ProteinâTannin Interactions of Tryptic Digests of Î±-Lactalbumin and

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Protein-Tannin Interactions of Tryptic Digests of #- Lactalbumin and Procyanidins Bei Wang, and Marina Heinonen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04256 • Publication Date (Web): 07 Dec 2016 Downloaded from http://pubs.acs.org on December 8, 2016

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Journal of Agricultural and Food Chemistry

Protein-Tannin Interactions of Tryptic Digests of α- Lactalbumin and Procyanidins

Bei Wang*, Marina Heinonen Department of Food and Environmental Sciences, Food Chemistry, P.O. Box 27, FI-00014 University of Helsinki, Finland

* Corresponding Author. Phone: +358-50 4485, E-mail: [email protected]

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ABSTRACT

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Protein-tannin interactions on a molecular level were investigated by using a model system

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containing peptides of α-lactalbumin and berry tannins (procyanidins). Oxidation of isolated tryptic

4

peptide LDQWLCEK (m/z 1034) with procyanidin B2 or procyanidin fraction (PF) isolated from

5

aronia juice was monitored by LC-ESI-MS. Procyanidin B2 and PF showed radical scavenging

6

activities toward oxidation of the peptide with the peptide also preventing procyanidin B2 from

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degradation. Oxidation enhanced the cleavage of peptide between tryptophan and glutamine.

8

Interaction products arising from WLCEK or WLCE residue and degradation product of

9

procyanidin B2 were also identified using both size exclusion chromatography and LC-MS.

10

Tryptophan and lysine were the amino acids most prone to interact with procyanidin B2. The study

11

shows that protein-tannin interaction takes place during oxidation leading to both degradation of the

12

parent compounds and formation of interaction products. This may in turn affect the quality of

13

protein and tannin containing food.

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KEYWORDS: protein-tannin interaction, procyanidin, aronia, α- lactalbumin, digestion, tryptic,

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protein oxidation, HPLC, LC-MS, SEC, size exclusion, whey protein

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INTRODUCTION

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Proteins are known to bind to polyphenols when present simultaneously such as in dairy foods with

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berry ingredients containing tannins. The consequent formation of soluble and insoluble protein-

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polyphenol interaction products may affect the quality of protein containing food.1 Phenolic

27

compounds can complex with proteins through non-covalent forces including hydrogen and

28

hydrophobic bonding or irreversibly through covalent bonds.2-6 The stability of the polyphenol-

29

protein complexes depends on not one but many reactive groups in the amino acids. The ability of

30

the protein to interact with phenolic compounds is related to the protein’s secondary structure. The

31

greater extent of hydrogen bonding depends on the increased accessibility of the peptide bond. In

32

addition, the carbonyl groups of tertiary amides are better than the carbonyl groups of primary or

33

secondary amides as a hydrogen bond acceptor. 7-11

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However, there is rather limited experimental data regarding the structural basis for protein-tannin

35

interactions. Our previous interaction experiments between specific isolated tryptic digests of β-

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lactoglobulin (β-Lg) and a dimeric ellagitannin isolated from berries support our hypothesis that

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oxidation of proteins may have a role in protein-tannin interactions as tannins are known to have

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antioxidant properties.12-16 This hypothesis is further investigated in the current study using a model

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system containing peptides isolated from dairy whey proteins and tannins isolated from a

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procyanidin rich berry source (aronia). Ortoquinone is formed by the oxidation of polyphenol,

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which could on one hand undergo Michael addition reaction with nucleophilic groups, or on the

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other hand, build the cross-link by Schiffs base formation.17-19

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Dairy whey proteins (WP) are one of the highly nutritious food ingredients available for

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commercial use not only because they contain high concentration of all the essential amino acids

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compared to any other natural food protein source, but also due to the high content of branched

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chain amino acids that contribute to the structure of food. 20-23 Whey proteins are also highly

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soluble over a wide pH range which enables stabilizing emulsions by creating interfacial films

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between hydrophilic and hydrophobic food components. 24 As well as with other proteins, the amino

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acids in whey proteins, such as α-lactalbumin (ALA) comprising of 20-25% of whey proteins 25 ,

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are succeptible to oxidation. With the molecular weight of 14.2 kDa ALA consists of 123 amino

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acid residues stabilized by four disulfide bonds and a tightly bound Ca2+.

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tryptophan (W, Trp) and lysine (K, Lys) are the amino acids prone to be oxidized by reactive

53

oxygen species (ROS).

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oxidation may occur with cysteine, which generates sulfenic acid (RSOH), sulfinic acid (RSO2H),

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sulfonic acid (RSO3H) forms of oxidation products. Hydroxytryptophan and N-formylkynurenine

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are the two oxidation products resulting by oxidation of tryptophan. The oxidation of tryptophan is

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irreversible, while it is reversible for cysteine.

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groups may be formed by lysine, such as amino-adipinicsemialdehyde.

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products of W, C, K in peptides, such as tryptic digests of ALA, could be used as markers to

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monitor both the oxidation of the peptide or protein as well as protein-phenolic interaction.

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The aim of the study was to investigate at a molecular level the antioxidant reactions and protein-

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tannin interactions by applying both LC-ESI-MS and size exclusion chromatography (SEC) to

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investigate oxidation, adduct formation, and binding reactions. A model system containing a

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selected tryptic peptide isolated from α-lactalbumin together with procyanidin B2 and a

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procyanidin fraction isolated from aronia juice was used in the oxidation experiments.

27, 28

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Cysteine (C, Cys),

Disulfur bonds may form between cysteines and the two-electron

29, 30

In addition, the ketone and aldehyde carbonyl 31

Therefore, oxidation

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MATERIALS AND METHODS

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Materials. Chromatographically purified and lyophilized α- Lactalbumin (ALA) from bovine milk

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was purchased from Sigma Aldrich, Inc. (St. Louis, MO, USA). Sequencing grade modified trypsin

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was obtained from Promega Corp. / BioFellows (Madison, WI, USA). Of the reagents used in the 4 ACS Paragon Plus Environment

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analyses ammonium bicarbonate, hydrogen peroxide (30% wt. solution in water) and iron(III)

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chloride (reagent grade, 97%) were purchased from Sigma-Aldrich (Steinheim, Germany) whereas

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L(+)-ascorbic acid was a product of Merck (Darmstadt, Germany), PIPES-buffer [piperazine-1,4-

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bis(2-ethanesulfonic acid)] from FlukaBioChemika (Buchs, Switzerland), and Sephadex LH-20

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from GE Healthcare (Sweden). Methanol, acetone and acetonitrile were of HPLC grade and

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purchased from Rathburn (Walkerburn, Scotland). All other chemicals used in the analyses were

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supplied by J.T. Baker (Deventer, The Netherlands) or Sigma-Aldrich (Steinheim, Germany) in

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either HPLC or reagent grade. Water used was always purified first by the Milli-Q system

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(Millipore Corp., Bedford, MA, USA). Procyanidin B2 was a product of Extransynthese (GENAY

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Cedex) and the procyanidin fraction was extracted from aronia juice (Nutrika, Vašazdrava, Serbia).

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Digestion and fractionation of ALA peptides. The in-liquid digestion of ALA was prepared using

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sequencing grade modified trypsin to obtain the cleavage to peptides which were later to be

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separated and collected using preparative HPLC according to the method described by Koivumäki

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et al. (2012) 32.

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Isolation and analysis of procyanidin fractions from aronia juice. The aronia juice procyanidins

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were fractionated in a 300 x 40 mm open glass column packed with Sephadex LH-20 that was first

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swollen in acetone. The procedure was modified from those of Hellström et al. (2007) 33 and Kylli

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et al. (2011) 34. The column was prepared with 30% methanol prior to introducing the aronia juice

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sample (15 mL, including 10% methanol, filtered with 5 µm AcrodiscVersapor 25 mm syringe

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filter). The column was then eluted with 3 x 300 mL of 30% methanol and the first fraction

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containing organic acids and sugars was discarded before collection of procyanidins while

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hydroxycinnamate and anthocyanin fractions remained. The procyanidin fraction was evaporated

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into dryness using vacuum. The dry procyanidin fractions from several repeated fractionation

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procedures were dissolved in 20% methanol, sampled for UHPLC analysis and frozen until use.



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Procyanidins (DP2-10) were analyzed from the isolated aronia juice fraction using a NP-HPLC

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method described by Hellström et al. 2007 33 with minor modifications by Kylli et al. 2011 34. The

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equipment consisted of a Waters 717 plus Autosampler, Waters 515 HPLC Pump, Waters996

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Photodiode Array Detector (PDA).The column was a 5 µm Phenomenex Silica Luna 250 x 4.6 mm

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with

pre-column

Phenomenex

silica,

4.0

x

3.0

mm.

The

eluents

consisted

of

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dichloromethane/methanol/water/acetic acid in ratios of 82:14:2:2 (v/v/v/v, eluent A) and 10:86:2:2

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(v/v/v/v, eluent B). The flow rate used was 1 mL/min, column temperature 35 °C and the gradient

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program is 13.5 % eluent B at 20 minutes, 29.2 % eluent B at 50 minutes then 100 % eluent B from

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55 to 65 minutes.

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The sample was diluted with methanol and filtered with 0.45 µm Acrodisc GHP syringe filters prior

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to analysis. The total procyanidin fraction and the percentual amounts of dimers, oligomers, and

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polymers in the aronia juice were quantified with PDA at 280 nm using procyanidin B2 as the

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standard compound.

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Oxidation of the peptide samples with procyanidin B2 or aronia juice procyanidin fraction

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(PF). The chosen peptide LDQWLCEK (m/z 1034) was prepared into separate oxidation samples

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with and without adding of procyanidin B2 or the aronia juice procyanidin fraction (PF).Regarding

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the nature of the adduct between peptide and the procyanidin B2, the molar ratio of peptide-to-

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tannin was chosen to be 1:1 and 10:1, which were considered the molecular weight of procyanidin

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fraction was the same as procyanidin B2 (m/z 577). After procyanidin B2 or procyanidin fraction

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was added to samples, H2O2-solution (final concentration 1 mM) was added into each sample just

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before the reaction in order to start the oxidation reactions. The samples in triplicates (in duplicates

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for samples with peptide LDQWLCEK and PF) were placed in an oven of +37 °C for 7 days with

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stirring on a magnetic tray. An aliquot of 200 µL of each sample was taken on days 0, 1, 4, and 7

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for analysis of reaction products. The sub samples were collected into Eppendorf tubes and stored at

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-20 °C until analysis. 6 ACS Paragon Plus Environment

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Analysis of the oxidation and interaction products by LC–MS. The LC–MS used in the analysis

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of all the oxidation samples was an Agilent 1100 HPLC including a binary pump, a degasser, an

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automated sample manager, a column heating unit and DAD and fluorescence detectors (Agilent

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Technologies, Santa Clara, CA, USA) all connected to a Bruker Esquire quadrupole ion trap mass

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spectrometer (QIT-MS, from Bremen, Germany) using electrospray ionization (ESI) in both

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positive and negative mode. The column was Waters XBridge BEH130 C18 (3.5 µm, 2.1 x 100 mm)

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together with a pre-column (both by Waters Corp., Wexford, Ireland). Injection volume was always

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10 µL and the column temperature was kept at +30 °C. Flow rate was 350 µL/min and the eluents

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consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B).

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The diode array detector was set to record at 214 nm and the fluorescence detector was set to 280

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nm (Ex) and 350 nm (Em). The gradient used as well as all the MS-parameters for the positive

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mode was consistent with the method presented by Koivumäki et al. (2012) 32. Only in the negative

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mode the MS-parameters were optimized using peptide LDQWLCEK and procyanidin B2 and were

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set to following: dry temperature +300 °C, dry gas 8.01/min, nebulizer 60.0 psi, capillary 3800 V,

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end plate offset -500, trap drive 102.5, capillary exit -178.5, lens-1 5.0, lens-2 60.0, and octopolerf

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amplitude 137.7 Vpp. For both positive and negative mode the mass spectra were recorded in the

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full-scan mode over the m/z-range of 200-2200 and analyzed by Bruker Daltonics Data Analysis

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software (Bremen, Germany).

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Analysis of the oxidation and interaction products by size exclusion chromatography (SEC).

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Size exclusion chromatography analyses were performed to analyze the oxidation product of

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peptide LDQWLCEK and interaction products between peptide LDQWLCEK and procyanidin B2

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or aronia procyanidin fraction. SEC analyses were operated in room temperature on a Waters

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system including Waters® 717 plus autosampler, 515 HPLC pump coupled with Waters 2475

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Fluorescence (FLR) Detector and Waters 996 photodiode array detector. Two prepacked columns

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Superdex 200 10/300 GL were used. The injection volume was 60 µL. Flow rate was 0.6 mL/min 7 ACS Paragon Plus Environment


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and the eluents consisted of 0.1% TFA, 30% acetonitrile in aqueous solution. The diode array

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detector was set to record at 210 nm and 280 nm and the fluorescence detector was set to 280 nm

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(Ex) and 350 nm (Em). The running time of each sample was set as 130 min.

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Statistical data analysis

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Analysis of variance (ANOVA) was carried out by Microsoft Excel 2013. Significance was defined

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as a p value ˂ 0.05.

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RESULTS AND DISCUSSION

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Identification of procyanidin B2 and isolated aronia juice procyanidin fractions

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The identity of procyanidin B2 standard was verified using both NP-HPLC analysis (Figure 2) and

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LC-MS for further comparisons with the analyses of tannin-peptide interaction products. The

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fragment m/z 578 is the molecular weight of procyanidin B2 and m/z 577 is the form [M-H] - in

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negative mode. The fragment 577 was further identified by MS/MS in negative mode (Figure 3).

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The fragment ions found, m/z 289, m/z 407, m/z 425, and m/z 451, comply with the literature. 35, 36

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A further fragment ion, m/z 1155.1, was identified as the dimer of procyanidin B2. 35, 36

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Aronia proanthocyanidins consist solely of (-)-epicatechin units connected by a C4 to C8 linkage.

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33, 37

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proportions of dimers, oligomers, polymers were 60.8%, 26.5% and 12.7%, respectively (Figure 2).

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The dimers included 4% of procyanidin B2. However, most peaks eluted prior to procyanidin B2,

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which indicates that these dimeric procyanidins isolated from aronia juice may be lower in

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molecular size as compared to the procyanidin B2.

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Oxidative stability of peptide LDQWLCEK with procyanidins

The amount of procyanidins in aronia juice in the present study was 1.1 mg/mL and the relative


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The ALA peptide LDQWLCEK (m/z 1034) with added procyanidin B2 in ratio of 1:1 was the

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significantly most stable as compared to oxidation of the peptide without added procyanidins or to

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peptide-tannin ratios of 10:1 (Figure 4). The higher ratio of the procyanidin resulted in less oxidized

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forms of LDQWLCEK. This is according to previous findings were berry procyanidins also have

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been found to be protective toward protein oxidation, however, as measured by oxidation products

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of tryptophan by HPLC or loss of tryptophan fluorescence in liposomes.

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most of the fragment ions (m/z 1034, 1050, 1098, 1114), representing various oxidized forms of the

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peptide, were significantly lower with procyanidins present than with those without (Figure 4). The

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peptide LDQWLCEK with added procyanidin B2 in ratio of 1:1 retained the highest amount of

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parent peptide at fragment ion m/z 1034 with the lowest amount of peptide oxidation products in

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fragment ions m/z 1050 (+16 amu) and m/z 1066 (+32 amu) formed.

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The oxidation products in fragment ion m/z 1050 are tentatively identified as sulfenic acid (RSOH)

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form derived from cysteine or hydroxytryptophan derived from tryptophan of peptide LDQWLCEK

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with a 16 amu shift based on the assumption that cysteine and tryptophan are among the amino

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acids most prone to oxidation in the peptide LDQWLCEK. The oxidation products in fragment ion

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m/z 1066 are likely to be the sulfinic acid (RSO2H) derived from cysteine or N-formylkynurenine

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derived from tryptophan of peptide LDQWLCEK with a 32 amu shift, or containing both sulfenic

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acid (RSOH) form and hydroxytryptophan, both with a 16 amu shift. Oxidation of sulfur-containing

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amino acids such as methionine in an isolated whey peptide has earlier been shown to yield

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sulfoxide (+16 amu) and sulfone (+32 amu).

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tryptophan is known to lead to formation of a carbonyl compound, formylkynurenine. 29

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Also PF in the ratio of 1:1 enhanced the stability of peptide LDQWLCEK (m/z 1034) toward

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oxidation as monitored by the loss of original peptide and formation of the oxidation products, m/z

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1050 and m/z 1066 (Figure 4). While procyanidin B2 at ratio of 1:1 most effectively prevented fast

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oxidation resulting in oxidation products of m/z 1050 and m/z 1066, the PF at ratio 1:1 performed

32

12, 13, 38

The amounts of

On the other hand, cleavage of the pyrrole ring of



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better in preventing later stage oxidation i.e. inhibiting formation of oxidation products of m/z 1082,

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1098, and 1114 (Figure 4). This result is in accordance with previous studies reporting that when

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monitoring protein oxidation by tryptophan fluorescence the dimeric and trimeric procyanidins of

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lingonberries were more effective than the monomers.

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of the lower DP phenolics have been exhausted by the rapid scavenging activity and interaction

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with peptide LDQWLCEK, that the oligomeric and polymeric procyanidins of aronia juice fraction

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may contribute more hydroxyl groups to the later stage oxidation reactions, thus showing higher

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later stage scavenging activity than the dimeric procyanidin B2. This effect of oligo- and polymeric

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berry procyanidins on the formation of later stage protein oxidation products has not been

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previously reported. Moreover, the higher the degree of polymerization is, the stronger antioxidant

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efficacy it will display due to the increasing electron delocalization of the phenoxyl radical by the

203

interflavan linkage. 40-43 These findings are coherent with the study of Von Staszewski et al. (2011)

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44

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with the same total polyphenol concentration. 44 For example, Bartolome et al. (2000) 45 studied the

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interaction between BSA and low molecular weight phenolics and found that the strongest

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interaction (BSA-binding affinity) was with 3, 4-dihydroxy benzoic and cinnamic acid, whereas p-

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hydroxybenzoic acid did not interact with BSA.

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Interaction between peptide LDQWLCEK and procyanidins

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Small molecular size products (1a, 1b) were detected by SEC already on day 0 (before oxidation) in

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the sample containing both parent peptide m/z 1034 and procyanidin B2 (Figure 5). The UV peak

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area of peak 1 displaying interaction between peptide and B2 on Day 7 (1c) was not changed

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compared to that on day 0 (1a), whereas the fluorescence of peak 1 on Day 7 (1d) was significantly

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increased. As the molecular size of this interaction product (peak 1) is smaller than that of either

215

procyanidin B2 or peptide LDQWLCEK, it indicates a breakdown product of peptide or its

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interaction product with degraded procyanidin B2. Most likely this breakdown product was due to

12, 13

In the present study it may be because

, which revealed that the degree of inhibition of antioxidant activity in each variety varied even


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degradation of the peptide to WLCEK and WLCE residues interacting with breakdown products of

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procyanidin B2. This conclusion is supported by LC-MS results showing that oxidation enhanced

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the cleavage of tryptophan and glutamine resulting in increase of the sodium product of peptide

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WLCEK residue m/z 699 (Figure 6, peak 2). The amount of peptide WLCE residue m/z 547 ([M-

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H]-) increased in the sample with procyanidins B2 as compared to without. This demonstrates that

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the cleavage between tryptophan and glutamine, as well as the cleavage between glutamate and

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lysine were enhanced by adding procyanidin B2 suggesting reaction between tryptophan and

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procyanidin, as well as reaction between lysine and procyanidin B2. Lysine has positive charge near

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neutral pH which makes it possible to react with the aromatic ring of procyanidin B2. The

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hydrophobic effect takes place simultaneously when dispersion interactions happen.

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other hand, the indole moiety of tryptophan is the most likely group to react with procyanidin B2

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based on studies showing that semiquinones or quinones of phenolic compounds may react with the

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heterocyclic nitrogen-atom of tryptophan. 48, 49

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The data shows that procyanidin B2 was degraded due to oxidation (Figures 5 and 6) making the

231

degradation products available for interaction reactions. It was also seen that peptide LDQWLCEK

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protected B2 from degradation as more of the procyanidin in the combination sample was still intact

233

after 7 days of oxidation (Figure 6, peak 1c) as compared to no presence of peptide (Figure 6b). The

234

amount of peptide LDQWLCEK (Figure 6 peak 19) was higher in the sample with procyanidin B2

235

on day 7 than without indicating that also the procyanidin may inhibit oxidation of the peptide.

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With the PF from aronia juice no protective effect toward oxidation could be seen as both the PF

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and the peptide were totally consumed in the combination sample (Figure 7). Neither were any

238

interaction products observed between PF and the peptide. Interaction between parent peptide ion

239

m/z 1034 and procyanidin B2 took place during oxidation also with yielding adducts with higher

240

molecular size (Figure 5, peaks 2a, 2b, 3a, 3b). These peaks are likely to be the interaction products

241

between procyanidin B2 and the oxidized forms of the peptide displaying higher molecular weights 11 ACS Paragon Plus Environment

46, 47

On the


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than the parent compounds. Figure 6 shows that peak 3 and peak 4 are the oxidation products of

243

peptide LDQWLCEK, which are m/z 1096 ([M-H]-) (peptide LDQWLCEK+4[O]) and m/z 1080

244

([M-H]-) (peptide LDQWLCEK+3[O]) respectively. The peptide and procyanidin B2 were also

245

each forming higher molecular products due to oxidation (Figure 5, peak 4). However, these

246

reactions did not lead to significant differences in the sample containing both peptides and

247

procyanidins.

248

Both SEC and LC-MS were used to detect protein-tannin interaction products. LC-ESI-MS proved

249

to be an efficient method for detection and characterization of protein-tannin interaction complexes.

250

16

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weights ranging from 10000 to 600000 Da was the first choice of method applied to the

252

characterization of the protein-tannin interaction adducts. Tryptophan, cysteine, and lysine were the

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most likely groups of ALA peptide LDQWLCEK to be oxidized, whereas oxidized tryptophan and

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lysine were especially prone to interact with procyanidin B2. Peptide LDQWLCEK showed the

255

property to prevent procyanidin B2 from oxidation and degradation during the oxidation process. In

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conclusion, the results show that protein-tannin interaction does take place during oxidation.

However, because of the detection limit of mass molecular size (2200 m/z), SEC with a molecular

257 258 259 260 261 262 263 264 265


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(1) Ozdal T.; Capanoglu E.; Altay F. A review on protein-phenolic interactions and associated

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Am. Chem. Soc. 2002, 124, 9899-9905.

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(3) Jöbstl, E.; O'Connell, J.; Fairclough, J.P.; Williamson, M.P. Molecular model for astringency

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(4) Poncet-Legrand, C.; Edelmann, A.; Putaux, J.; Cartalade, D.; Sarni-Manchado, P.; Vernhet, A.

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(9) Murray, N.; Williamson, M.; Lilley, T. and Haslam, E. Study of the interaction between salivary

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FIGURE CAPTIONS

398

399

Figure 1. Chemical structure of procyanidin B2.

400

401

Figure 2. NP-HPLC chromatogram monitored at 280 nm of procyanidin B2 and procyanidin

402

fraction (PF) isolated from aronia juice. The regions marked dimers, oligomers, and polymers is

403

according to Hellström et. al. 2007.

404

405

Figure 3. LC-MS profile of procyanidin B2 standard in negative mode showing m/z 577 as the [M-

406

H]-form and the fragment ions m/z 289, 407, 425, and 451. Elution of procyanidin B2 is also shown

407

as monitored by UV-absorbance and fluorescence.

408

409

Figure 4. Changes of the amounts of peptide LDQWLCEK (m/z 1034) and its oxidation products

410

in the samples with and without procyanidin B2 and procyanidin fraction (PF) isolated from aronia

411

juice in positive mode MS-detection. (a) Amounts of peptide LDQWLCEK (m/z 1034) from day 0

412

to day 7. (b) Amounts of oxidized product of peptide LDQWLCEK (m/z 1050) from day 0 to day 7.

413

(c) Amounts of oxidized product of peptide LDQWLCEK (m/z 1066) from day 0 to day 7. (d)

414

Amounts of oxidized product of peptide LDQWLCEK (m/z 1082) from day 0 to day 7. (e)

415

Amounts of oxidized product of peptide LDQWLCEK (m/z 1098) from day 0 to day 7. (f) Amounts

416

of oxidized product of peptide LDQWLCEK (m/z 1114) from day 0 to day 7. The samples of

417

peptide LDQWLCEK with and without procyanidin B2 (run in triplicates) are displayed with error

418

bars in percentage, which were less than 5%.

419 19 ACS Paragon Plus Environment


420

Figure 5. Size exclusion chromatography chromatogram of peptide LDQWLCEK with and without

421

procyanidin B2 on day 0 and day 7 of oxidation as monitored by UV-absorbance (210 nm) and by

422

lfuorescence. The circled areas with numbers correspond to the changes during oxidation, and are

423

explained in the text.

424

425

Figure 6. LC-MS and fluorescence chromatograms in negative mode: (a) Total ion chromatogram

426

(TIC) of procyanidin B2 on day 0, (b) TIC of procyanidin B2 on day 7, (c) TIC of TIC of peptide

427

LDQWLCEK on day 0, (d) TIC of peptide LDQWLCEK on day 7, (e) TIC of peptide

428

LDQWLCEK with procyanidin B2 sample on day 0, and (f) TIC of peptide LDQWLCEK with

429

procyanidin B2 sample on day 7.

430

Figure 7. Size exclusion chromatography chromatogram of peptide LDQWLCEK sample with and

431

without procyanidin fraction (PF) isolated from aronia juice on day 0 and day 7 as monitored by

432

UV-absorbance (210 nm) and fluorescence.


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Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Figure 6


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Figure 7



TOC graphic


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ProteinâTannin Interactions of Tryptic Digests of Î±-Lactalbumin and

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