolic acids levels of about 20–30% in breads in comparison to start material – enriched flour (4.44 lmol phenolic acid/g flour). However, whereas interactions of phenolic compounds with proline-rich proteins (especially the saliva proline-rich protein) (Bennick, 2000) and ‘‘pure’’ proteins have been extensively studied (see Introduction), interactions of phenolic compounds with food matrix proteins have not been studied in detail. Our study is the first to show that a decrease in the antioxidant potential of phenol-enriched breads is not bound with thermal deactivation of bioactive supplements, but is mainly due to the formation of indigestible complexes with bread proteins. In model systems, the evidence of interactions between phenolics (especially flavonoids) and protein was confirmed by Sivam et al. (2011), Siebert, Troukhanova, and Lynn (1996) and Arts

et al. (2002). The influence of the derivatization on the secondary and tertiary structure of proteins was also observed. Wheat bread enriched with polyphenols gained b-sheets in the protein’s secondary conformation at the expense of the b-turn, and contained more unordered conformations (Sivam et al., 2011). It should be noted that flavonoids also interact with starch (Singh, Dartois, & Kaur, 2010). Zhang, Yang, Li, and Gao (2011) characterized the solid complex of starch with quercetin and showed that this led to a reduction in starch digestibility via formation of resistant starch. In the light of the presented results researchers suggest that these interactions may significantly influence the bioactivity of phenolic antioxidants and the bioaccessibility of starch and proteins in in vivo conditions. Phenolic antioxidants can form complexes with proteins and/or polysaccharides through different mechanisms, such as hydrophobic and ionic interactions, hydrogen and covalent bindings. The binding parameters are influenced by different factors, where, e.g., increasing temperature and ionic strength as well as decreasing pH cause a diminished binding. The derivatization of soy protein with selected flavonoids caused a reduction of lysine, cysteine and tryptophan residues in the proteins (Rawel, Rohn, & Kroll, 2002a). A statistically significant reduction of free amino group levels in enriched breads was also determined in our studies; however, there were no changes in thiol content (data not shown). Also, in comparison to control breads, an increase in the chromatogram areas obtained for both the studied extracts (BE

M. S´wieca et al. / Food Chemistry 141 (2013) 451–458

456

0,14

1

Flavonoids

0,9 0,7

0,08

0,6

Flawonoids [A430]

0,8

0,10

0,5 0,06

0,4

0,04

0,3 0,2

0,02

0,1

0,00

0 1

B

5

9

13

0,14

17

21

25

29

33

Fraction no.

37 0,003

Proteins

0,12

Flavonoids

Proteins [A280]

0,1

0,002

0,08 0,0015 0,06 0,001

0,04

0,0005

0,02 0

0 1

C

5

9

13

17

21

25

29

Fraction no.

0,35

33

37

0,12

Proteins Flavonoids

0,3

0,1

0,25 Proteins [A280]

0,0025

Flawonoids [A430]

Proteins [A280]

0,12

Proteins

0,08

0,2 0,06 0,15 0,04

0,1

Flawonoids [A430]

A

0,02

0,05 0

0 1

5

9

13

17

21

25

29

Fraction no

D 0,35

33

37

Proteins Flavonoids

0,3

0,018 0,016

Proteins [A280]

0,012

0,2

0,01

0,15

0,008

Flawonoids [A430]

0,014 0,25

0,006

0,1

0,004 0,05

0,002

0

0 1

9

13

0,4

17 21 Fraction no.

25

29

33

37 0,06

0,35

0,05

Proteins [A280]

0,3

Proteins Flavonoids

0,25

0,04

0,2

0,03

0,15

0,02

Flawonoids [A430]

E

5

0,1 0,01

0,05 0

0 1

5

9

13

17

21

25

29

33

37

Fraction no.

Fig. 2. Sephadex G-15 elution profiles of bread extracts after digestion in vitro A BC, control breads; B-, C-, D-, E- breads with onion skin supplement B1–B4% (1–4% of onion skin), respectively.

and GE) was observed. This observation clearly indicates the presence of interaction between phenolics and bread ingredients. This

statement is confirmed by results obtained by Han and Koh (2011b). SE-HPLC profiles of SDS-soluble gluten proteins of hard wheat breads with and without phenolics acids showed that the addition of caffeic and ferulic acids increased the area of peaks corresponding with high (HMW) and low (LMW) molecular weight proteins when compared with the control breads. The cited researchers also suggest that the increase in HMW proteins with the addition of caffeic and ferulic acids indicates that the phenolic acid was involved in reducing protein cross-linking and in releasing the HMW proteins that had been insoluble in SDS extraction buffer. Also, the study performed by Sivam et al. (2011) concluded that the added pectin and/or phenolic extract had influenced on the bread dough cross-linking microstructure and bread properties through being involved in the interactions with bread components, such as wheat proteins, during dough development and bread baking. The interaction, resulting in protein–polyphenol complexes, can be both reversible and irreversible depending on pH, temperature, and protein and flavonoid concentrations (Arts et al. 2002). The cited investigators stated that the fate of these complexes in the gastrointestinal tract is not known; however, in our studies it was observed that OS flavonoids form indigestible complexes with bread proteins. The presence of indigestible phenolic–protein complexes was also observed in the studies performed by Rawel, Kroll, and Riese (2000) and Rawel, Czajka, Rohn, and Kroll (2002). Researchers have proved that derivation of soy glycinin with selected phenolic acids and flavonoids have influenced the in vitro digestibility with trypsin, chymotrypsin, pepsin and pancreatin. A consequence of the protein interaction might be a decrease in protein digestibility and a consequent reduction in their bioavailability. Generally, the measured antioxidant activity of enriched-breads is mainly derived from the phenolics, rather than other compounds, present in the bread (Gawlik-Dziki, S´wieca, et al., 2012; Han & Koh, 2011a; Sivam et al., 2011); thus, their bioaccessibility is the most important factor determining food bioactivity. It was found that the antioxidant capacities in model systems used for protein–phenolic interaction study (Arts et al., 2001; Arts et al., 2002; Bohin, Vincken, van der Hijden, & Gruppen, 2012; Rawel et al., 2000; Rawel et al. 2002) and also in studies of phenolic-enriched food products is less than expected. Binding also decreases the concentration of free antioxidant. In our studies performed on the complete food matrix system a decrease in phenolic and the masking antioxidant potential of enriched breads was observed. The degree of this masking depends on the amount of OS addition and was also observed after digestion in vitro. Sivam and co-workers (2011) suggest that reduced phenolic recovery from enrichedbreads was affected by the stability of phenolics during breadmaking and the extractability of these phenolics from the bread matrix system; phenolics might have formed polyphenol–protein or polyphenol–polysaccharide complexes via hydrogen bonding and/or hydrophobic interactions. On the other hand, in the whole food system, Han and Koh (2011a) observed a reduction of phenolic content and subsequent antioxidant activity at each step of bread preparation, which seems to indicate a key role of protein–phenolic interactions. In conclusion, the interaction of phenolics (flavonoids) has a multiplying effect on food quality. They affect the antioxidant efficacy of flavonoid and protein digestibility in enriched breads. The bioaccessibility of food components is strongly dependent on interaction between them. It should be kept in mind that the potential bioactivity of enriched food is determined by many factors and there is no simple method for designing functional products with a predict nutritional and nutraceutical quality.

M. S´wieca et al. / Food Chemistry 141 (2013) 451–458

mA

457

BC B1% B2%B3%B4%

A

25.0

BC B1% B2%B3%B4% MW kDa

5

116.0 BC B1% B2%B3%B4%

66.2

4

14.4

45.0 35.0

3 25.0

B4%

2

18.4

B3%

14.4

B2%

1

B1% BC onion skin

0 bcd

a

5 B

e

f

10

g

h

15

20

25

30

minutes 35

7,00E+06 6,00E+06

d

d

1,6

C

1,4

c

5,00E+06

b

ab

ab

ab

a

1,2

4,00E+06

3,00E+06 2,00E+06

1

[mmol/ g DW]

Free -NH2 groups

Peaks area

b

0,8

0,6 0,4

a 1,00E+06

0,2

0,00E+00

0

BC

B1%

B2%

B3%

B4%

BC

B1%

B2%

B3%

B4%

Fig. 3. Chromatographic analysis of protein–phenolic interactions in extracts after digestion in vitro. A – size-exclusion chromatography (elution profiles; molecular mass markers (kDa): a, 102; b, 42; c, 35; d, 22; e, 18; f, 6.5; g, 3; h, 1.5.) and electrophoresis, B – size-exclusion chromatography (peaks area), C – free amino group contents Means followed by different small letters are significantly different at a < 0.05. BC – control breads, B1–B4% – breads enriched with 1–4% of onion skin, respectively.

Acknowledgements This scientific study was financed by the Polish Ministry of Scientific Research and Higher Education (grant NN312233738).

References Arts, M. J. T. J., Haenen, G. R. M. M., Voss, H.-P., & Bast, A. (2001). Masking of antioxidant capacity by the interaction of flavonoids with protein. Food Chemistry and Toxicology, 39, 787–791. Arts, M. J. T. J., Haenen, G. R. M. M., Wilms, L. C., Beetstra, S. A. J. N., Heijnen, C. G. M., Voss, H., & Bast, A. (2002). Interactions between flavonoids and proteins: Effect on the total antioxidant capacity. Journal of Agricultural and Food Chemistry, 50(5), 1184–1187. Balestra, F., Cocci, E., Pinnavaia, G. G., & Romani, S. (2011). Evaluation of antioxidant, rheological and sensorial properties of wheat flour dough and bread containing ginger powder. LWT – Food Science and Technology, 44, 700–705. Bennick, A. (2000). Interaction of plant polyphenols with salivary proteins. Critical Reviews in Oral Biology & Medicine, 13(2), 184–196. Bohin, M. C., Vincken, J. P., van der Hijden, H. T. W. M., & Gruppen, H. (2012). Efficacy of food proteins as carriers for flavonoids. Journal of Agricultural and Food Chemistry, 60, 4136–4143. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein – dye binding. Analytical Biochemistry, 7(72), 248–254. Charlton, A. J., Baxter, N. J., Khan, M. L., Moir, A. J., Haslam, E., Davies, A. P., & Williamson, M. P. (2002). Polyphenol/peptide binding and precipitation. Journal of Agricultural and Food Chemistry, 50, 1593–1601. Gawlik-Dziki, U., Dziki, D., Baraniak, B., & Lin, R. (2009). The effect of simulated digestion in vitro on bioactivity of wheat bread with Tartary buckwheat flavones addition. LWT – Food Science and Technology, 42, 137–143.

_ _ J. (2012a). Gawlik-Dziki, U., Jezyna, M., S´wieca, M., Dziki, D., Baraniak, B., & Czyz, Effect of bioaccessibility of phenolic compounds on in vitro anticancer activity of broccoli sprouts. Food Research International, 49, 469–476. _ J. (2012b). Gawlik-Dziki, U., S´wieca, M., Dziki, D., Baraniak, B., Tomiło, J., & Czyz, Quality and antioxidant properties of breads enriched with dry onion (Allium cepa L.) skin. Food Chemistry, 138(2), 1621–1628. Gawlik-Dziki, U., S´wieca, M., Sugier, D., & Cichocka, J. (2011). Comparison of in vitro lipoxygenase, xanthine oxidase inhibitory and antioxidant activity of Arnica montana and Arnica chamissonis tinctures. Acta Scientiarum Polonorum, Hortorum Cultus, 10, 15–27. Glei, M., Kirmse, A., Habermann, N., Persin, C., & Pool-Zobel, B. L. (2006). Bread enriched with green coffee extract has chemoprotective and antigenotoxic activities in human cells. Nutrition and Cancer, 56, 182–192. Habeeb, A. (1966). Determination of free amino groups in proteins by trinitrobenzenosulfonic acid. Analytical Biochemistry, 14, 328–336. Han, H.-M., & Koh, B.-K. (2011a). Antioxidant activity of hard wheat flour, dough and bread prepared using various processes with the addition of different phenolic acids. Journal of the Science of Food and Agriculture, 91, 604–608. Han, H.-M., & Koh, B.-K. (2011b). Effect of phenolic acids on the rheological properties and proteins of hard wheat flour dough and bread. Journal of the Science of Food and Agriculture, 91, 2495–2499. Kroll, J., Rawel, H. M., Rohn, S., & Czajka, D. (2001). Interactions of glycinin with plant phenols – influence on chemical properties and proteolytic degradation of the proteins. Nahrung/Food, 45, 388–389. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacterio-phage T4. Nature, 227, 680–685. Lamaison, J. L. C., & Carnet, A. (1990). Teneurs en principaux flavonoids des fleurs de Crataegeus monogyna Jacq et de Crataegeus laevigata (Poiret D. C) en fonction de la vegetation. Pharmaceutica Acta Helvetiae, 65, 315–320. Peng, X., Ma, J., Cheng, K.-W., Jiang, Y., Chen, F., & Wang, M. (2010). The effects of grape seed extract fortification on the antioxidant activity and quality attributes of bread. Food Chemistry, 119, 49–53. Rawel, H. M., Czajka, D., Rohn, S., & Kroll, J. (2002). Interactions of different phenolic acids and flavonoids with soy proteins. International Journal of Biological Macromolecules, 30, 137–150.

458

M. S´wieca et al. / Food Chemistry 141 (2013) 451–458

Rawel, H. M., Kroll, J., & Riese, B. (2000). Reactions of chlorogenic acid with lysozyme: physicochemical characterization and proteolytic digestion of the derivatives. Journal of Food Science, 65, 1091–1098. Rawel, H. M., Rohn, S., & Kroll, J. (2002). Inhibitory effects of plant phenols on the activity of selected enzymes. Journal of Agricultural and Food Chemistry, 50, 3566–3571. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26, 1231–1237. Siebert, K. J., Troukhanova, N. V., & Lynn, P. Y. (1996). Nature of polyphenol–protein interactions. Journal of Agricultural and Food Chemistry, 44(1), 80–85. Singh, J., Dartois, A., & Kaur, L. (2010). Starch digestibility in food matrix: a review. Trends in Food Science & Technology, 21, 168–180. Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstics acid reagents. American Journal of Enology and Viticulture, 16, 144–158.

Sivam, A. S., Sun-Waterhouse, D., Perera, C. O., & Waterhouse, G. I. N. (2011). Application of FT-IR and Ramanspectroscopy for the study of biopolymers in breads fortified with fibre and polyphenols. Food Research International, 50(2), 574–585. Sivam, A. S., Sun-Waterhouse, D., Quek, S., & Perera, C. O. (2010). Properties of bread dough with added fiber polysaccharides and phenolic antioxidants: A review. Journal of Food Science, 75, 163–174. Wiczkowski, W., Romaszko, J., Bucinski, A., Szawara-Nowak, D., Honke, J., Zielinski, H., & Piskula, M. K. (2008). Quercetin from shallots (Allium cepa L var. aggregatum) is more bioavailable than its glucosides. The Journal of Nutrition, 138, 885–888. Zhang, L., Yang, X., Li, S., & Gao, W. (2011). Preparation, physicochemical characterization and in vitro digestibility on solid complex of maize starches with quercetin. LWT – Food Science and Technology, 44, 787–792.