Interactions of free and encapsulated

LWT - Food Science and Technology 65 (2016) 823e831

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Interactions of free and encapsulated hydroxycinnamic acids from green coffee with egg ovalbumin, whey and soy protein hydrolysates _ ska a, Bartłomiej Pałecz b, Danuta Rachwał-Rosiak a, Grazyna Budryn a, *, Donata Zaczyn ~ a-García c, Horacio Pe rez-Sa nchez c, 1 Sylwia Belica b, Helena den-Haan c, Jorge Pen a

Institute of Chemical Food Technology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, 90-924, Lodz, Poland Department of Physical Chemistry, Faculty of Chemistry, University of Lodz, 90-131, Lodz, Poland lica San Antonio de Murcia Bioinformatics and High Performance Computing Research Group (BIO-HPC), Computer Science Department, Universidad Cato (UCAM), Guadalupe, Murcia, Spain b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 April 2015 Received in revised form 5 August 2015 Accepted 3 September 2015 Available online 9 September 2015

The interactions of hydroxycinnamic and chlorogenic acids (all described as CHAs) from green coffee with hydrolysates of proteins from egg ovalbumin (EOH), whey (WPH) and soy (SPH) were studied depending on temperature (25 and 90 C) and CHAs form (free or included in b-cyclodextrin (b-CD)). The binding degree was determined by liquid chromatography with Q-Exactive tandem mass spectrometry. The interactions were confirmed by liquid chromatography with ultrahigh resolution hybrid quadrupletime-of-flight mass spectrometry. As the result of binding, the content of CHAs in protein hydrolysates (PHs) ranged from 12.87 to 15.15 g/100 g. Inclusion of CHAs with b-CD strongly limited these interactions to a level of 0.48e1.32 g/100 g. Thermodynamic parameters of peptide-ligand interactions were determined by isothermal titration calorimetry and energetics of interactions at the atomic level by molecular modelling. The amount of CHAs released during proteolytic digestion was at a relatively low level of 0.06 e1.14 g/100 g, and the availability for absorption of CHAs after proteolysis ranged from 1.26 to 4.05 g/ 100 g for free form and from 14.67 to 15.39 g/100 g for included form, indicating that encapsulating of CHAs in b-CD significantly increased their availability from processed food containing PHs after digestion. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Green coffee b-cyclodextrin Peptide-polyphenol interactions Molecular modelling

1. Introduction Hydroxycinnamic acids, such as caffeic acid and its methoxyderivative ferulic acid, as well as their esters with quinic acid, referred to as chlorogenic acids (CHAs), are one of the main groups of phenolic compounds. They have a broad range of biological activities such as: anti-bacterial, anti-fungal, hepatoprotective, antithrombotic, anti-inflammatory, hypoglycemic and antioxidant (Bassoli et al., 2008; El-Medany, Bassiouni, Khattab, & Mahesar, 2011; Lou, Wang, Zhu, Ma, & Wang, 2011; Satake, Kamiya, An, Oishi, & Yamamoto, 2007; Sato et al., 2011; Shi et al., 2013; Sung & Lee, 2010). Their high antioxidant activity contributes to the reduction

* Corresponding author. Institute of Food Technology and Analysis, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Poland. E-mail addresses: [email protected] (G. Budryn), [email protected] rez-Sa nchez). (H. Pe 1 To whom all molecular modelling related Correspondence should be addressed: lica San Antonio de Murcia Computer Science Department, Universidad Cato (UCAM), Guadalupe, Murcia, Spain. http://dx.doi.org/10.1016/j.lwt.2015.09.001 0023-6438/© 2015 Elsevier Ltd. All rights reserved.

of the risk of several oxidative stress-related diseases, including atherosclerosis, some kinds of cancer and Alzheimer's disease (Cheng, Dai, Zhou, Yang, & Liu, 2007; Lee & Lee, 2006; Silva, Ferreres, Malva, & Dias, 2005). Coffee is one of the plants that accumulate CHAs in quantities sufficient to have physiological effects (Kim, Shang, & Um, 2010). Its green beans contain 4e10% of CHAs. Coffee water extract which contains high concentration of these compounds can be used as food component or food supplement. In novel foods containing different health-promoting components such as protein hydrolysates (PHs) as well as plant extracts containing polyphenols, it is possible that peptides and phenolics interact and as a consequence bioactivity of both decreases ndez-Ledesma & de Lumen, 2008; Mann (Clemente, 2000; Herna et al., 2015; Rawel & Rohn, 2010; Yu et al., 2012; Yuksel, Avci, & Erdem, 2010). To avoid such interactions it may be beneficial to encapsulate CHAs by forming inclusion complexes with b-cyclodextrin (b-CD) and adding such complexes to food products (Nasirullah, Kumar, & Shariff, 2011; Szejtli & Szente, 2005).

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Encapsulation of CHAs with b-CD presumably does not limit their bioavailability and antioxidant activity (Paramera, Konteles, & Karathanos, 2011). The aim of this study was to characterize the degree of interactions of CHAs in free or included form with egg ovalbumin, soy and whey PHs and to assess stability of interactions during proteolysis. Studies conducted earlier by other research groups reported mainly interactions of polyphenols with proteins or individual amino acids (Gallo, Vinci, Graziani, De Simone, & Ferranti, 2013; ndez-Jarabela et al., 2015; Papadopoulou & Frazier, 2004; Herna Prigent et al., 2008; Shen et al., 2014). Till now, very few published reports concerned limiting of interactions between proteins and polyphenols as a result of encapsulation in b-CD. So far the limitation of interactions of polyphenol oxidase with inclusion complex of 5-caffeoylquinic acid (5-CQA) was confirmed and the authors proved reduction of interactions of CHAs from green coffee extract with food protein isolates as an effect of inclusion and for peptides such research so far has not been conducted at all (Budryn et al., 2015; Gacche, Zore, & Ghole, 2003). 2. Materials and methods 2.1. Chemicals and reagents Analytical-grade ethanol and ethyl acetate were purchased from Poch (Gliwice, Poland), HPLC-grade acetonitrile from Fluka (St. Louis, MO, USA), 5-caffeoylqunic acid (5-CQA, ~99%), caffeic acid (CA, ~99%), ferulic acid (FA, ~99%), benzoic acid (BA, ~99%) and bcyclodextrin (b-CD, ~98), pepsin (~99%), trypsin (~99%), chymotrypsin (~99%), from Sigma Aldrich (St. Louis, MO, USA), phosphate buffer pH 6.45 from BioChrom (Cambridge, UK), and nylon syringe filters from Chromacol (Herts, UK). Ultrapure water (resistivity 18.2 MU cm) was obtained from a Millipore Milli-Q Plus purification system (Bedford, MA, USA). Protein hydrolysates (PHs): whey protein hydrolysate Amino 4500 (WPH) from Trec Nutrition (Gdynia, Poland), egg ovalbumin hydrolysate A 6710 (EOH) from Sigma (St. Luis, MO, USA) and soy protein hydrolysate S 1674 (SPH) from Fluka (St. Luis, USA), were used for interactions with coffee phenolics.

suspension was centrifuged in MIKRO 22R centrifuge from Hettich (Kirchlengern, Germany) at 4 C for 20 min at 10,000 g. The precipitate contained a mixture of b-CD complexes with particular HCAs and was characterized by ESI-MS/MS method. The molar ratio of b-CD to CHA in the obtained complexes was 1:1. The procedure was described with details in the previous work (Budryn et al., 2014a). 2.4. Interactions of CHAs with PHs The interactions were conducted at a weight ratio of PHs to CHAs of 1:0.15. For this purpose 0.10 g of WPH, EOH or SPH and 0.015 g of CHAs were suspended in 3 mL of phosphate buffer, pH 6.45. CHAs were added as 0.028 g of GCE or 0.075 g of b-CD:CHAs inclusion complexes, which contained 0.015 g CHAs. PHs (0.10 g) were additionally reacted with 0.01 g of standards: 5-CQA, CA and FA. The solutions were reacted for 1.5 h at 25 or 90 C in a Pierce Reacti-Therm TS-18821 reactor I 18821, Thermo Scientific (Palo Alto, CA, USA) and centrifuged in MIKRO 22R centrifuge, Hettich (Kirchlengern, Germany) at 4 C for 20 min at 10,000 g. The concentration of unbound CHAs in supernatant was determined to calculate the degree of peptide-CHAs binding as: (concentration before interactions (cbi) e concentration of unbound after in, Estrella, & Hern teractions)/cbi (Altunkaya, 2011; Bartolome andez, 2000). 2.5. LC-Orbitrap-MS/MS analysis of CHAs The supernatant was diluted in ultrapure water and internal

2.2. Preparation and purification of green coffee extract (GCE) In the study green Robusta coffee beans (Coffea canephora L.), Conilon variety, harvested in Brazil in 2012 from Bero Polska (Gdynia, Poland) were used. The crude extract was prepared as described in the previous work (Budryn et al., 2014a; Budryn, _ zelewicz, _ Nebesny, Zy & Oracz, 2014b). Briefly to purify GCE the centrifugal partition chromatography (CPC) method was used with , SPOT Prep II 50 chromatograph from Armen Instrument (Saint-Ave France). The two-phase system of solvents was prepared from three solvents: water, ethanol and ethyl acetate in the ratio of 5:1:4 (v/v/v). 2.3. Preparation of inclusion complexes of b-CD with CHAs from GCE Inclusion complexes of b-CD with CHAs from GCE were obtained using average molar ratio of substrates 1:2 (b-CD:CHAs, where CHAs were calculated based on 5-CQA equivalent), thus with the excess of polyphenols to maximize the efficacy of complexation. Therefore, 0.1135 g of b-CD and 0.1303 g of GCE, which contained 0.0708 g of HCAs were dissolved in 2 mL of water. The complexation was conducted for 2 h at 50 C in a Pierce Reacti-Therm TS-18821 reactor from Thermo Scientific (Palo Alto, CA, USA). After complexation the solution was left for 24 h at 0 C and the formed

Fig. 1. ITC plots: (A) - raw data - the exothermic heat released by injecting 93 portions of 3-ml aliquots of ferulic acid solution (4.29 mM) into the solution of soy proteins hydrolysate (0.015 mM); (B) enthalpy change per mol of injectant (FA solution).


standard (BA) was added. Then the solution was filtered through a 0.20 mm nylon syringe filter. The identification and quantitative analysis of CHAs was performed by TurboFlow UHPLC-DAD-ESIMS/MS method in a Transcend TLX-1 chromatograph equipped with diode array detector (DAD) and Q-Exactive tandem mass spectrometer from Thermo Scientific (Palo Alto, CA, USA). The UHPLC system consisted of purifying TurboFlow cyclone-P 50 0.5 mm column and analytical Hypersil GOLD 50 2.1 mm, 1.9 mm column, both from Thermo Scientific (Palo Alto, CA, USA), each supplied with a loading or analytical pump, respectively. Peptides bound with CHAs that remained soluble after interactions were separated from the unbound CHAs at the first step of the analysis on the TurboFlow column (Couchman, 2012). The conditions of CHAs identification and quantitative analysis were consistent with Budryn et al., (2014a).

2.6. LC-Q-TOF-MS/MS analysis of peptideeCHAs interaction products The freeze-dried precipitate from the Section 2.4 was considered as interaction products. This material contained only peptides insoluble after interactions. It was dissolved in water/acetonitrile 80/20 (v/v). Chromatographic analysis was carried out using

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UHPLCþ Ultimate 3000 system with a diode array detector (UV/ DAD, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA), and an ultrahigh resolution hybrid quadrupole/time of-flight mass spectrometer (UHR-Q-TOF-MS/MS, Bruker Daltonics GmbH, Bremen, Germany) using ESI source according to Budryn et al., (2015). The interaction products were ionized in positive and negative ion polarity mode. Full-scan MS and auto MS/MS spectra were obtained by scanning m/z from 50 to 3000. MS/MS spectra were obtained in collision-induced dissociation (CID) mode using nitrogen as the collision gas. Instrument control, data acquisition and evaluation were done with the HyStar 3.2, Chromeleon 6.8.1 Chromatography Data System, and OTOFControl 3.2 software, respectively.

2.7. Proteolytic digestibility The interaction products were subjected to proteolytic digestion (Rawel, Kroll, & Hohl, 2001). This step was performed to hydrolyse polypeptides and to release a part of CHAs as a result of peptides molecular mass, their secondary structure and polarity changes, which may favour interactions before hydrolysis. The details were described in the previous work by Budryn et al., 2015. Briefly 6 mg of protein hydrolysate was dissolved in 1.5 mL of pH 2.0 buffer and 100 mL of pepsin (1 mg mL1) was added. Samples were incubated

Table 1A Content of hydroxycinnamic and chlorogenic acids (CHAs) from green coffee in whey proteins hydrolysate (g/100 g peptides). CHAs

Interactions temperature and CHAs form 25 C GCE

Bound after interactions 3-CQA 2.37 ± 0.34a 5-CQA 3.93 ± 0.27a 3-FQA 0.51 ± 0.04a 4-CQA 3.09 ± 0.23a CA 0.63 ± 0.11a 5-FQA 0.36 ± 0.06a 4-FQA 0.96 ± 0.08a FA 0.09 ± 0.02a 3,4-diCQA 0.63 ± 0.09a 3,5-diCQ 0.21 ± 0.03a 4,5-diCQA 0.45 ± 0.03a Total 13.23 ± 0.87a Released from the bonds after proteolytic digestion 3-CQA 0.27 ± 0.04a 5-CQA 0.30 ± 0.03a 3-FQA 0.06 ± 0.01a 4-CQA 0.24 ± 0.05a CA nd 5-FQA 0.06 ± 0.01 4-FQA 0.06 ± 0.02a FA nd 3,4-diCQA 0.03 ± 0.00 3,5-diCQ nd 4,5-diCQA nd Total 1.02 ± 0.08a Available (released after proteolytic digestion þ unbound) 3-CQA 0.93 ± 0.07 5-CQA 1.02 ± 0.09 3-FQA 0.21 ± 0.03 4-CQA 0.81 ± 0.05 CA 0.09 ± 0.02 5-FQA 0.24 ± 0.04 4-FQA 0.12 ± 0.02 FA nd 3,4-diCQA 0.09 ± 0.03 3,5-diCQ nd 4,5-diCQA 0.03 ± 0.00 Total 3.57 ± 0.19a

90 C GCE

90 C b-CD:CHAs

2.52 ± 0.45a 3.78 ± 0.18a 0.57 ± 0.04a 3.21 ± 0.04a 0.57 ± 0.03a 0.33 ± 0.02a 0.99 ± 0.06a 0.09 ± 0.02a 0.60 ± 0.02a 0.21 ± 0.01a 0.42 ± 0.01a 13.32 ± 0.53a

0.18 0.27 0.03 0.18 0.18 0.03 0.03 nd 0.03 0.03 0.06 0.99

0.24 0.36 0.06 0.21 nd 0.09 0.06 nd 0.12 nd nd 1.14

± ± ± ±

0.75 1.23 0.15 0.66 0.15 0.30 0.09 nd 0.21 nd 0.06 3.60

± ± ± ± ± ± ±

0.03a 0.06a 0.01a 0.04a

± 0.02 ± 0.01a ± 0.03

± 0.09a 0.04 0.08 0.02 0.05 0.01 0.04 0.01

± 0.01 ± 0.02 ± 0.22a

90 C 5-CQA or CA or FA

± ± ± ± ± ± ±

0.03 0.02 0.00 0.01 0.02 0.01 0.00

± ± ± ±

0.01 0.01 0.01 0.10

e 6.61 ± 0.48 e e 2.64 ± 0.33 e 4.08 ± 0.56 e e e e e

nd 0.03 ± 0.01 nd nd nd nd nd nd nd nd nd 0.03 ± 0.01

e 2.89 ± 0.19 e e 0.93 ± 0.07 e e 1.39 ± 0.11 e e e e

2.85 ± 0.44 4.41 ± 0.48 0.63 ± 0.08 3.48 ± 0.29 0.54 ± 0.08 0.51 ± 0.08 0.99 ± 0.12 0.09 ± 0.03 0.66 ± 0.04 0.18 ± 0.03 0.42 ± 0.03 14.85 ± 0.87

e 6.66 e e 8.67 e e 7.69 e e e 6.66

± 0.46

± 0.91

± 0.61

± 0.46

3-CQA e 3-caffeoylquinic acid; 4-CQA e4-caffeoylquinic acid; 5-CQA e 5-caffeoylquinic acid; 3-FQA e 3-feruloylquinic acid; 4-FQA e 4-feruloylquinic acid; 5-FQA e 5feruloylquinic acid; 3,4-diCQA e 3,4-dicaffeoylquinic acid; 3,5-diCQA e 3,5-dicaffeoylquinic acid; 4,5-diCQA e 4,5-dicaffeoylquinic acid; CA e caffeic acid; FA e ferulic acid; ±SD; nd e not detected; n ¼ 6; the same letter in one row corresponds to lack of significant differences (p > 0.05).

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for 2 h at 36 C. Subsequently 1.5 mL of pH 8.2 buffer together with 100 mL (1 mg mL1) of both trypsin and chymotrypsin were added. Prepared samples were re-incubated for 24 h at 36 C. The next step was the quantitative analysis of CHAs released during proteolysis (as described in the Section 2.5). 2.8. Thermodynamic analysis of peptideseCHAs interactions Calorimetric measurements of interactions between PHs and CHAs standards (CA, FA and 5-CQA) were conducted using isothermal titration calorimeter VP-ITC MicroCal (Northampton, MA, USA). The analysis was conducted according to Budryn et al., (2015). Typical ITC raw data and enthalpy changes per mole of injectant are shown in Fig. 1. The plot was used to determine the thermodynamic parameters of interactions between PHs and CHA. 2.9. Molecular modelling Molecular modelling studies were performed in order to obtain information about the interactions between the CHAs and the different PHs at the atomic level. Docking simulations were performed, since their predictions can reveal useful information about how these interactions are established (electrostatic, van der Waals,

hydrogen bonds, hydrophobic, etc), and concerning which residues ndez et al., 2012). The of the peptide are involved (Navarro-Ferna most probable sequences for the hydrolysates were taken for EOH as YPILPEYLQCVK, for WPH as SQSKVLPVPQ, and for SPH as AIPSEVLAHSYNLR (Mann et al., 2015; Monaci et al., 2014). The fullatom models of the peptides used in this study were prepared venet et al., submitting their sequences to the PEP-FOLD server (The 2012), and atomic models of the CHAs were built manually using the software package Molecular Operating Environment (MOE) (Chemical Computing Group Inc., Montreal, Canada). Afterwards atomic partial charges using the AMBER99 forcefield were added to the peptide models and the ligands using MOE (Wang, Cieplak, & Kollman, 2000). The docking of CHAs to the prepared peptide models and the detailed binding energy calculations were performed with the Lead Finder v 1.1.10 software using default configuration parameters (Stroganov, Novikov, Stroylov, Kulkov, & Chikov, 2008). The size of the grid box for ligand docking was set to extend 30 Å in each direction from the geometric centre of each peptide structure. The dG-score produced by Lead Finder was taken as the predicted value of the ligand binding energy. Only the top-ranked poses resulting from the blind docking simulations of each ligand across the whole peptide molecular surface were used for structural and energetic

Table 1B Content of hydroxycinnamic and chlorogenic acids (CHAs) from green coffee in egg ovalbumin hydrolysate (g/100 g peptides). CHAs


Bound after interactions 3-CQA 2.43 ± 0.19a 5-CQA 3.63 ± 0.21a 3-FQA 0.54 ± 0.08a 4-CQA 3.15 ± 0.15a CA 0.57 ± 0.04a 5-FQA 0.30 ± 0.06a 4-FQA 0.96 ± 0.11a FA 0.09 ± 0.02a 3,4-diCQA 0.57 ± 0.06a 3,5-diCQ 0.21 ± 0.04a 4,5-diCQA 0.42 ± 0.07a Total 12.87 ± 0.64a Released from the bonds after proteolytic digestion 3-CQA 0.18 ± 0.04 5-CQA 0.24 ± 0.06 3-FQA 0.06 ± 0.01a 4-CQA 0.18 ± 0.03 CA nd 5-FQA 0.06 ± 0.02 4-FQA 0.03 ± 0.01 FA nd 3,4-diCQA 0.03 ± 0.00 3,5-diCQ nd 4,5-diCQA nd Total 0.78 ± 0.09 Available (released after proteolytic digestion þ unbound) 3-CQA 0.75 ± 0.06 5-CQA 0.96 ± 0.05 3-FQA 0.18 ± 0.03a 4-CQA 0.63 ± 0.05 CA 0.18 ± 0.04a 5-FQA 0.24 ± 0.03 4-FQA 0.06 ± 0.01 FA nd 3,4-diCQA 0.09 ± 0.02 3,5-diCQ 0.03 ± 0.01 4,5-diCQA 0.03 ± 0.01 Total 3.18 ± 0.48

90 C GCE

90 C b-CD:CHAs

2.46 ± 0.20a 3.93 ± 0.17a 0.54 ± 0.03a 3.21 ± 0.38a 0.54 ± 0.05a 0.36 ± 0.08a 0.99 ± 0.06a 0.09 ± 0.01a 0.63 ± 0.03a 0.18 ± 0.03a 0.45 ± 0.05a 13.38 ± 0.75a

0.06 0.09 nd 0.06 0.21 0.03 nd nd nd nd 0.03 0.48

± 0.01 ± 0.02

0.03 0.03 nd 0.03 nd nd nd nd nd nd nd 0.09

± 0.01 ± 0.00

0.33 0.48 0.06 nd nd 0.12 0.06 nd 0.06 nd 0.03 1.14 0.93 1.50 0.18 0.51 0.15 0.36 0.12 nd 0.18 nd 0.09 4.05

± 0.05 ± 0.04 ± 0.02a

± 0.02 ± 0.01 ± 0.03 ± 0.01 ± 0.10 ± ± ± ± ± ± ±

0.06 0.09 0.02a 0.05 0.04a 0.02 0.02

± 0.01 ± 0.02 ± 0.50

± 0.01 ± 0.04 ± 0.01

± 0.01 ± 0.06

± 0.00

± 0.01

3.00 ± 0.43 4.59 ± 0.38 0.66 ± 0.03 3.63 ± 0.18 0.51 ± 0.40 0.51 ± 0.09 1.02 ± 0.08 0.09 ± 0.02 0.69 ± 0.08 0.21 ± 0.03 0.45 ± 0.02 15.39 ± 1.05


e 5.92 ± 0.38 e e 1.18 ± 0.09 e e 3.30 ± 0.25 e e e e e 0.90 ± 0.04 e e 0.33 ± 0.02 e e 0.48 ± 0.02 e e e _ e 5.36 ± 0.39 e e 9.53 ± 0.65 e e 7.56 ± 0.38 e e e e

3-CQA e 3-caffeoylquinic acid; 4-CQA e4-caffeoylquinic acid; 5-CQA e 5-caffeoylquinic acid; 3-FQA e 3-feruloylquinic acid; 4-FQA e 4-feruloylquinic acid; 5-FQA e 5feruloylquinic acid; 3,4-diCQA e 3,4-dicaffeoylquinic acid; 3,5-diCQA e 3,5-dicaffeoylquinic acid; 4,5-diCQA e 4,5-dicaffeoylquinic acid; CA e caffeic acid; FA e ferulic acid; ±SD; nd e not detected; n ¼ 6; the same letter in one row corresponds to lack of significant differences (p > 0.05).


ndez et al., 2012). For the visualization of analyses (Navarro-Ferna the three dimensional structures of the top-ranked poses the PYMOL software (http://pymol.org) was used, and for the 2D representation of the peptide-ligand interactions, the Poseview server was used (Stierand & Rarey, 2010). 2.10. Statistical analysis The protein hydrolysate-CHAs interactions were conducted twice. Analyses were carried out in triplicate and their results were subjected to statistical analysis. It comprised determination of average values of six measurements and their standard deviation as well as one-way ANOVA (analysis of variation) using Statistica 10.0 software at the significance level p < 0.05. 3. Results and discussion 3.1. Binding degree of CHAs and PHs Purified and freeze-dried GCE containing CHAs or inclusion complexes of CHAs with b-CD were interacted with three different PHs, commonly used to enrich novel food products i.e. WPH, EOH and SPH. As a result of the interactions a decrease of free or included CHAs concentration in the solution due to binding of CHAs to peptides was observed. Analysis of concentration of unbound CHAs in the solutions after interactions was performed to evaluate the binding degree of each CHA and the used protein hydrolysate (Table 1A,B,C) (Friedman & Jürgens, 2000). As the result of binding of CHAs to peptides, in case when they were added in the free form, the content of CHAs in PHs amounted 14.94 and 15.15 g/100 g for SPH at 25 and 90 C respectively, 13.23 and 13.32 g/100 g for WPH as well as 12.87 and 13.38 g/100 g for EOH. The binding degree of CHAs in studied models was relatively high (excluding inclusion complexes b-CD:CHAs) and amounted 94.68 and 96.01% with SPH for 25 and 90 C, respectively, 83.84 and 84.41% with WPH and 81.56 and 84.79% with EOH. As molecular modelling results showed (Fig. 2, and Supplemental Figs. 1e7), main interactions were related to hydrogen bonds, aromatic stabilization and hydrophobic interactions. The affinity of CHAs to hydrolysates followed the order: EOH < WPH < SPH. Both average peptide length and amino acid composition could have an effect on binding affinity and interactions with CHAs. Tang, Peng, Zhen and Chen (2009) demonstrated that along with an increase of a degree of hydrolysis and with shortening of average peptide length the content of polyphenols in buckwheat protein hydrolysate decreased. It was confirmed in our study by the preliminary average molecular mass analysis of PHs (data not shown), where the masses amounted about 4, 11 and 15 kDa for EOH, WPH and SPH respectively and that order agreed with that of degree of interactions. Although the structure of peptide is the second important factor influencing the interactions. The high content of amino acids such as: arginine, phenylalanine and proline may increase the degree of interactions (Budryn & Rachwał-Rosiak, 2013). The study of amino acids profile of the used PH (research in progress) showed the highest concentration of the above amino acids in SPH, while in WPH and EOH it was on a similar level. It could be the another reason for the highest affinity of CHAs with SPH. Comparing interactions of PHs with the analogous food proteins the degree of interaction was greater only for soy protein derived hydrolysate (Budryn et al., 2015). Such results are probably an effect of the greater availability of hydrophobic groups in the hydrolysate (as shown in Supplemental Figs. 1e7) as a consequence of the destruction of the soy protein at the stage of commercial hydrolysis. For the remaining two hydrolysates statistically the same level of interactions as for initial proteins was obtained.

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Table 1C Content of hydroxycinnamic and chlorogenic acids (CHAs) from green coffee in soy proteins hydrolysate (g/100 g peptides). CHAs


90 C GCE

90 C b-CD:CHAs

Bound after interactions 3-CQA 2.88 ± 0.31a 2.82 ± 0.14a 0.24 ± 0.04 5-CQA 4.32 ± 0.35a 4.44 ± 0.55a 0.33 ± 0.03 3-FQA 0.63 ± 0.04a 0.60 ± 0.04a 0.03 ± 0.00 4-CQA 3.57 ± 0.29a 3.57 ± 0.43a 0.24 ± 0.01 CA 0.69 ± 0.05a 0.69 ± 0.08a 0.27 ± 0.02 5-FQA 0.45 ± 0.05a 0.48 ± 0.05a 0.03 ± 0.01 a a 4-FQA 1.02 ± 0.08 1.02 ± 0.05 0.03 ± 0.00 a a 0.09 ± 0.01 nd FA 0.09 ± 0.01 a a 3,4-diCQA 0.66 ± 0.02 0.69 ± 0.05 0.06 ± 0.02 0.21 ± 0.01a 0.06 ± 0.01 3,5-diCQ 0.21 ± 0.04a 4,5-diCQA 0.48 ± 0.04a 0.48 ± 0.03a 0.06 ± 0.01 Total 14.94 ± 0.76a 15.15 ± 072a 1.32 ± 0.09 Released from the bonds after proteolytic digestion 3-CQA 0.15 ± 0.02 0.21 ± 0.04 0.06 ± 0.01 5-CQA 0.21 ± 0.01 0.48 ± 0.03 0.09 ± 0.03 3-FQA 0.03 ± 0.01 0.06 ± 0.02 nd 4-CQA 0.15 ± 0.00 0.21 ± 0.02 0.06 ± 0.02 CA nd nd nd 5-FQA nd 0.06 ± 0.01 nd 4-FQA 0.03 ± 0.01a 0.03 ± 0.01a nd FA nd nd nd 3,4-diCQA nd nd nd 3,5-diCQ nd nd nd 4,5-diCQA nd nd nd Total 0.63 ± 0.04 1.11 ± 0.07 0.21 ± 0.05 Available (released after proteolytic digestion þ unbound) 3-CQA 0.36 ± 0.05a 0.36 ± 0.02a 2.85 ± 0.19 5-CQA 0.42 ± 0.03 0.81 ± 0.05 4.41 ± 0.22 a a 3-FQA 0.09 ± 0.02 0.09 ± 0.01 0.63 ± 0.05 4-CQA 0.24 ± 0.02 0.30 ± 0.05 3.48 ± 0.39 CA 0.03 ± 0.01a 0.03 ± 0.00a 0.45 ± 0.03 5-FQA 0.06 ± 0.00 0.15 ± 0.01 0.51 ± 0.05 4-FQA 0.03 ± 0.01a 0.03 ± 0.01a 0.99 ± 0.12 FA nd nd 0.09 ± 0.01 3,4-diCQA nd 0.03 ± 0.00 0.63 ± 0.02 3,5-diCQ nd nd 0.15 ± 0.04 4,5-diCQA nd nd 0.42 ± 0.03 Total 1.26 ± 0.09 1.95 ± 0.11 14.67 ± 1.05


e 8.13 ± 0.67 e e 3.16 ± 0.40 e e 5.02 ± 0.32 e e e e e 1.86 ± 0.12 e e 0.45 ± 0.03 e e 0.75 ± 0.08 e e e e e 4.11 ± 0.29 e e 7.67 ± 0.61 e e 6.11 ± 0.43 e e e e

3-CQA e 3-caffeoylquinic acid; 4-CQA e4-caffeoylquinic acid; 5-CQA e 5caffeoylquinic acid; 3-FQA e 3-feruloylquinic acid; 4-FQA e 4-feruloylquinic acid; 5-FQA e 5-feruloylquinic acid; 3,4-diCQA e 3,4-dicaffeoylquinic acid; 3,5-diCQA e 3,5-dicaffeoylquinic acid; 4,5-diCQA e 4,5-dicaffeoylquinic acid; CA e caffeic acid; FA e ferulic acid; ±SD; nd e not detected; n ¼ 6; the same letter in one row corresponds to lack of significant differences (p > 0.05).

The interactions were conducted at pH 6.45. It is known that at pH from 5 to 7 hydrophobic and electrostatic interactions are predominant (Budryn & Rachwał-Rosiak, 2013). In the light of this data these two kinds of interactions were expected and this was confirmed by molecular modelling results, as shown in Fig. 2 and Supplemental Figs. 1e7. The degree of interactions of specific CHAs from GCE increased in the following order: FQAs < CQAS < diCQAs < FA < CA. An analogous study of protein interactions was carried out with the CA, 5-CQA and FA standards. This choice of standards made it possible to observe the effects of hydroxyl group methylation on the aromatic ring (CA vs. FA) or its esterification with quinic acid (CA vs. CQA) on the degree of interaction with PHs. FA is capable of interacting with proteins in a similar manner as FQAs found in GCE because both have methoxylated one hydroxyl moiety on the phenolic ring. The degree of interactions of CHAs in the form of standard substances with peptides was different than for those derived from GCE and added as a mixture. In the case of standards the content of CHAs in PHs was significantly lower and ranged from

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Fig. 2. Results obtained from docking simulations for the lowest energy pose obtained from blind docking simulations: (A) 5-CQA (yellow) and peptide SQSKVLPVPQ (purple); (B) FA (pink) and peptide SQSKVLPVPQ (Purple); (C) 5-CQA (yellow) and peptide YPILPEYLQCVK (light brown); (D) FA (pink) and peptide YPILPEYLQCVK (light brown); (E) 5-CQA (yellow) and peptide AIPSEVLAHSYNLR (green); (F) FA (pink) and peptide AIPSEVLAHSYNLR (green); (G) CA (light green) and peptide AIPSEVLAHSYNLR (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

1.18 (CA with EOH) to 8.13 g/100 g of peptide (5-CQA with SPH). The lowest degree of interactions was observed for CA, then for FA, and the largest for 5-CQA. This is probably due to the fact that 5-CQA has more hydroxyl groups available for establishing hydrogen bonds, as our molecular modelling results showed in Fig. 2 and Supplemental Figs. 1e7. Thus, both the presence of a quinic acid moiety and methoxylation of one of the hydroxyl groups at the phenolic ring intensified the interactions, since they increase the hydrophobicity of polyphenols and the hydrophobic interactions play also an important role in polyphenol-peptide binding, as was shown in Fig. 2 and Supplemental Figs. 1e7. Similar observations were made by Xiao and Kai (2012) in the study on plasma proteinsepolyphenols interactions, The degree of interactions of standard CHAs with different PHs increased in the following order:

WPH < EOH < SPH. Molecular modelling results showed that average peptide length was the largest for SPH and thus the capacity of the peptide for establishing hydrogen bonds with the CHAs, and in a lesser extent, for hydrophobic interactions due to slightly larger contact area was the highest for that PH. For CHAs included in b-CD the binding degree with PHs was substantially lower, in the range of 0.48e1.32 g/100 g of peptides (Table 1A,B,C). It is suspected that this limitation of interactions is caused by lower availability of the aromatic ring, which is fixed in the cavity of b-CD. Therefore as a result of inclusion complex formation it is possible not only to increase oxidative stability of CHAs during food processing but also to limit their interactions with PHs. The reaction temperature statistically did not affected the intensity of peptides-CHAs interactions so the effect of temperature rise on

Fig. 3. LC/MS2 analysis of interaction products of whey proteins hydrolysate with hydroxycinnamic and chlorogenic acids from green coffee extract at pH 6.45 and 90 C: (A) - All MS of interaction products at negative mode; (B) - MS spectra of interaction products at 19 min with molecular ions M at m/z 353.21 (CQA) and M at m/z 421.20 (interaction product of peptide fragment and CQA); (C) e MS2 spectra of molecular ion M at m/z 421.20 at 19 min with molecular ion M at m/z 353.21 (CQAs).

830


reduction of electrostatic interactions postulated by Prigent et al. (2003) was not observed. This may indicate the major importance of hydrophobic interactions. LC-Q-TOF-MS/MS analysis confirmed the formation of peptidesCHAs adducts. Fig. 3(A) presents exemplary LC/MS chromatogram of WPH and CHAs after interactions at 25 C. CQAs occurred in interaction products in the largest concentrations, so their presence in MS and MS/MS spectra was relatively simple to observe. The chromatogram was searched for M ion at m/z 353 characteristic for CQAs. These ion was found for example at 19 min of analysis. It was then observed in MS spectrum (Fig. 3(B)), indicating that CQAs were bound with peptides by hydrophobic interactions that were easily broken down already during ionization. Additionally this ion was found in MS/MS spectrum after fragmentation of ion M at m/z 421 (Fig. 3(C)), indicating that CQAs were bound with peptides also by electrostatic bonds that were broken down only under the influence of fragmentation. Chromatograms of other PHs, i.e. EOH and SPH gave similar observations. Afterwards CA, 5-CQA, and FA standards were used in ITC assay to assess the binding parameters of the PHs with the chosen CHAs standards. The maximum number of binding sites (bs) for each protein hydrolysate with particular CHAs was evaluated. For WPH binding of four FA molecules and five 5-CQA was observed, and for CA the recorded interactions were unstable and it was not possible to calculate its binding parameters (Table 2). Interactions of EOH with CA were also difficult to estimate, while 5-CQA and FA were bound in a number of six and three molecules, respectively. For SPI binding of three CA, one of FA and five molecules of 5-CQA was observed. Taking into account the average molecular mass of the PHs, the maximum content of CHAs standards in PH amounted from 3.48 to as high as 47.88 g/100 g of peptides and it increased in the order: WPH < SPH < EOH. Free energy change (DG), which is the measure of the affinity of CHAs to PHs indicated that for WPH the highest affinity was observed for 5-CQA, whereas for EOH and SPH the highest affinity was marked for FA. Comparing CA and FA in terms of their affinity to PHs was possible only for SPH, where it was higher for FA. Therefore methoxylation of hydroxyl moiety had a positive effect on binding affinity that indicated hydrophobic interactions as the most intense for this protein hydrolysate. Docking predictions for binding affinity are included in Table 2. They only in part confirmed the results obtained by ITC. It might be influenced by the fact that only one type of peptide selected from the literature was used for docking predictions instead of the mixture of peptides titrated in the ITC assay. Enthalpy change (DH) was lower than entropy change (DS) for the analysed interactions, which indicated entropic interaction mechanism (Yuksel et al., 2010). Binding constant (k) of the analysed PHs with CHAs ranged from 0.76 to 2920 103 1/M and

was the highest for WPH. Docking predictions showed the high flexibility of peptide chosen from WPH for molecular modelling that could promoted the high value of k for this PH by high matching of functional groups, comparing to the remaining two peptides from EOH and SPH. The ITC analysis showed that it is possible to obtain very high concentrations of CHAs bound to PHs during food processing. The effect of the interactions on the nutritional value of food products depends on susceptibility of formed interactions to be broken down during digestion and it could depend among others from the k value. Thus the next step was to evaluate the stability of interactions during proteolytic digestion. 3.2. Availability for absorption of CHAs after interactions with proteins and proteolysis The amount of released CHAs was analysed after proteolytic digestion. It was at a level of 4.20e16.67% of previously bound CHAs and statistically FQAs were released in higher amounts then CQAs (Table 1A,B,C). Free CA and FA were not detected in the solution after proteolysis and diCQAs (except 3,4-diCQAs) were almost not found among the released phenolics. Bound CHAs caused decrease of availability of peptide bonds for proteolytic enzymes (see Fig. 2 and Supplemental Figs. 1e7) and, consequently, more difficult peptide digestion that finally prevented the release of CHAs from hydrophobic and electrostatic interactions. Although the temperature statistically had no effect on interactions degree, after proteolysis higher amounts of free CHAs were determined in case of interaction products obtained at 90 C. Considering the type of protein hydrolysate the increase of CHAs released after proteolysis followed the order: SPH < EOH < WPH. In the test with standards of CHAs the phenolics were released after proteolysis in the amounts from 14.24 to 43.72% of previously bound. PHs interacted with bCD:CHAs inclusion complexes in negligible extent thus after digestion of peptides reacted with b-CD:CHAs release of CHAs was almost not found. The amount of available for absorption CHAs may be considered as the sum of those unbound and released after proteolytic digestion. It ranged 22.61 and 22.81% for WPH, 20.15 and 25.67% for EOH, as well as 7.98 and 12.36% for SPH interacted at 25 and 90 C, respectively. The profile of peptides and free amino acids released after proteolytic digestion will be the subject of further studies. In case of CHAs standards used for interactions the level of available for absorption phenolics was in the range from 35.55 to 83.53% and they were available in the largest amounts after interactions with EOH, while in the smallest with SPH. Nevertheless for b-CD:CHAs percentage of CHAs available after interactions and digestion was in the range of 92.11e97.53%, assuming that they could be completely

Table 2 Binding parameters of interactions of whey proteins hydrolysate (WPH) egg white ovalbumin hydrolysate (EOH) and soy proteins hydrolysate (SPH) with caffeic, ferulic and chlorogenic acids standards (CHAs) using isothermal titration calorimetry (ITC); n ¼ 6. Docking predictions for binding affinity are also included. Protein hydrolysate

CHAs

bs e number of binding sites

WPH

CA FA 5-CQA CA FA 5-CQA CA FA 5-CQA

unstable interactions 4.43 ± 0.06 3.48 4.84 ± 0.02a 3.53 unstable interactions 3.38 ± 0.13 15.81 5.61 ± 0.18b 47.88 4.40 ± 0.62 16.91 1.38 ± 0.11 5.72 5.23 ± 1.85a,b 39.52

EOH

SPH

Concentration of CHAs g/100 g of peptides

K (x103 1/M)

± 0.05 ± 0.02

2920 ± 450 1310 ± 320

± ± ± ± ±

11.90 2.46 1.98 7.19 0.76

0.61 1.54a 2.38 0.44 13.98a

± ± ± ± ±

2.02 0.65 1.10 0.92 0.57

DH (kJ/mol)

2.60 ± 0.07 2.12 ± 0.02 5.00 5.90 8.04 5.95 11.30

± ± ± ± ±

0.32 0.58a 2.51 0.59a 7.54

DS (J/mol K)

DG (kJ/mol)

DGpred (kJ/mol)

76.62 110.11

25.45 34.95

18.82 20.50

61.13 45.22 36.17 54.01 17.04

23.22 19.38 18.29 22.05 16.38

16.73 23.01 19.66 20.08 27.19

CA e caffeic acid; 5-CQA e 5-caffeoylquinic acid. FA-ferulic acid; ±SD; different letters at the same compound for different protein hydrolysates in one column correspond to significant differences (p > 0.05).


released from the complexes in the gut. It indicated that inclusion of CHAs with b-CD might be an effective method of preventing high nutritional value of PHs and CHAs in functional foods supplemented with phenolics and peptide formulations. 4. Conclusions Relatively high degree of interactions of CHAs with hydrolysates of food proteins from egg white, whey and soy in close to the natural pH, at room temperature and 90 C was detected in this study. The character of interactions was mostly hydrophobic and electrostatic and they were largely stable under proteolytic digestion. Inclusion of CHAs with b-cyclodextrin limited interactions with protein hydrolysates. Acknowledgments Authors are grateful for the financial support provided by Narodowe Centrum Nauki (Project UMO-2011/03/B/NZ9/00745) and n Se neca del Centro de Coordinacio n de la Investigacio n by Fundacio n de Murcia (Project 18946/JLI/13). de la Regio Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.lwt.2015.09.001. References Altunkaya, A. (2011). Effect of whey protein concentrate on phenolic profile and browning of fresh-cut lettuce (Lactuca Sativa). Food Chemistry, 128, 754e760. , B., Estrella, I., & Herna ndez, M. T. J. (2000). Interaction of low molecular Bartolome weight phenolics with proteins (BSA). Journal of Food Science, 65, 617e621. Bassoli, B. K., Cassolla, P., Borba-Murad, G. R., Constantin, J., SalgueiroPagadigorria, C. L., Bazotte, R. B., et al. (2008). Chlorogenic acid reduces the plasma glucose peak in the oral glucose tolerance test: effects on hepatic glucose release and glycaemia. Cell Biochemistry and Function, 26, 320e328. Budryn, G., Nebesny, E., Rachwał-Rosiak, D., Pałecz, B., Hodurek, P., Miskiewicz, K., et al. (2014a). Inclusion complexes of b-cyclodextrin with chlorogenic acids from crude and purified aqueous extracts from green Robusta coffee beans (Caffea canephora L.). Food Research International, 61, 202e213. _ zelewicz, _ Budryn, G., Nebesny, E., Zy D., & Oracz, J. (2014b). Properties of model systems of sunflower oil and green coffee extract after heat treatment and storage. LWT e Food Science and Technology, 59, 467e478. ska, D., Belica, S., et al. Budryn, G., Pałecz, B., Rachwał-Rosiak, D., Oracz, J., Zaczyn (2015). Effect of inclusion of hydroxycinnamic and chlorogenic acids from green coffee bean in b-cyclodextrin on their interactions with whey, egg white and soy protein isolates. Food Chemistry, 168, 276e287. Budryn, G., & Rachwał-Rosiak, D. (2013). Interactions of hydroxycinnamic acids with proteins and their technological and nutritional implications. Food Reviews International, 29, 217e230. Cheng, J. C., Dai, F., Zhou, B., Yang, L., & Liu, Z. L. (2007). Antioxidant activity of hydroxycinnamic acid derivatives in human low density lipoprotein: mechanism and structure-activity relationship. Food Chemistry, 104, 132e139. Clemente, A. (2000). Enzymatic protein hydrolysates in human nutrition. Trends in Food Science and Technology, 11, 254e262. Couchman, L. (2012). Turbulent flow chromatography in bioanalysis: a review. Biomedical Chromatography, 26, 892e905. El-Medany, A., Bassiouni, Y., Khattab, M., & Mahesar. (2011). Chlorogenic acid as potential anti-inflammatory analgesic agent: an investigation of the possible role of nitrogen-based radicals in rats. International Journal of Pharmacology and Toxicology Science, 1, 24e33. Friedman, M., & Jürgens, H. S. (2000). Effect of pH on the stability of plant phenolic compounds. Journal of Agricultural and Food Chemistry, 48, 2101e2110. Gacche, R. N., Zore, G. B., & Ghole, V. S. (2003). Kinetics of inhibition of polyphenol oxidase mediated browning in apple juice by b-cyclodextrin and L-ascorbate-2triphosphate. Journal of Enzyme Inhibition and Medicinal Chemistry, 18, 1e5. Gallo, M., Vinci, G., Graziani, G., De Simone, C., & Ferranti, P. (2013). The interaction of cocoa polyphenols with milk proteins studied by proteomic techniques. Food Research International, 54, 406e415. ndez-Jabalera, A., Corte s-Giraldo, I., Da vila-Ortíz, G., Vioque, J., Alaiz, M., Herna n-Calle, H., et al. (2015). Influence of peptides-phenolics interaction on the Giro antioxidant profile of protein hydrolysates from Brassica napus. Food Chemistry, 178, 346e357. ndez-Ledesma, B., & de Lumen, B. O. (2008). Lunasin: a novel cancer preHerna ventive seed peptide. Perspectives in Medical Chemistry, 2, 75e80.

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