Formation of whey protein isolate (WPI)-maltodextrin conjugates in

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Formation of whey protein isolate (WPI)-maltodextrin conjugates in fibers produced by needleless electrospinning Ines Kutzli, Monika Gibis, Stefan K. Baier, and Jochen Weiss J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02104 • Publication Date (Web): 12 Sep 2018 Downloaded from http://pubs.acs.org on September 13, 2018

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Journal of Agricultural and Food Chemistry

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Formation of whey protein isolate (WPI)-maltodextrin conjugates in fibers produced by

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needleless electrospinning

3 Ines Kutzlia, Monika Gibisa, Stefan K. Baierb, Jochen Weissa,*

4 5 a

6 7

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Department of Food Physics and Meat Science, Institute of Food Science and

Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany b

PepsiCo Global Functions Governance and Compliance, Measurement Science, 3 Skyline

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Drive, Hawthorne, NY 10532

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Submitted to Journal of Agricultural and Food Chemistry March 2018

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*Corresponding author

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E-mail address: [email protected]

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Tel. +49 711 459-24415; Fax: +49 711 459-24446

1 ACS Paragon Plus Environment


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Abstract

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Glycation of proteins via the first stage of the Maillard reaction is capable of improving their

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stability but not economically feasible yet. This work reports the glycation of whey protein

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isolate (WPI) with maltodextrin at a high yield after heating electrospun fibers made from the

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reactants. Glycoconjugates were characterized by Fourier transform infrared spectroscopy

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(FTIR) and SDS-PAGE. The binding ratio between WPI and maltodextrin was assessed via

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the free amino groups. The molecular weight of the conjugates and the reaction yield were

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studied by size exclusion chromatography. The impact of different mass ratios between WPI

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and maltodextrin in the fibers (5:95; 10:90; 20:80 and 25:75 w/w) was investigated. With

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increasing WPI content, the binding ratio of maltodextrin decreased from ~2.1 to ~1.2.

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Preferably small polysaccharides (2-13 kDa) from the maltodextrin reacted. Protein specific

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reaction yields of up to 44.52 ± 7.46% w/w were demonstrated in all WPI-maltodextrin fibers

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after heating.

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Keywords: glycoconjugate, maltodextrin, whey protein isolate, Maillard reaction, needleless

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electrospinning

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1. Introduction

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Whey protein isolate (WPI) is widely used in food industry due to its nutritional value,

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high technofunctionality, and abundance, being a by-product of cheese manufacturing.1

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However, thermally induced denaturation above ~62 °C and aggregation close to the

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isoelectric point (pI = 4.8-5.2)2 of the main proteins α-lactalbumin (α-la) and β-lactoglobulin

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(β-lg) are limiting the use of WPI in applications such as functional protein beverages.3

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The functional properties of proteins can be protected and improved upon the covalent

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bonding with polysaccharides through the first stage of the Maillard reaction.4-6 Carbonyl

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functions of the polysaccharide react with free ε-amino groups of the lysine residues, which

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are the primary glycation site of the proteins to form glycoconjugates.7 No additional

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chemicals are required for this reaction.8 Glycoconjugates produced from WPI and

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maltodextrin were reported in numerous studies to show improved emulsifying ability,4,

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higher foam stability,10 increased solubility,11-12 increased heat stability,8, 13-14 and stability

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over a larger pH range12, 14 compared to native WPI.

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However, the commercial use of these glycoconjugates is not possible yet since no

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economically feasible method of manufacturing is available.15 Heating the protein-

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polysaccharide mixture in dry or wet state either requires expensive freeze-drying, lacks

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control of unwanted Maillard reaction products or delivers only low reaction yields.16-17

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Electrospinning of dispersions made from WPI and polysaccharide prior to the Maillard

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reaction is supposed to enable glycation in shorter time with a higher yield and at lower cost

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than previously used dry and wet state methods, thus making glycosylated proteins

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commercially available.18 The determining factor is the close molecular contact of the two

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polymers inside the electrospun fibers.18 This effect is achieved by a molecular alignment as a

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result of the stretching and bending motions during electrospinning, by the prevention of



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micro-phase separation due to the rapid evaporation of the solvent, and by the close packaging

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of the molecules inside the very fine fibers.18

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In needleless electrospinning, multiple fibers are produced simultaneously from the

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surface of a polymer solution which is loaded onto a rotating metal roller that is connected to

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a high voltage source. As in needle electrospinning, jets are ejected if the electrical forces

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overcome the surface tension of the polymer solution.19 Fibers are collected on a grounded

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collector located above the spinneret. Compared to needle electrospinning, much higher

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production rates can be achieved with needleless electrospinning.20 Furthermore, the problem

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of clogged needles has been eliminated.21

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The purpose of the high molecular weight polysaccharides in this method is not only to

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provide the reducing group for the Maillard reaction. The entanglement of the high molecular

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weight polysaccharide chains is also a key factor for the formation of stable jets in the

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electrospinning process.22-23 Previously, conjugated whey proteins were successfully

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produced from electrospun fibers made from WPI and dextran with a molecular weight of

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70 kDa.24 In our previous study, we were able to determine concentrations and molecular

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weight distributions that enabled the successful production of fibers from WPI and

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maltodextrin in a needleless setup.25 Compared to dextran, the starch degradation product

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maltodextrin is very cost-efficient and of food-grade quality since dextran is mainly used for

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cosmetic or pharmaceutical applications.

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The objective of this study was to evaluate the feasibility of producing glycoconjugates

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from electrospun WPI-maltodextrin fibers and to evaluate how the mass ratio of WPI to

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maltodextrin influences the conjugation reaction. WPI-maltodextrin dispersions were

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electrospun at different mass ratios and the fibers were subsequently heated. The fibers were

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tested for glycoconjugate formation. Glycoconjugates were characterized with regard to their

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molecular weight and the binding ratio of maltodextrin and WPI. It was hypothesized that the


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mixing ratio would affect the reaction yield of the glycation as well as the conjugate structure

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since a molar excess of carbonyl groups had already been reported to promote the Maillard

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reaction.10, 13

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2. Materials and Methods

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2.1 Materials

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WPI with a protein content of 92.00 ± 0.06% w/w (98.67 ± 0.17% w/w dry weight basis)

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was obtained from Fonterra (product name: WPI 895, Fonterra Co-Operative Group Ltd.,

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Auckland, New Zealand). The protein content was determined in triplicate using the Dumas

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flash combustion method (Dumatherm® DT N Pro, C. Gerhardt GmbH & Co. KG,

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Königswinter, Germany) with a protein conversion factor of 6.25. Maltodextrin (MD)

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(Eliane™ MD 2, batch 709805), produced by enzymatic hydrolysis of waxy potato starch,

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was kindly provided by Avebe (Avebe U.A., GK Veendam, Netherlands). As previously

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determined, the maltodextrin had a dextrose equivalent (DE-value) of 2.10 ± 0.08 and a

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weight average molecular weight of Mw = 129.6 kDa and a number average molecular weight

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of Mn = 18.0 kDa (polydispersity index PDI = 7.2).

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TRIS (buffer grade) was obtained from AppliChem (AppliChem GmbH, Darmstadt,

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Germany). Ethanol (≥99.8%), ortho-phthaldialdehyde (OPA) (≥99%), sodium dodecyl sulfate

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(SDS) (≥99%), TRIS-HCl (≥99%) and methanol (HPLC ultra gradient grade) were purchased

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from Carl Roth (Carl Roth GmbH & Co. KG, Karlsruhe, Germany). Acetic acid,

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bromophenol blue, and sodium tetraborate (>98%) were obtained from Merck (Merck KGaA,

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Darmstadt, Germany). Glycine (≥99%), β-mercaptoethanol (≥98%) and NaCl (≥99.5%) were

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purchased from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

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Glycerol (bidistilled, 99.5%) was obtained from VWR (VWR International S.A.S., Fontenay-

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sous-Bois, France). 5 ACS Paragon Plus Environment


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2.2 Preparation of WPI-maltodextrin fibers

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Based on previous work,25 dispersions of WPI and maltodextrin (MD) were prepared at

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four different ratios: 5:95, 10:90, 20:80 and 25:75 w/w (WPI5MD95, WPI10MD90, WPI20MD80,

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WPI25MD75) with a total solids concentration of csolids = 1 g/mL in deionized water. They were

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stirred on a magnetic stirrer overnight at ambient temperature to ensure complete hydration.

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Dispersions were spun in a needleless upward roller electrospinning apparatus. The dispersion

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was charged (60 kV) with a high voltage power supply (SL 60, Spellman, Hauppauge, NY,

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USA) and distributed onto a stainless steel cylinder spinneret by rotation (80 rpm). Fibers

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were collected on a grounded, rotating (250 rpm) stainless steel cylinder. The distance

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between spinneret and collector was 19.5 cm. Four independently prepared samples were used

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for each WPI-maltodextrin ratio.

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2.3 Scanning electron microscopy (SEM)

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The morphology of the fibers was investigated using a scanning electron microscope

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(DSM 940, Carl Zeiss Microscopy, Jena, Germany) at an acceleration voltage of 5 kV at a

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high vacuum. The required fibers were collected under spinning conditions for approximately

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30 seconds on a conductive tab that was attached to the collector of the electrospinning

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machine and subsequently sputtered with a gold/palladium mixture prior to microscopy. The

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average fiber diameters were obtained by image analysis (Image J by Wayne Rasband,

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National Institutes of Health, Bethesda, MD, USA) using the measurement of 100 fibers for

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each sample.

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2.4 Preparation of WPI-maltodextrin conjugates

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A desiccator containing saturated NaCl solution was pre-equilibrated to 60 °C and

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74.02 ±0.61% relative humidity (RH). The electrospun fibers were then placed inside the

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desiccator on a perforated plate above the saturated salt solution and heated in an Innova® 6 ACS Paragon Plus Environment

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incubator shaker (Eppendorf AG, Hamburg, Germany) for 48 h. Temperature and RH were

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recorded during the conjugation with a data logger (ALMEMO 2590-4AS, Ahlborn GmbH,

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Holzkirchen, Germany). Four independently prepared samples were used per WPI-

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maltodextrin ratio.

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2.5 Visual appearance of heated fibers

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To visually evaluate the degree of browning, pictures of the heated fibers were taken with

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a Canon PowerShot G10 with a built-in flash (Canon Inc., Tokyo, Japan).

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2.6 Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy

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FTIR spectra of unheated and heated WPI-maltodextrin fibers were recorded using a

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Spectrum 100 (Perkin Elmer, Beaconsfield, UK) equipped with a universal attenuated total

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reflectance accessory. Spectra were collected in transmission mode within the wavenumber

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range of 4000-650 cm-1 (resolution 4 cm-1) and averaged over 64 scans. Conversion to

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absorbance, baseline correction and normalization of the spectra were executed with the

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software Spectrum Std v6.3 (Perkin Elmer, Beaconsfield, UK). The amide I and II region

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(1500 to 1800 cm-1) of the spectra were analyzed.

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2.7 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

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SDS-PAGE was performed on a Mini-PROTEAN® Tetra Cell (Bio-Rad Laboratories,

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Hercules CA, USA) under reducing conditions according to Laemmli.26 Unheated and heated

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fibers were diluted in purified water in order to obtain a protein concentration of 1 mg/mL for

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protein staining and 2 mg/mL for glycoprotein staining. An amount of 10 µL per sample was

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loaded onto 4-20% Mini-PROTEAN® TGX™ precast gels (Bio-Rad Laboratories, Hercules

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CA, USA). Electrophoresis was run for 35 min at 200 V in 0.025 M Tris-HCl buffer solution

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(pH 8.3, including 0.192 M glycine and 0.1 %w/w SDS) at room temperature. After

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electrophoresis, the gels were stained for proteins and glycoproteins, respectively. Coomassie 7 ACS Paragon Plus Environment


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Brilliant Blue R-250 Staining Solution (Bio-Rad Laboratories) was used for protein staining.

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Destaining was done with 10% v/v acetic acid containing 15% v/v methanol. For glycoprotein

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staining, the Pierce® Glycoprotein Staining Kit (Thermo Fisher Scientific Inc., Rockford IL,

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USA) was used according to the protocol of the manufacturer.

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2.8 Degree of glycation and binding ratio by chemically available lysine

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Chemically available lysine in unheated and heated fibers was analyzed by the ortho-

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phthaldialdehyde (OPA) method according to the slightly modified microtiter plate assay of

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Barba, et al.27 The OPA reagent was prepared fresh daily by mixing 80 mg OPA (dissolved in

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2 mL ethanol), 50 mL 0.1 M sodium tetraborate buffer (pH 9.7-10), 5 mL 20% w/v SDS

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solution and 0.2 mL β-mercaptoethanol. 25 µL of sample (approx. protein concentration of

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10 mg/mL in purified water) were mixed with 475 µL purified water and 500 µL 12% w/v

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SDS solution and stored overnight at 4 °C. 8 µL sample/blank, 8 µL 0.1 M sodium tetraborate

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buffer (pH 9), and 250 µL OPA reagent were pipetted into 96-well black plates (Brand GmbH

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& Co. KG, Wertheim, Germany). After a delay of 2 min, plates were read in a Synergy HT

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microtiter plate reader (BioTek Instruments GmbH, Bad Friedrichshall, Germany) at

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λex = 340 nm and λem = 455 nm. A six-point WPI calibration curve (c = 0.1-24 mg/mL) was

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used for quantification. Two independently prepared samples were pipetted into the plate four

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times per WPI-maltodextrin ratio.

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The average number of maltodextrin molecules attached per mole of WPI was calculated

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according to Wooster and Augustin.28 An average value of 15.25 available lysine groups per

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mole WPI was used based on the molecular ratio of the main whey proteins α-la and β-lg

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(1:2.7) and the number of their lysine groups (one terminal NH2 and 12-15 lysine residues).29-

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in one binding site per molecule maltodextrin.32-33

It was assumed that the polysaccharide component has one reducing sugar group, resulting

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2.9 High performance size exclusion chromatography (HP-SEC)

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The molecular weight distribution of the WPI-maltodextrin conjugates was analyzed by SEC

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performed on the liquid chromatography system Agilent HP Series 1100 (Agilent

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Technologies GmbH & Co. KG, Waldbronn, Germany) with the software ChemStation for

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LC, Rev. B.04.03 [16]. The silica packed columns TSK-Gel 4000SWXL and TSK-Gel

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2000SWXL (TOSOH Bioscience, Tokyo, Japan) were protected by a guard column SWXL

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(TOSOH Bioscience, Tokyo, Japan) and tempered to 25 °C. The flow rate was 0.6 mL/min

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and the injection volume of the samples was 20 µL. Samples were prepared by dissolving

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unheated and heated WPI-maltodextrin fibers in the mobile phase (5 mM acetic acid

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containing 0.25 M NaCl) and filtering the solutions with 0.45 µm regenerated cellulose

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syringe adapter filters (Macherey-Nagel GmbH & Co. KG, Düren, Germany). Samples were

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detected with an Agilent 1260 Infinity diode array detector (DAD) at 280 nm followed by an

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Agilent 1100 refractive index detector (RID) (Agilent Technologies GmbH & Co. KG,

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Waldbronn, Germany). The WPI content of the samples was determined according to a six-

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point standard calibration curve of WPI (c = 0.2-2.0 mg/mL). The protein specific yield of the

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glycation reaction was calculated from the WPI content of the unheated fibers (cWPI UF) and

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heated fibers (cWPI HF) with Eq. 1.

Yield = ( − )⁄ ∙ 100

(1)

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Six analytical dextran standards with molecular weights of 12, 25, 50, 80, 150 and

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270 kDa (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) were used to analyze the

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molecular weight distribution of the samples. Four independently prepared samples were used

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per WPI-maltodextrin ratio.

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2.10

Statistical analysis

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At least two independent replicates were used in all experiments. Means, standard deviations,

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and the significance of determination were calculated from the measurements using Excel

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(Microsoft, 98052 Redmond, WA, USA) and SigmaPlot 12.5 (Systat Software Inc., 95110

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San Jose, CA, USA). A statistical analysis of the results of the protein specific reaction yield

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and the reacted NH2 groups after heating was performed using Tukey's test in IBM SPSS

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Statistics 24 (IBM, Armonk, NY, USA). Significant differences (p < 0.05) were labeled with

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different letters.

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3. Results and Discussion

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3.1 Characterization of electrospun fibers

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Protein content

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In order to evaluate if the protein content in the spinning dispersions corresponded to the

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protein content in the spun fibers, the concentration of protein in the fibers was quantified by

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using HP-SEC. The protein content of the fibers was lower than the used WPI concentration

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in the spinning dispersions (Table 1). The ratio between the fiber protein content and the

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percentage of WPI ranged from 1:0.87 to 1:0.93. These differences were expected since the

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protein content of the WPI was 92.00 ± 0.06% w/w (98.67 ± 0.17% w/w dry weight basis).

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Consequently, it could be shown that the major part of the protein in the spinning dispersions

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was incorporated in the electrospun fibers.

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Fiber morphology

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Distribution of fiber diameters calculated from SEM images using image analysis software

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indicated that the average fiber diameters were affected by the ratio of WPI and maltodextrin

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in the spinning dispersions (Table 1). The statistically significantly lowest diameter of

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1.21 ± 0.29 µm was observed for WPI5MD95 fibers. With 1.56 ± 0.44 µm, WPI10MD90 fibers


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had the statistically significantly highest diameter. No clear trend could be observed between

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the fiber diameter and the composition of the spinning dispersions. A reason for this might be

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the usage of a needleless electrospinning setup in this study. Needleless electrospinning has

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been shown to produce broader fiber diameter distributions compared to needle

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electrospinning.34 Possible reasons are the different geometries of the electric field and the

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fact that jets can eject from droplets of various sizes from an open surface, which leads to

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various fiber diameters.34 It could be seen from the SEM images (Figure 1) that the fiber

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structure was mostly even and only interrupted by a few, unsystematic bead defects and

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agglomerated material. The smooth fiber surfaces allowed the conclusion that the whey

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proteins were homogeneously incorporated into the maltodextrin fibers. According to Baier,

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et al.,18 the stretching and bending motions of the jet during spinning result in a uniform

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distribution of the proteins and the polysaccharides in the fibers. The small diameter of the

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fibers assures a dense packing of the molecules. This state has been described as the

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prerequisite for a fast glycation reaction with a high yield.18

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Visual appearance of heated fibers

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While the products of the initial condensation stage of the Maillard reaction are colorless, the

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second stage produces yellow compounds35 and the third stage delivers the browning products

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melanoidins.7 Due to functionality and nutritional properties, the reaction conditions for the

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glycation need to be chosen in order to minimize the formation of advanced Maillard reaction

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products.36 After heating the WPI-maltodextrin fibers at 60 °C and 75% RH for 48 h, no color

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formation could be visually observed (Figure 2). The fibers remained white. Consequently,

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the chosen reaction conditions were considered mild enough to prevent the second and third

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stages of the Maillard reaction. Similar reaction conditions have previously been reported for

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the successful glycation of whey proteins with maltodextrin in dry state reaction by Wooster

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and Augustin13 and Liu and Zhong.8



257 258

3.2 Characterization of conjugates FTIR

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FTIR spectra of the unheated and heated WPI-maltodextrin fibers were used to detect changes

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in their chemical composition. The technique has been previously shown to be able to detect

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Maillard reaction products.37 Figure 3 shows the FTIR spectra of the unheated and heated

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WPI-maltodextrin fibers. Proteins were represented by the peaks around 1640 and 1540 cm-1,

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which belong to the amide I and amide II regions, respectively.38 The amide I band is

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composed of the stretching vibrations of the C=O and C-N groups.39 The band of the amide II

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region originates from in-plane N-H bending and the stretching vibrations of the C-N group

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and C-C.39 After heating the fibers, the amide I absorption band of WPI shifted toward lower

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wavenumbers in all samples, namely, from 1644 cm-1 to 1640 cm-1 for WPI5MD95, from

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1643 cm-1 to 1635 cm-1 for WPI10MD90, from 1643 cm-1 to 1635 cm-1 for WPI80MD20, and

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from 1640 cm-1 to 1635 cm-1 for WPI25MD75. This shift was previously attributed to glycation

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via the first stage of the Maillard reaction during which amino groups are consumed while

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Schiff bases are formed. The reported wavenumber shifts were 1640 cm-1 to 1634 cm-1,8

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1654 cm-1 to 1637 cm-1,37 and 1644 cm-1 to 1630 cm-1.36 It had also been reported that a shift

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of the amide II region from 1596 cm-1 to 1581 cm-1 would occur after glycation.8 However,

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the shift in the amide II region only became visible for WPI25MD75 fibers. Due to the lower

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protein content of the other samples, the peak of the amide II region was less distinct and it

276

was not possible to observe a shift in the wavenumber. Since neither a clear increase nor

277

decrease of absorbance in all the normalized spectra could be observed, it was assumed that

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WPI is not distributed completely homogeneously in the fibers, and therefore no statement

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about the intensity of the peaks could be made.

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SDS-PAGE profiles of conjugates

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SDS-PAGE was carried out to verify the covalent coupling of whey proteins to maltodextrin

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and to examine the molecular weight of the conjugates. Protein components were stained with

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Coomassie blue and polysaccharide components with a PAS stain (Figure 4). When stained

285

for proteins, the main whey proteins α-la (14 kDa) and β-lg (18 kDa)2 were evidently shown

286

in the unheated fibers. In the heated fiber samples, the α-la and β-lg bands were faded.

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Furthermore, a broad band at a molecular weight range higher than that of β-lg appeared

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(~18-23 kDa). This band was attributed to glycated whey proteins. The polydispersity of the

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glycoconjugate band was a possible result of the broad molecular weight distribution of the

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maltodextrin.40 Furthermore, the weight of the glycated proteins was low compared to the

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average molecular weight of the maltodextrin (Mw = 129.6 kDa, Mn = 18.0 kDa). However,

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the maltodextrin’s molecular weight distribution ranged from 2 kDa to almost 900 kDa (data

293

not shown). It was concluded that preferably very small polysaccharides of the maltodextrin

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had reacted with the whey proteins due to less steric hindrance. The decrease of α-la and β-lg

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and the presence of high molecular weight protein species after heating became more evident

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for the fibers with a ratio of 5:95 w/w WPI:maltodextrin (WPI5MD95) and generally became

297

less intense with a higher WPI content. The results indicate that a lower protein-to-

298

polysaccharide (i.e., a lower NH2-to-carbonyl group) ratio is favorable for glycation. This

299

finding was consistent with findings by Wooster and Augustin13 and Martinez-Alvarenga, et

300

al.,10 who reported that an increase in carbonyl groups in the molar ratio of reactants promotes

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the formation of glycoproteins. However, the bands of α-la and β-lg did not completely

302

disappear for any sample, which indicated that the glycation reaction did not completely

303

occur.

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When stained for glycoproteins, WPI remained unstained, while maltodextrin showed positive

305

staining in the molecular weight range above 123 kDa. This is consistent with the fact that the



306

PAS staining method can also be used to stain carbohydrates.41 Unheated samples were

307

stained only in the molecular weight range in which the maltodextrin was stained. Heated

308

samples showed a positive smear stain above ~19 kDa, indicating the formation of

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glycoproteins. The polydispersity of the glycoproteins was again attributed to the broad

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molecular weight distribution of the maltodextrin. The effect of a promoted glycation upon a

311

lower WPI-maltodextrin ratio became visible again. The glycoprotein band appeared less

312

intense for the heated fibers with higher protein content.

313

Molecular weight distribution and glycation yield by SEC

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The molecular weight of the glycoconjugates was investigated in detail by size exclusion

315

chromatography (Figure 5). Chromatograms of unheated fibers showed the characteristic

316

peaks for the main whey proteins α-la (14 kDa at 23.6 mL) and β-lg (18 kDa at 22.5 mL).

317

After heating, the intensity of the whey protein peaks decreased in all samples. Conjugates

318

were detected at a lower elution volume (i.e., a higher hydrodynamic volume) than the WPI.

319

The peak of the glycoconjugates had shoulders at the elution volumes of α-la and β-lg,

320

respectively, indicating an incomplete glycation.

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At the peak maximum (~22 mL), the molecular weight of the conjugates was determined

322

with ~19.5 kDa. Conjugates were detected up to a molecular weight of ~30 kDa. These

323

findings agreed with the results of SDS-PAGE where the main band of the conjugates in the

324

heated fibers became visible in protein staining around 19-23 kDa. The broad peaks of SEC

325

and the smeared bands in SDS-PAGE gel suggested that proteins had reacted with a broad

326

range of maltodextrins of different molecular weights but preferably formed conjugates with

327

lower molecular weight maltodextrin components with weights of 2 to 13 kDa.

328

Reaction yields of the glycation reaction were calculated from integrating the SEC peaks

329

of α-la and β-lg before and after glycation. The results of the protein specific reaction yield

330

are shown in Table 2. The amount of WPI which was linked to at least one maltodextrin


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molecule lay between 31.22 ± 3.89% w/w for WPI25MD75 fibers and 44.52 ± 7.46% w/w for

332

WPI10MD90 fibers. No clear trend could be observed for a relation between the protein-

333

polysaccharide-ratio and the protein specific reaction yield. According to the hypothesis, a

334

higher ratio of reducing carbonyl groups to NH2 groups would promote the glycation

335

reaction10, 13 and lead to a higher protein specific yield. However, the steric hindrance of the

336

larger maltodextrin molecules at a higher maltodextrin concentration might have counteracted

337

this effect and slowed down the glycation reaction.42-43 Extent of glycation and binding ratio

338 339

The extent of conjugation of maltodextrin and WPI via the first stage of the Maillard

340

reaction was also reflected by a decrease in available amino groups as determined by the OPA

341

fluorometric assay (Table 3). With lower maltodextrin content in the fibers (i.e., higher

342

protein content), more NH2 groups were thus available after heating. The extent of glycation

343

was therefore decreased. The highest extent of the conjugation reaction could be achieved

344

with the lowest protein concentration tested (WPI5MD95, i.e., 1:0.16 NH2:carbonyl groups)

345

when 86.44 ± 1.53% of the amino groups were still chemically available after heating. A

346

binding ratio of 2.1 ± 0.2 maltodextrin molecules per molecule WPI was calculated for this

347

sample.

348

The lowest extent of conjugation was observed in samples with the highest protein

349

concentration

and

the

lowest

maltodextrin

concentration

(WPI20MD80,

350

1:0.03 NH2:carbonyl groups). 92.27 ± 0.99% of the amino groups were still chemically

351

available after heating. For this sample, the lowest binding ratio of 1.2 ± 0.2 attached

352

molecules of maltodextrin to one molecule of WPI was calculated.

i.e.,

353

According to the molar ratio of NH2 to reducing carbonyl groups, maltodextrin was the

354

limiting component of the Maillard reaction in all samples tested. The findings were

355

consistent with previous research where an excess of reducing carbonyl groups in a whey



356

protein-maltodextrin mixture promoted the Maillard reaction and led to increased glycation.10,

357

13

358

does not differentiate between unreacted and reacted molecules.28

359

Wooster and Augustin13 calculated binding ratios of maltodextrin (DE 4, Mn = 1.9 kDa) per

360

mole β-lg for the molar ratios 1:0:17 and 1:0.09 NH2:carbonyl groups with 1.6 and 1.1

361

molecules. Although their maltodextrin had a lower molecular weight, their findings

362

confirmed the trend of a lower binding ratio with higher protein content as demonstrated in

363

Table 3.

However, the extent of the reaction can only be seen as an average since the OPA assay

364

Based on the calculated binding ratio of 1.2-2.1 (Table 3), an average molecular weight of

365

17 kDa for WPI and the findings of SEC, where conjugates were detected up to a molecular

366

weight of ~30 kDa (Figure 5), it was theorized that the molecular weight of the bound

367

maltodextrin varied between 2 and 13 kDa. It was previously reported that low molecular

368

weight reducing saccharides have a higher glycation rate than high molecular weight

369

polysaccharides.44-45 The reason is the steric hindrance of high molecular weight molecules.44

370

It was previously shown that the stabilization of an emulsion by glycoconjugates is a

371

function of both the molecular weight and the number of polysaccharide molecules attached

372

to the protein.13 For a maltodextrin with a molecular weight of ~900 Da, 6 molecules needed

373

to be attached to one β-lg in order to enhance emulsion stability against salt and heat induced

374

flocculation.13 In the case of a maltodextrin with ~1900 Da, only 1.6 attached maltodextrin

375

molecules were sufficient to achieve the same stability.13 According to these results, the WPI-

376

maltodextrin conjugates prepared from electrospun fibers seem to be promising emulsifying

377

agents.

378

In our study, in which the glycoconjugates were produced from electrospun protein-

379

polysaccharide fibers, a high reaction yield without browning of the fibers was achieved.

380

Thus, glycation was facilitated by the fibrous structure. In their patent for the formation of


Page 16 of 39

Page 17 of 39


381

conjugated protein from electrospun fibers, Baier, et al.18 described the high molecule-to-

382

molecule contact in the fibers as the key factor for a high reaction yield. Further reasons are

383

the uniform distribution of molecules due to the stretching and bending motions of the jet

384

during spinning and the prevention of phase separation by the rapid evaporation of the

385

solvent.46 The high surface area allows the reaction to take place under mild reaction

386

conditions.

387

In conclusion, it was shown that electrospun fibers made from WPI and maltodextrins at

388

different mass ratios can serve as a promising starting material for the production of glycated

389

whey proteins. The determining factor for the successful formation of glycated whey proteins

390

incorporated in the fibers is the close molecular contact of the two reactants. The high surface

391

area and great porosity of the electospun fibers are caused by the three-dimensional web

392

structure and lead to a fast dissolution behavior of the fibers47 so that the glycated proteins can

393

be used in form of the fibers right away. Glycoconjugate formation at a high protein specific

394

reaction yield of up to 44.52 ± 7.46% w/w was demonstrated in all WPI-maltodextrin fibers

395

after heating. Previous studies about glycoconjugate formation under wet heating conditions

396

reported only low reaction yields of

Formation of whey protein isolate (WPI)-maltodextrin conjugates in

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