Accepted Manuscript Structural changes and functional properties of highly concentrated whey protein isolate-citrus pectin blends after defined, high temperature treatments L. Koch, L. Hummel, H.P. Schuchmann, M.A. Emin PII:
S0023-6438(17)30430-9
DOI:
10.1016/j.lwt.2017.06.026
Reference:
YFSTL 6321
To appear in:
LWT - Food Science and Technology
Received Date: 18 January 2017 Revised Date:
11 June 2017
Accepted Date: 12 June 2017
Please cite this article as: Koch, L., Hummel, L., Schuchmann, H.P., Emin, M.A., Structural changes and functional properties of highly concentrated whey protein isolate-citrus pectin blends after defined, high temperature treatments, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.06.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Structural changes and functional properties of highly concentrated whey protein isolate-citrus
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pectin blends after defined, high temperature treatments
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L. Kocha, L. Hummela, H. P. Schuchmanna, M. A. Emina
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a
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Institute of Technology, Karlsruhe, Germany
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Email-address: L. Koch (
[email protected]), M. A. Emin (
[email protected]) H. P. Schuchmann
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(
[email protected])
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Corresponding author: M. A. Emin, Email-address:
[email protected], Tel: +49 (0)721 608-48311,
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Fax: +49 (0)721 608-45967
M AN U
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Institute of Process Engineering in Life Sciences, Section of Food Process Engineering, Karlsruhe
10 Abstract
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Extrusion processing can be used for conjugation of whey proteins with citrus pectins. However,
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many reactions can take place simultaneously, and so far less is known about the influence of the
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extrusion parameters on the structural and functional properties of the reaction products. This study
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focuses on the influence of elevated temperatures on the structural and functional properties of
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highly concentrated whey protein isolate-citrus pectin blends. Defined temperature treatments were
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performed by using a closed-cavity rheometer. Structural changes due to non-disulfide, covalent
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cross-links, and the formation of fluorescent compounds were analyzed. Functional properties (e.g.
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viscosity, emulsifying capacity) were determined of selected samples. The results showed that the
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emulsifying capacity can be improved by defined temperature treatments at 120 °C and 140 °C.
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Samples with an improved emulsifying capacity also exhibited higher maximum fluorescence
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intensity indicating the formation of Maillard reaction products (e.g. conjugates).
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Keywords: Whey protein; Citrus pectin; Maillard reaction; Heat treatment; Emulsion
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1. Introduction
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With an ever-expanding consumer awareness of health and nutrition, the use of natural food
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ingredients has gained increasing importance. Biopolymers such as whey proteins are often used as
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natural emulsifiers in bakery, meat, dairy and other food products (Morr & Ha, 1993). However, the
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industrial application is limited due to the sensitivity of whey proteins against changes in
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temperature, concentration of ions and pH (Damodaran, 2007). Therefore, the functionalization of
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whey proteins has been the focus of many researchers during the last years. It is well-known that
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mixing and heating of whey proteins with high molecular polysaccharides result in covalently linked
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molecules with improved functional properties e.g. emulsifying capacity and stabilizing properties
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(Akhtar & Dickinson, 2003; Einhorn-Stoll, Ulbrich, Sever, & Kunzek, 2005; Neirynck, Van der Meeren,
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P, Bayarri Gorbe, Dierckx, & Dewettinck, 2004; Schmidt et al., 2016). These molecules are referred to
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as protein-polysaccharide-conjugates and are formed during the initial stage of the Maillard reaction
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(Hodge, 1953; Maillard, 1912).
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The most commonly used methods for conjugation are incubation of lyophilized protein-
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polysaccharide powders (referred to as dry heating) and of protein-polysaccharide aqueous solutions
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(referred to as wet heating) under controlled conditions. Both processes can only be run batch wise
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and exhibit reaction times of several hours up to a few days. Further methods for conjugation with
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shorter reactions times (seconds to a few minutes) are pulsed electric field (Sun, Yu, Zeng, Yang, &
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Jia, 2011) and ultrasonic treatments (Li, Xue, Chen, Ding, & Wang, 2014), as well as extrusion
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processing (Guerrero, Beatty, Kerry, & Caba, 2012; Koch, Emin, & Schuchmann, 2017). Extrusion is a
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continuous process with reaction times of a few minutes. It is well-known that the Maillard reaction
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can take place during extrusion processing (Ames, Guy, & Kipping, 2001; Camire, 1991; Davidek,
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Illmann, Rytz, & Blank, 2013). However, up to now only a few studies focused on conjugation during
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extrusion processing and on the functional properties of the reaction products (Bueno, Pereira,
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Menegassi, Arêas, & Castro, 2009; Guerrero et al., 2012; Koch et al., 2017).
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Besides the Maillard reaction many other reactions can proceed during heat treatments of whey
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protein with citrus pectin. These reactions can involve denaturation (Areas, J. A. G., 1992; Mulvihill &
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Donovan, 1987), degradation (Burgess & Stanley, 1976), and polymerization of proteins (Areas, J. A.
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G., 1992; Burgess & Stanley, 1976), as well as degradation of pectin and thus caramelization (Axelos
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& Branger, 1993; Morris, Foster, & Harding, 2002; Schols & Voragen, 2002). Up to now, very little is
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known about the influence of extrusion parameters such as mechanical stress, reaction time and
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temperature on the reactions taking place in highly concentrated systems. This information;
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however, is essential to control the reactions. During extrusion, the material is exposed to broadly
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distributed thermal and mechanical stresses simultaneously. Moreover, these stresses are also
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coupled to each other which make extrusion trials not suitable to consider the effect of these
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stresses separately. Defined elevated temperatures and mechanical stresses can be applied by using
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a specific rheometer e.g. closed-cavity rheometer (CCR) (Emin & Schuchmann, 2016; Habeych, van
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der Goot, & Boom, 2009; Madeka & Kokini, 1994; Madeka & Kokini, 1996; Pommet, Morel, Redl, &
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Guilbert, 2004). This provides the opportunity to consider elevated temperatures and mechanical
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stresses in highly concentrated systems independently. Moreover, water evaporation can be
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avoided.
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This study aims at gaining a better understanding on the impact of elevated temperatures on
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structural changes (i.e. non-disulfide, covalent links) and functional properties (i.e. water solubility,
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viscosity and emulsifying activity) of highly concentrated whey protein isolate-citrus pectin blends.
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2. Material and methods
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2.1. Material
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Whey protein isolate (WPI), German Prot 9000, with 90 g/100g protein on dry matter was obtained
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from Sachsenmilch Leppersdorf GmbH (Leppersdorf, Germany). According to the supplier, the WPI
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contains typically ≤ 3 g/100g lactose, ≤ 1 g/100g fat and an ash content of 4 g/100g. The moisture
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content is stated to be ≤ 5 g/100g. Highly methylated citrus pectin (HMCP), Classic CU-L 009/13, was
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provided by Herbstreith & Fox (Neuenbuerg/Enz, Germany). According to the manufacturer, the
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HMCP exhibits a protein content of 3 g/100g, a moisture content of 7.5 g/100g and an average
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molecular weight of about 79.8 kDa determined by intrinsic viscometry. For the preparation of the
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emulsions, pure rapeseed oil (Bernhard Schell, Lichtenau, Germany) was used.
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2.2. Sample preparation and temperature treatments
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A blend of WPI and HMCP (1:1 g:g) was adjusted to a moisture content of 28 g/100g, and mixed in a
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Thermomix (Vorwerk, Wuppertal, Germany) for 3 min. To achieve a homogeneous water
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distribution, the samples were stored for 3 days at 8 °C. Subsequently, temperature treatments were
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performed by using a closed-cavity rheometer (RPA flex, TA Instruments, New Castle, Delaware,
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USA). Figure 1 shows a schematic illustration of the device. The biconical test chamber consists of
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two cones in opposite. The grooved cones, to prevent slippage, are thermoregulated by direct
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heating and forced air cooling. To prevent water evaporation the test cavity is sealed, and can be
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pressurized up to 8000 kPa. During the experiments the lower cone is driven by a motor and
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oscillates with a defined frequency and amplitude. At a constant strain and frequency of 1% and 1 Hz
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(linear viscoelastic region), respectively, samples were heated at 80 °C, 100 °C, 120 °C and 140 °C for
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0.75 min, 2 min, 6 min and 10 min. Two samples were prepared for each condition. Subsequently,
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samples were dried in a vacuum dryer (Heraeus, Hanau, Germany) at 40 °C and 8 kPa and milled
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(POLYMIX® PX-MFC 90 D, Kinematica, Luzern, Switzerland) to a particle size < 500 µm.
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2.3. Size-exclusion chromatography
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Size-exclusion chromatography (SEC) experiments were performed to analyze changes in the
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molecular size distribution of non-disulfide, covalent cross-links by HPLC (Shimadzu, Kyoto, Japan).
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Therefore, samples (1 mg/ml) were solved in a 0.2 mol/L phosphate buffer (pH 7) consisting of
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0.033 mol/L SDS, 0.54 mol/L NaCl, 8 mol/L Urea, and 0.002 mol/L dithiothreitol (DTT). Experiments
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showed a maximum in solubility when solving for 7 days. Afterwards, samples were centrifuged at
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4637 x g for 30 min ensuring that no insoluble fractions contaminate the analysis. For the same
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purpose, the mobile phase, a 0.2 mol/L phosphate buffer with 2 mol/L Urea (pH 7), was filtered
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through 0.2 µm cellulose acetate filters (Sartorius, Goettingen, Germany) and degassed. Finally,
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100 µl of the soluble sample fraction was injected by an auto sampler to the TSKgel G3000SWXL
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column (Tosoh Bioscience, King of Prussia, USA). The measurements were conducted with a flow rate
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of 0.5 ml/min at 25 °C and the absorbance of the samples was recorded by an UV/VIS detector at
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280 nm. The analyses were performed in triplicate for each sample.
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2.4. Fluorescence spectroscopy
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Fluorescence spectroscopy was performed to detect fluorescent compounds that are formed during
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the Maillard reaction (Leclère & Birlouez-Aragon, 2001; Matiacevich & Pilar Buera, 2006). An Infinite
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200 Pro microplate reader (Tecan, Crailsheim, Germany) was used to determine the fluorescence
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spectra of the samples. The sample solutions were prepared by solving 25 mg in 10 ml of
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0.2 mol/L phosphate buffer containing 0.033 mol/L SDS, 0.54 mol/L NaCl, 8 mol/L Urea and
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0.002 mol/L dithiothreitol (DTT) for 7 days. The excitation spectra from 300 to 400 nm were scanned
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at an emission wavelength of 420 nm. For the emission spectra, the maximum in excitation at
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368 nm was set constant, and emission spectra were recorded from 400 to 580 nm. Excitation and
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emission slits were set to 2 nm. The spectrum of each sample was recorded in triplicate. Maximum
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fluorescence intensity of the emission spectra at 488 ± 5 nm was used as a measure for the amount
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of fluorescent compounds formed during heat treatments.
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2.5. Determination of water solubility
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Water solubility was determined by a gravimetric analysis. Therefore, 0.2 g of each sample was
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solved in 19.8 g demineralized water, mixed with a vortex mixer for 1 min, and shaked for 24 h at
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room temperature. Thereafter, sample solutions were centrifuged at 4637 x g for 30 min, and the
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supernatant was carefully removed to separate it from the precipitate. The precipitate was dried in a
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vacuum dryer (Heraeus, Hanau, Germany) at 40 °C and 8 kPa for at least 2 days. The precipitate was
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weighed (mprecipitate), and the solubility (S) was calculated as follow:
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S (g/100g) = (0.2 g – mprecipitate)*100/ 0.2 g
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Analyses were carried out in triplicate for each sample.
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2.6. Determination of pH-values
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The pH-value was measured of sample solutions consisting of 2 g sample solved in 198 g
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demineralized water. To achieve 1 g/100g solutions, the solubility of each sample was considered.
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Measurements were conducted in triplicate.
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2.7. Preparation of the continuous phase and O/W emulsions
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Emulsions were prepared by dispersing 30 g/100g rapeseed oil in 70 g/100g continuous phase. The
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continuous phase consists of a 1 g/100g sample solution. The solutions with constant soluble sample
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content are prepared by weighing the samples according to their solubility and solving them in
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demineralized water by using a rotor-stator system (Ultraturrax T 25 digital, IKA-Werke GmbH & CO.
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KG, Staufen, Germany) at a rotational speed of 6.28 m/s for 2 min. The gap width was set to
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0.159 mm. Subsequently, solutions were adjusted to pH 5 at approx. 22 °C, and stored over night at
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8 °C. Thereafter, they were centrifuged at 4637 x g for 30 min. The supernatant was taken and the oil
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phase was dispersed to prepare the emulsion premix by using a rotor-stator system (Ultraturrax T 25
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digital, IKA-Werke GmbH & CO. KG, Staufen, Germany) at a rotational speed of 3.14 m/s for 3 min.
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Thereafter, the emulsion premix was emulsified by a colloid mill (magic LAB, IKA-Werke GmbH & CO.
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KG, Staufen, Germany) at a rotational speed of 21.60 m/s for 3 min. Emulsification experiments were
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performed in duplicate for each sample.
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2.8. Viscosity measurements
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The viscosity of the emulsion, and of the continuous phase were measured with a rheometer (MCR
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301 by Anton Paar, Graz, Austria) equipped with Couette geometry CC-27. The rotational
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measurements were performed at 25 °C by applying a shear rate of 1 s-1 to 120 s-1. The viscosity was
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measured at least three times.
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2.9. Measurement of droplet size distributions
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The droplet size distribution (DSD) was measured immediately after emulsification by using a laser
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diffraction particle size analyzer (LS 13 320, Beckman Coulter, Inc., Miami, FL, USA). The
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measurements were performed by polar intensity differential scattering (PIDS) technology.
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Therefore, the emulsions were diluted approx. 1:20 with demineralized water. The optical model
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used also cover the Mie region. For the water and oil phase, it was set a refractive index of 1.333 and
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1.47, respectively. As both phases are transparent, the imaginary parts were set to zero. Emulsion
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samples were always measured in triplicate and evaluation of scattering patterns followed the Mie
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model. The Sauter mean diameter (d3,2) was calculated from the cumulative volumetric droplet size
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distribution and used as characteristic value to describe the droplet size distribution.
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Emulsion stability was evaluated by changes in the DSD after 15 days, as no creaming could be
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observed for all samples. Finally, emulsion stability can be described by a stability index (SI) defined
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as:
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SI (-) = d3,2(day 15)/d3,2(day 0)
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where d3,2(day 15) is the Sauter mean diameter after 15 day and d3,2 (day 0) the Sauter mean diameter
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immediately after emulsification.
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3. Results and Discussion
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3.1. Structural changes
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The influence of temperature on structural changes resulting from non-disulfide, covalent cross-links
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is depicted in figure 2 for treatment periods of 0.75 min (a), 2 min (b), 6 min (c), and 10 min (d). The
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chromatograms show two main peaks for the untreated sample. The first peak (14.8 min to 20.5 min)
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corresponds to the native protein fractions and the second peak (24 min to 27 min) corresponds to
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the buffer peak which is caused by DTT. The results show that for all treatment periods the peaks of
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the native protein fractions decreased with temperature. This result can be explained by an
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accelerated reaction of the protein fractions with increasing temperature. A reduced absorbance of
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the native protein peak area is caused by the formation of insoluble reaction products that are too
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large to dissolve and/or soluble reaction products appearing in a new peak. These reaction products
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are stabilized by non-disulfide, covalent cross-links. The results further show that the native protein
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peak decreased with time which is related to an increase in insoluble reaction products. Moreover, a
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new peak at approximately 11 min appeared for the samples treated at 140 °C for all treatment
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periods. This peak is attributed to soluble, high molecular reaction products which are formed due to
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non-disulfide, covalent cross-links. The intensity, and therefore the concentration of these reaction
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products increased for up to 6 min (figure 2a), whereas a treatment period of 10 min (figure 2d)
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resulted in a lower concentration of reaction products. This might result from a further increase in
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the molecule size of the reaction products.
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Figure 2d further shows that small molecular compounds were formed for the sample treated at
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140°°C for 10 min which can be observed in a higher intensity at elution times above 20.5 min. This
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might result from degradation reactions of the native protein fractions and of the reaction products.
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Additionally, degradation of pectin and further reactions such as caramelization can also result in
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high absorbing molecules which can be detected at elution times above 20.5 min (Bornik & Kroh,
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2013; Scott, Moore, Effland, & Millett, 1967). For the samples treated at 100 °C and 120 °C for 10 min
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(figure 2d), a peak appeared at 11 min, while for a temperature treatment of 80 °C no additional
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peak can be observed for all treatment periods. Finally, the results show that changes in non-
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disulfide, covalent cross-links were accelerated with higher treatment temperatures.
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Fluorescence spectroscopy was carried out in order to determine the formation of fluorescent
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compounds, e.g., formed during the Maillard reaction (Baisier & Labuza, 1992; Matiacevich & Pilar
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Buera, 2006). The Maillard reaction can also lead to the formation of brown pigments; however,
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these can also be formed during heat treatments of pectin (e.g., caramelization products). The
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maximum fluorescence intensity (a) and a picture of the appearance (b) of the samples treated at
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different temperatures for various time periods are depicted in figure 3.
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The sample treated at 80 °C exhibited a significant increase in the maximum fluorescence intensity by
200
increasing treatment time from 6 to 10 min (figure 3a). No visible color development can be
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observed for the samples treated at 80 °C as shown in figure 3b. It has been reported that
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fluorescent compounds formed during the Maillard reaction can be detected prior to the formation
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of visible brown pigments (Baisier & Labuza, 1992). Therefore, the formation of Maillard reaction
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products can be the reason for the increase in maximum fluorescence intensity at 80 °C from 6 to
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10 min for the samples without any visible color change.
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The maximum fluorescence intensity of the samples treated at 100 °C increased with treatment time
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for up to 6 min. The sample treated for 10 min exhibited similar maximum fluorescence intensity as
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the sample treated for 6 min. However, the maximum fluorescence intensity was lower compared to
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that of the sample treated at 80 °C for 10 min. To explain lower intensity for a higher temperature;
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however, more information on the reaction products, and therefore on the structure of these
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fluorescent compounds is needed. Treatments at 100 °C for up to 10 min also resulted in no visible
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color development (figure 3b) indicating that the increase in maximum fluorescence intensity can be
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related to the formation of Maillard reaction products.
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When increasing the treatment temperature to 120 °C, first changes in color can be observed after
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2 min where the sample showed a darker yellow color (figure 3b). Treatment periods of 6 min and
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10 min resulted in a reddish brown color which became darker with time. The reddish brown color is
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characteristic for temperature treated D-galacturonic acids (Bornik & Kroh, 2013) which are the main
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components of the pectin backbone (Vries, Rombouts, Voragen, & Pilnik, 1984). Browning can also
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be the result of the Maillard reaction. The maximum fluorescence intensity for the sample treated at
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120 °C was always higher compared to that of a treatment temperature at 100 °C (figure 3a).
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Furthermore, the maximum fluorescence intensity increased from 0.75 min to 2 min (figure 3a). A
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treatment period of 6 min exhibited similar fluorescence intensity compared to 2 min, while after
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10 min maximum fluorescence intensity increased again. Temperature treatments at 140 °C showed
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the highest maximum fluorescence intensity and a similar progression as for 120 °C until 6 min
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(figure 3a). However, after 10 min the intensity decreased. In figure 3b can be seen that for the
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samples treated at 140 °C, the reddish brown color became darker with increasing treatment time.
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High maximum fluorescence intensity can be observed for samples showing no visible changes in
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color (e.g. 80 °C/10 min). This result indicates that fluorescent compounds appeared in highly
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concentrated WPI-HMCP systems prior to the formation of visible color changes (Baisier & Labuza,
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1992). This is expected to be the result of early stage Maillard reactions products showing
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fluorescent properties with no visible coloring effect (Matiacevich & Pilar Buera, 2006; Morales & van
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Boekel, 1997). Moreover, same maximum fluorescence intensity can be determined by samples
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showing significantly different colors (e.g. 80 °C/10 min vs. 140 °C/10 min). This indicates that the
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formation of fluorescent compounds is independent of color development.
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3.2. Functional properties
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Functional properties were investigated for the samples treated at 80 °C, 120 °C and 140 °C for
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0.75 min and 2 min, and are discussed in the following sections. These conditions were chosen
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corresponding to residence times determined in extrusion trials (Koch et al., 2017). The pH of the
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untreated sample is 4.4 ± 0.2 and did not change significantly due to temperature treatments.
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3.2.1. Water solubility
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The effect of temperature on the solubility of the samples treated for 0.75 min and 2 min and of the
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untreated sample is depicted in figure 4. The untreated sample exhibited a solubility of 96 ±
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1 g/100g. The solubility of the treated samples decreased as a function of treatment temperature
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and time. This can be the result of an accelerated protein aggregation resulting in insoluble
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molecules and the formation of insoluble reaction products. In contrast to the structural analyses in
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section 3.1., also non-covalent and disulfide interactions can lead to water insoluble molecules.
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3.2.2. Viscosity of the continuous phase
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Shear rheological behavior of the continuous phases are depicted in figure 5. Newtonian behavior
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(i.e. viscosity is independent of shear rate) can be observed for the continuous phase prepared with
250
the untreated sample, and with all samples treated for 0.75 min (figure 5a). The continuous phase
251
prepared with the samples treated for 2 min at 80 °C and 140 °C (figure 5b) also exhibited Newtonian
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behavior. Shear thinning behavior (i.e. viscosity decrease with increasing shear rate) can be
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monitored for the continuous phase of the sample treated at 120 °C for 2 min (figure 5b). Moreover,
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this sample exhibited the highest viscosity of the continuous phase. The viscosity of the continuous
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phase prepared with the treated samples was always higher compared to that prepared with the
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untreated sample. For the samples treated for 0.75 min, it can be observed that the viscosity
257
increased with treatment temperature (figure 5a). This can be due to an increase in the molecular
258
size, as the viscosity is influenced by that.
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The continuous phase prepared with the samples treated at 140 °C for 2 min resulted in lower
260
viscosity compared to that prepared by 120 °C and 80 °C, for the same treatment period (figure 5b).
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However, the viscosity was still slightly higher compared to that of the untreated sample. Moreover,
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it can be seen that the continuous phase prepared by the sample treated at 140 °C and 2 min was
263
lower compared to that prepared by the sample treated at 140 °C and 0.75 min. This can be the
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result of a higher concentration of small molecular compounds (e.g. due to degradation of pectin,
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protein fractions and reaction products). For samples treated at 80 °C, the viscosity of the continuous
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phase increased with treatment period of the samples. The viscosity of the continuous phase
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prepared by the samples treated at 120 °C did not change with treatment period.
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3.2.3. Emulsifying capacity
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The emulsifying capacity of the samples was evaluated at pH 5 which is close to the isoelectric point
270
of whey protein (pI ~ 4.8). Therefore, the net charge of the whey protein molecules is close to zero
271
leading to less electrostatic repulsions and thus precipitation of the molecules. This results in large
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droplets and poor emulsion stability. The emulsifying capacity of the samples treated at 80 °C, 120 °C
273
and 140 °C for 0.75 min and 2 min was assessed by the Sauter mean diameter depicted in figure 6.
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The dashed line and grey band show the Sauter mean diameter and the standard deviation,
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respectively, of the emulsions prepared with the untreated sample. Emulsions prepared with the
276
samples treated at 80 °C, exhibited Sauter mean diameters similar to that of the untreated sample
277
with 5.7 µm, regardless of the treatment period. Sauter mean diameters being smaller compared to
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that of the untreated sample can be observed for emulsions prepared with the samples treated at
279
120 °C and 140 °C. The emulsion with the smallest Sauter mean diameter was produced with the
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sample treated at 120 °C for 0.75 min. When increasing treatment period of the sample treated at
281
120 °C to 2 min, Sauter mean diameter increased by 0.4 µm. Treatment periods of the samples
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treated at 140 °C had no significant influence on the Sauter mean diameter of the emulsions. Their
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Sauter mean diameters were around 4.8 µm.
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The resulting droplet sizes are influenced by two mechanisms: droplet break up and droplet
285
stabilization. Both mechanisms are affected by the molecular properties e.g. interfacial properties
286
and the capacity to increase the viscosity (Arai, Konno, Matunaga, & Saito, 1977a; Armbruster, 1990).
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Disruptive forces, affecting droplet break up, are transmitted by the viscosity of the droplet
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surrounding phase (Arai et al., 1977a). Therefore, not only the interfacial properties of the treated
289
samples, but also their effect on the viscosity must be considered to explain the resulting droplet
290
sizes of the emulsions (figure 7).
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The viscosity of the emulsions prepared with the samples treated for 0.75 min at 120 °C and 140 °C
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was 2-fold higher compared to that of the untreated sample (figure 7a). During emulsification of the
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samples with a higher emulsion viscosity, higher disruptive forces can be transmitted onto the
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droplets which can be a reason for smaller Sauter mean diameters. The sample treated at 140 °C for
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2 min (figure 7b) exhibited a similar characteristic droplet size compared to the sample treated for
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0.75 min (figure 7a), although, the viscosity was significantly lower. This indicates that changes in the
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interfacial properties improved the emulsifying capacity of the sample with a lower viscosity
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(140 °C/2min).
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The samples treated at 80 °C for 0.75 min (figure 7a) and 2 min (figure 7b) showed similar viscosities
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for their emulsions being slightly higher compared to that of the untreated sample. Sauter mean
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diameters (figure 6) were in the same range as of the untreated sample indicating that molecular
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changes (e.g. increased viscosity) are not affecting the resulting Sauter mean diameter.
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The stability index, to evaluate the long term stability of emulsions, can be seen in figure 8a. The
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dashed line and grey band mark the stability index of the untreated sample. Stability indexes larger
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than 1 signify an increase in Sauter mean diameter measured after 15 days compared to the Sauter
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mean diameter measured immediately after emulsification. The results show a stability index close
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to 1 for all emulsions. Moreover, all emulsions showed no visible changes over a period of 15 days.
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As an example, figure 8b shows pictures of the emulsion prepared with the sample treated at 120 °C
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for 2 min immediately after emulsification and after 15 days (figure 8b). Consequently, all emulsions
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were physically stable over a period of 15 days.
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4. Conclusion
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It could be shown that elevated temperature treatments of highly concentrated whey protein-pectin
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blends can result in molecular changes exhibiting improved emulsifying capacity. This can be due to
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changes in the molecular properties causing a higher viscosity of the emulsions, enhancing droplet
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break up and droplet stabilization. Furthermore, the results indicate that an improved emulsifying
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capacity is due to changes in the interfacial properties. Structural analyses showed that the samples
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with an improved emulsion capacity exhibited higher concentration of fluorescent compounds which
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might be the result of Maillard reaction products (e.g. conjugates). These results give insight in
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reactions taking place in highly concentrated WPI-HMCP blends at elevated temperature and their
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kinetics. For a better process design of the extrusion trials, further work will focus on the influence of
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shear rates on the structural and functional properties of highly concentrated whey protein isolate-
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citrus pectin.
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This research project was supported by the German Ministry of Economics and Technology (via AiF)
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and the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn). Project AIF 18070 N.
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Figure 1: Schematic illustration of the closed-cavity rheometer. Figure 2: Structural changes resulting from non-disulfide, covalent cross-links. Size-exclusion chromatogram of the untreated ( ), and 140 °C (
) and treated samples at 80 °C (
), 100 °C (
), 120 °C (
) for 0.75 min (a), 2 min (b), 6 min (c), and 10 min (d).
Figure 3: Fluorescence spectroscopy and color changes. Maximum fluorescence intensity at
140 °C (
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488 ± 5 nm (a) and a picture (b) of the samples treated at 80 °C ( ), 100 °C ( ) for various time periods.
Figure 4: Water solubility. Effect of treatment temperature of the untreated (
), and
), and treated
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Figure 5: Viscosity of the continuous phase. Viscosity is depicted as a function of shear rate for the untreated ( ), and treated samples at 80 °C ( ), 120 °C ( symbols) and 2 min (b; half-open symbols).
), and 140 °C ( ) for 0.75 min (a; closed
Figure 6: Effect of treatment temperature on Sauter mean diameter. Sauter mean diameter is depicted for samples treated for 0.75 min ( ), and 2 min ( ) and for the untreated sample (
).
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), and 140 °C ( ) for
0.75 min (a; closed symbols) and 2 min (b; half-open symbols).
untreated sample (
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Figure 8: Long-term stability of the emulsions. Stability index of the emulsion prepared with the ), and with the samples treated at 80 °C, 120 °C and 140 °C for 0.75 min (
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Structural analyses indicate the formation of Maillard reaction products
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Improved emulsifying capacity after temperature treatments at 120 °C and 140 °C
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Emulsifying capacity apparently depends on fluorescence intensity
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Physically stable emulsions over a period of 15 days
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