Influence of shear stress, pectin type and calcium chloride on the process stability of thermally stabilised whey protein–pectin complexes

Accepted Manuscript Title: Influence of shear stress, pectin type and calcium chloride on the process stability of thermally stabilised whey protein-pectin complexes Author: Kristin Protte Thomas Ruf Zeynep Atamer Alina Sonne Jochen Weiss J¨org Hinrichs PII: DOI: Reference:

S2213-3291(17)30023-0 http://dx.doi.org/doi:10.1016/j.foostr.2017.06.007 FOOSTR 83

To appear in: Received date: Revised date: Accepted date:

17-3-2017 28-6-2017 29-6-2017

Please cite this article as: Kristin Protte, Thomas Ruf, Zeynep Atamer, Alina Sonne, Jochen Weiss, Jddotorg Hinrichs, Influence of shear stress, pectin type and calcium chloride on the process stability of thermally stabilised whey protein-pectin complexes, (2017), http://dx.doi.org/10.1016/j.foostr.2017.06.007 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.

Highlights

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• Stability of WPI-pectin complexes versus shear and heating during upscaling is shown • Degree of blockiness and shear rate are main influences on complex structure and size • CaCl2 and biopolymer concentration can compensate defragmenting effects of shear • Process parameters to tailor process-stable whey protein-pectin complexes are defined

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Influence of shear stress, pectin type and calcium chloride on the process stability of thermally stabilised whey protein-pectin complexes

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Kristin Prottea,∗, Thomas Rufb , Zeynep Atamera , Alina Sonnec , Jochen Weissd , J¨org Hinrichsa a University

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of Hohenheim, Department of Soft Matter Science and Dairy Technology, Garbenstraße 21, 70599 Stuttgart, Germany b Nestl´ e Health Science GmbH, Dr.-Wander-Straße 11, 67574 Osthofen, Germany c Weleda AG,M¨ ohlerstraße 3, 73525 Schw¨ abisch Gm¨ und, Germany d University of Hohenheim, Department of Food Physics and Meat Science, Garbenstraße 23, 70599 Stuttgart, Germany

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Abstract

There is a strong demand for fat-reduced foods due to an increasing incidence of overweight and obesity. To overcome the sensory deficiencies going along

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with a fat-reduction, process stable fat replacers with tailored properties, such as whey protein-pectin complexes, are necessary. In this study, the effects of

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process (shear rate: 0, 150, 500 s−1 ; scale: lab / pilot plant) and composition (pectin type: high / low degree of blockiness; CaCl2 : 0, 15, 30 mM; biopolymer

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concentration: chigh = 5.0 % WPI + 1.0 % pectin, cmed = 2.75 % + 0.55 %; clow = 0.5 % + 0.1 %) parameters on micro- and macro-structural characteristics of whey protein-pectin (WPI-pectin) complexes were investigated. Thermomechanical treatments of WPI-pectin suspensions in a high-pressure double gap geometry revealed a high shear-stability during complex generation at 500 s−1 . The degree of blockiness (DB) of pectin was identified as critical parameter influencing complex structure and biopolymer system stability. WPIpectin complexes with a high DB pectin had a fragile structure, low DB resulted in a compact structure. The shear rate was the main parameter to adjust yield and particle size, both on the lab and pilot scale. A higher shear rate led to ∗ corresponding

author: [email protected]

Preprint submitted to Innovative Food Science and Emerging Technologies

June 28, 2017

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a higher yield consisting of smaller particles. This effect could be partially compensated by medium CaCl2 concentrations (≤ 10 mM) or high biopolymer

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concentrations (≤ 5.0 % (w/w) WPI + 1.0 % (w/w) pectin). Modelling the

parameter effects resulted in sets of processing and composition parameters

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suitable for the generation of WPI-pectin complexes, owning the potential as fat replacers.

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Keywords: Whey protein · pectin · process stability · calcium chloride · shear · fat replacer

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1. Introduction

In the past years, there has been a strong demand for calorie-reduced foods

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due to increasing incidence of overweight and obesity (Wharton, 2016). Apart from the minimisation of sugar, reduction of fat is a powerful tool to reduce the 5

overall calorie content as it contains the most calories per unit. Even though

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many fat-reduced or fat-free products are available, their consumer acceptance is still very low, since they often lack textural attributes such as creaminess or

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an appropriate mouthfeeling (Folkenberg & Martens, 2003; Cayot et al., 2008). A common approach to overcome these deficiencies is the application of fat replacers, which substitute or mimic the sensory and structural properties of fat

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in calorie-reduced food systems. Besides fat- and carbohydrate-based replacers (Lucca & Tepper, 1994), most research focused on protein-based fat replacers due to their complementary nutritional benefits (Patel, 2015). Among proteinbased fat replacers, special attention was drawn to whey protein-pectin com-

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plexes due to their high nutritional (Hamaker & Tuncil, 2014; Patel, 2015) and functional properties (Tolstoguzov, 1997; Jones & McClements, 2010; Schmitt & Turgeon, 2011; Dickinson, 2013). By complexation of whey proteins with pectin, the heat-induced aggregation of the proteins is limited without suffering losses in structuring abilities (Jones & McClements, 2010). Studies conducted

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on incorporation of formed complexes in low-fat yoghurt showed that the complexes act as active filler between the microgel particles by forming interparticle

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cross-links, which resulted in a creamy perception resembling that of full-fat yoghurt (Krzeminski et al., 2014).

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Whey protein-pectin complexes have been studied thoroughly, including the

impact of biopolymer ratio (Neirynck et al., 2007), pH (Jones & McClements,

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2008), heating temperature (Gent´es et al., 2010), ionic environment (Hirt & Jones, 2014) and low shear conditions (Thongkaew et al., 2015). Depending on

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the chosen parameters such as particle size, hardness and surface structure, characteristics of thermally stabilised complexes can be tailored for an application in 30

fat replacement. Among these, particle size is a good predictor for creaminess, a

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central attribute in fat-replacement (Laiho et al., 2017; Krzeminski et al., 2013). In dairy products, particle sizes from 1 to 10 µm was shown to create the highest creaminess attributes (Krzeminski et al., 2014).

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However, when it comes to a specific process application, studies on the effects of typical processing parameters are missing. Most of the studies were carried out under static or low shear conditions which are not reflecting real

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processing conditions. Still, it was shown that the effect of higher shear rates

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(≥100 s−1 ) on aggregation or complexation should not be neglected (Erabit et al., 2013; Thongkaew et al., 2015). Moreover, calcium ions play an important role during thermomechanical treatment of pure whey protein suspensions and

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during whey protein-pectin complex formation, affecting the micro-structure of the aggregates and complexes (Erabit et al., 2013, 2015; Wagoner et al., 2016). High biopolymer concentrations are desirable for higher process yields and efficiencies. Yet, very few information related to application of higher protein

45

concentrations is available (Wagoner et al., 2017), as the majority of studies were conducted at very low concentration levels. Furthermore, to our best knowledge, most studies focused on lab scale experiments, overlooking typical problems in up-scaling related to shear rates and heating. For the generation of thermally stabilised whey protein-pectin complexes as

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fat replacers on a large scale, primary requirements are achieving stability during thermomechanical treatment, robustness towards environmental parameters and particle sizes with the potential to substitute fat. The application of a statistical 3

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design of experiments allows furthermore to identify critical parameters and parameter ranges within a limited amount of experiments. The first objective of this study was to investigate the process stability of

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whey protein-pectin complexes with special emphasis on shear rates and calcium

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sensitivity. Secondly, the effect of different process scales (lab and pilot plant)

on the micro- and macro-structure of whey protein-pectin complexes was stud-

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ied. Based on this, parameter sets for controlling macro-structural properties of thermally stabilised whey protein-pectin complexes were explored.

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2. Materials and Methods 2.1. Materials

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Native whey protein isolate (WPI 895), obtained from sweet whey, was purchased by Fonterra co-operative Group Ltd. (Auckland, New Zealand). As 65

stated by the manufacturer, the protein fractions were as follows: 69.2 % β-

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lactoglobulin (β-Lg), 14.2 % α-lactalbumin, 3.3 % bovine serum albumin, 2.1 % immunoglobulin G, 1.6 % glycomacropeptide and 1.2 % proteose peptone 5. The

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total protein content of the samples was calculated according to the nitrogen content as determined by a nitrogen analyser (Dumatherm, C. Gerhardt GmbH & Co. KG, K¨ onigswinter, Germany) following the Dumas method (Interna-

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tional Dairy Federation 185:2002). The conversion factor 6.38 was used. The total protein content determined was 93.9 ± 0.2 %, which was used for further

calculations.

Two unstandardised high-methoxylated citrus pectins with different distribu-

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tion of esterified groups (degree of blockiness, DB) were kindly provided by Herbstreith & Fox (Neuenb¨ urg, Germany) and used without further purification. According to the distribution of esterified groups, pectins are subdivided into statistically deesterified with a low DB (HMP) and block-wise deesterified pectins with a high DB (HMPb). As stated by the manufacturer, the degree

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of esterification (DE), the DB and the apparent molecular weight (MW, determined by capillary viscosimetry) of the citrus pectins were as follows: 71 % DE,

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low DB, 85 kDa (CU 201; HMP), and 68 % DE, high DB, 85 kDa (CU-L 024/15; HMPb).

grade and purchased by Carl Roth, Karlsruhe, Germany.

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If not stated elsewhere, all other chemicals and reagents were of analytical

2.2. Preparation of WPI-pectin suspensions

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Stock suspensions of WPI and pectins were prepared in percent weight per weight as described in Protte et al. (2016), resulting in final concentrations of 10.0, 5.5 and 1.0 % protein and 2.0, 1.1 and 0.2 % pectin, respectively. Calcium

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chloride dihydrate (CaCl2 · 2H2 O, p.a.) was added to the protein suspensions, resulting in concentrations of 0, 30 and 60 mM CaCl2 .

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Stock suspensions of WPI and pectin were mixed by dropwise addition of pectin suspensions to WPI suspensions during stirring at 450 rpm. The weight ratio 95

of protein to pectin was kept at 5:1, which was shown optimal for achieving

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complete saturation of proteins with pectins (Stenger et al., 2016; Krzeminski

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et al., 2014a). Suspensions of protein and pectin were prepared at three different levels (5.0 + 1.0 % (chigh ), 2.75 + 0.55 % (cmed ), 0.5 + 0.1 % (clow )) , covering a wide range of process relevant concentrations. The final suspensions had pH values of 6.00 ± 0.50 and CaCl2 concentrations of 0, 15 and 30 mM. pH changes

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due to CaCl2 addition were not readjusted. Thereby, protein and pectin suspensions at different concentrations, 5.0 +

1.0 % (chigh ), 2.75 + 0.55 % (cmed ), 0.5 + 0.1 % (clow ), were obtained at pH 6.10 ± 0.05.

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2.3. Calcium concentration Calcium concentrations were determined complexometrically in treated and

untreated samples according to the VDLUFA-method C10.6.8 (2003).

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2.4. Thermomechanical treatments

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2.4.1. High pressure double gap cylinder geometry In the first experimental set-up (DG), 6.8 mL of unheated WPI-pectin sus-

pensions were given in a high-pressure double gap cylinder geometry (B-DG

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35.12/PR-STD; inner gap width: 0.80 mm, outer gap width: 0.88 mm) attached to a MCR 302 rheometer (both Anton Paar GmbH, Graz, Austria). During the

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thermomechanical treatment, the samples were sheared at a constant rate of 500 s−1 . A temperature profile (inserted in Fig. 3) was applied by connecting two water baths (at 19 and 93 ◦ C) to the rheometer. Thereby, a heat treatment

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similar to previous experiments (Protte et al., 2016) was achieved including a holding phase at 90.0 ± 1.5 ◦ C for 250 s, ensuring a protein denaturation ≥ 90 % (Dannenberg & Kessler, 1988; Kessler & Beyer, 1991). During the treatments, the viscosity of the suspensions was recorded.

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2.4.2. High pressure single gap cylinder geometry

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In the second experimental set-up (SG), 10.0 mL of unheated WPI-pectin suspensions were given in a high pressure single gap geometry equipped with a conical rotor (gap width: 0.5 mm), attached to an AR2000 rheometer (both TA-

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Instruments, New Castle, USA) as presented by Stoeckel et al. (2013). During the thermomechanical treatment, the samples were sheared at constant rates of 0, 150 and 500 s−1 . In parallel, a temperature treatment was applied at 90.0 ± 1.5 ◦ C for 250 s (Fig. 1, left).

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2.4.3. Scraped surface heat exchanger In the third experimental set-up (SSHE), 130 mL of unheated WPI-pectin

suspensions were given in a lab-scale scraped surface heat exchanger (Technical workshop of the University of Hohenheim, Stuttgart, Germany) (Fig. 2). The samples were sheared at constant rates of 150 or 500 s−1 , respectively. The

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water inlet and outlet were connected to two water baths (at 10 and 90 ◦ C) to

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supply either cold or hot water. A temperature profile similar to the previous experiments was achieved (Fig. 1, right).

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Placeholder for Fig. 1left and 1right Placeholder for Fig. 2 2.5. Intrinsic fluorescence spectroscopy

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Intrinsic fluorescence measurements were performed using a LS 50B fluores-

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cence spectrometer (Perkin Elmer Inc., Waltham, USA). Tryptophan (Trp) fluorescence determinations were performed at an excitation wavelength of 295 nm

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to ensure selective excitation of Trp residues. Emission spectra were recorded at 300-500 nm. Slit widths were set to 5 nm, both for excitation and emission. Samples were diluted to a final protein concentration of 0.01 %(w/w) with a

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50 mM phosphate buffer (pH 6.0). The two buffer stock suspensions were prepared using dipotassium hydrogen phosphate, p.a. (Merck KgA, Darmstadt, Germany) and potassium dihydrogen phosphate, p.a.. All measurements were performed at 25.0 ◦ C. SSHE-samples were measured six-fold, all other samples

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were measured three-fold

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The intensity ratio (IR) was calculated from the spectra by dividing the fluorescence intensity I350

at λ350

nm

by the fluorescence intensity I330

nm

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(Bhattacharjee & Das, 2000). The impact of variations in environ-

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λ330

nm

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nm

mental parameters is reflected in this quantity and thus serves information on the relation between parameter variations and micro-structure of the complexes.

2.6. Yield of manually separable complexes The yield of manually separable complexes was determined by differential

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weighing of the initial sample, the fluid and the deposited complexes after the thermomechanical treatment. The fluid was removed by letting it drip off into a flask. The remaining deposited complexes were removed manually using a bevelled teflon spatula. Due to the scraping blades, any yield of manually separable complexes was detectable in the SSHE samples.

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2.7. Particle size determination

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Particle size distributions of WPI-pectin complexes were determined using a LS-230 laser scattering particle size analyzer (BeckmanCoulter, Brea,USA). The calculations are based on the Mie theory allowing a particle detection within a

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range of 0.01 - 3000 µm. Datasets were evaluated based on a logarithmic density

distribution (Post et al., 2012; Sommer, 2001). A refractive index of 1.42 was

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used for the measurements (Krzeminski et al., 2014a). All measurements were performed at room temperature. The particle sizes from the third experimental set up (section 3.3.1) were further evaluated by applying a least squares model

set.

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(SigmaPlot 12.5; Systat Inc., San Jose, USA) to the statistically designed data

2.8. Optical Microscopy

Light microscopy images were taken with a Canon Power Shot G16 (Canon,

MicroImaging GmbH, G¨ ottingen, Germany). One drop of sample was placed on

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Tokyo, Japan) mounted on an Axio Scope optical microscope (A1, Carl Zeiss

an objective slide and carefully closed with a cover slip. Images of the samples

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were taken with 20-fold magnification at room temperature. From each sample, a total number of 9 pictures were taken by randomly picking areas for imaging, ensuring a good representation of the investigated structures.

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2.9. Experimental design and statistical analysis The effects of the variables, biopolymer concentration level c, shear rate γ˙

and calcium concentration level CaCl2 on the micro- and macro-structural prop-

erties of WPI-pectin suspensions were studied using a 3-factorial experimental design applied to the mentioned (see 2.4) three different experimental set-ups

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(Table 1). The experiments of the first set-up (DG) were performed in one block. Experiments of the second set-up (SG) were repeated in three independent blocks. In order to get a more detailed understanding of the impacts of a high-methoxylated 8

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Table 1: Design of experiments of the three experimental set-ups with the corresponding analysis methods CaCl2

(-)

(mM)

clow

high pressure 1

DG

double gap

cmed

geometry

2

SG

single gap

SSHE

heat

x

30

x

0 30 0

cmed

surface

chigh

exchanger

x

0

Ca2+

fluorescence

x

viscosity

x

x

x

x

x

x

x

fluorescence

x

x

x

Ca2+

yield

x x

Ca2+

15

x

x

fluorescence

0

x

x

particle size

x

x

microscopy

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3

x x

15

chigh

scraped

x x

30

cmed

geometry

Analysis 500

0

0

clow

150

30 15

chigh

high pressure

0

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Abbr.

γ˙ (s−1 )

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Biopolymer conc.

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No

Device

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Set-up

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DG: high-pressure double gap geometry; SG: high-pressure single gap geometry; SSHE: scraped surface heat exchanger; WPI: whey protein isolate; chigh : 5.0 % (w/w) WPI + 1.0 %

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pectin; cmed : 2.75 % (w/w) WPI + 0.55 % pectin; clow : 0.5 % (w/w) WPI + 0.1 % pectin

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pectin with a high calcium sensitivity on the complex structure, the experiments of the second set-up were performed additionally with WPI-HMPb suspensions.

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The third experimental set-up was performed in three independent blocks. All experiments were performed in randomised order. The micro- and macrostructural properties were determined at least three-fold, if not stated elsewhere. Results were analysed statistically using SAS software (version 9.4, SAS Insti-

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tute Inc., Cary, USA). Significant differences in micro- and macro-structural properties (p < 0.05) were evaluated.

3. Results and Discussion 3.1. Pressure cell experiments - double gap 3.1.1. Shear stability

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The influence of a thermomechanical treatment on the relative viscosity ηrel of pure biopolymer suspensions (WPI, HMP) and of WPI-pectin suspensions 9

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(exemplary shown for chigh , 0 mM CaCl2 ) is shown as a function of time in Fig. 3. Both pure biopolymer suspensions, WPI and HMP, showed a temperature

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related change in the viscosity, inversely proportional to the applied temper-

ature profile. This is typical for low concentrated biopolymer suspensions, in

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which the viscous behaviour is dominated by the viscosity of the solvent, here

water (Kestin et al., 1978). However, the pectin suspension did not recover

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its starting viscosity. Such a reduction in viscosity can be due to a decrease in molecular weight (Vithanage et al., 2010), often resulting from a shear treatment 215

(Shpigelman et al., 2015).

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In the first 100 s, ηrel of the WPI-HMP suspension showed a similar behaviour as ηrel of the pure HMP suspension, suggesting that pectin is dominating the viscous behaviour throughout this time span. After 225 s, ηrel of

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the WPI-HMP suspension increased, unlike ηrel of the pure biopolymer suspensions. During this time span, 80-85 ◦ C were reached, which is above the typical denaturation temperature of β-Lg (83 ◦ C) (Paulsson et al., 1993; Petit

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et al., 2016). Above their denaturation temperature, globular proteins such as

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β-Lg, can either denature and aggregate (Dannenberg & Kessler, 1988; Tolkach & Kulozik, 2007) or form thermally stabilised complexes if a suitable reactant, e.g. pectin, is present (Wagoner et al., 2016). Thus, we hypothesise that this

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increase in ηrel is related to a complex formation between WPI and pectin as no such an increase is apparent for the pure biopolymer suspensions. During the subsequent treatment, ηrel of WPI-HMP suspensions remained at the elevated,

parallel level, no decrease due to structural break down was apparent (Wu &

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McClements, 2015). Hence, it is likely that the generated complexes were shear stable under the applied conditions. The specific impact of biopolymer concentration level and CaCl2 concentra-

tion on ηrel increase is shown in Table 2. Addition of CaCl2 caused an increase in ∆ηrel (more than doubling) and the slope m. As the viscosity is related to 235

the hydrodynamic volume of particles (N¨obel et al., 2012), this increase points towards the formation of a more hydrated, expanded complex structures. This expansion in structure might be caused by the addition of CaCl2 as an in10

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CaCl2

∆ηrel

m · 10−3

(-)

(mM)

(-)

(-)

clow

0 30 15 0 30

0.10 0.21 0.38 0.07 0.15

0.8 0.6 1.8 0.2 0.3

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cmed chigh

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Biopolymer conc.

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Table 2: Viscosity difference ∆ηrel after 400 s and slope m of the viscosity increase of the WPI+pectin suspensions measured at 80 - 90 ◦ C. Three different biopolymer concentration levels (clow , cmed , chigh ) are presented. All suspensions have undergone a thermomechanical treatment in a pressure cell (DG) (90 ◦ C, 250 s; γ˙ = 500 s−1 ).

∆ηrel = ηrel (WPI+HMP) - ηrel (HMP); WPI: whey protein isolate; HMP: statistically deesterified high-methoxylated pectin; chigh : 5.0 % (w/w) WPI + 1.0 % (w/w) pectin; cmed : 2.75 %

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(w/w) WPI + 0.55 % (w/w) pectin; clow : 0.5 % (w/w) WPI + 0.1 % (w/w) pectin

crease in ionic strength in β-LG-pectin mixtures increases the size of formed

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microgel particles (Jones & McClements, 2008, 2010). However, an increase in biopolymer concentration level had a contrary effect as both parameters decreased. Protte et al. (2016) showed, that changes in biopolymer concentration

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level result in different complex structures. Due to the observed decline in both

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parameters (viscosity and slope), one can speculate that the increase in biopolymer concentration level resulted in a more condensed complex structure. The most expanded structure seemed to be reached at cmed and 15 mM CaCl2 as m

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and ∆ηrel reached their maximum. Placeholder for Fig. 3

3.2. Pressure cell experiments - single gap 3.2.1. Manually separable complexes

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Biological material can deposit and have substantial impacts on the process

design as it affects heat-transfer, process efficiency and yield amongst others (Fryer, 2012; Wallh¨ ausser et al., 2012; Iritani & Katagiri, 2016). Thus, the formation of deposited material during thermomechanical treatments of WPIpectin suspensions is of deeper interest.

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In general, the thermomechanical treatments of the second set-up in a pressure cell (SG) resulted in deposited material only for systems at chigh . Systems 11

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at clow and cmed did not show manually separable deposited material indicating that a minimum concentration level is necessary for the formation of deposited

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complexes. If the results would be transferred to process applications, systems

at clow and cmed could be treated in fouling sensitive devices such as plate

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heat-exchangers.

To get a deeper understanding of the underlying mechanisms, the deposited

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material from systems at chigh were further studied (Fig. 4). The amount of manually separable complexes of WPI-HMP suspensions increased with increas265

ing shear rate, indicating an improved heat transfer due to stronger mixing at

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higher shear rates (Park et al., 2014). A damping impact of CaCl2 became apparent at higher shear rates. At low shear rates (≤ 150 s−1 ), the addition of CaCl2 increases the probability of the formation of stable large aggregates after

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a collision (Erabit et al., 2013). At high shear rates (≥ 500 s−1 ), the probability of collisions increases generating small, not deposited complexes due to fragmentation. Hence, higher CaCl2 concentrations might have caused the formation of

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large complexes which in turn had a higher probability of collision and thus of

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fragmentation due to the high shear rates. By contrast, WPI-HMPb suspensions showed a distinct dependency on variations in CaCl2 concentration and a minor one on shear rates. This difference

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between the two systems might be due to the different DB of the pectins. Previous studies showed that the main characteristics of the pectin, namely source, DE and DB, have a large impact on micro- and macro-structure of WPI-pectin complexes (Sperber et al., 2009, 2010). The DB is a measure for the size of

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the non-methoxylated blocks of galacturonic acid on the pectin molecule (Daas et al., 1999; Sperber et al., 2009). Pectins with a high DB possess a higher binding affinity to β-Lg due to a lower electrostatic repulsion (Sperber et al.,

2009, 2010) resulting in an enhanced complex formation and thus in an overall higher amount of deposited material for WPI-HMPb suspensions. Moreover, 285

pectins with a high DB possess an enhanced sensitivity towards cations due to their patches of high local charge density (Daas et al., 1999). It is possible that the calcium ions interacted with the HMPb and thus deposited more. Measure12

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ments of the Ca2+ concentration in the deposited material (data not shown) support this hypothesis as the Ca2+ concentration in the deposited material of WPI-HMPb suspensions was up to 5 % higher than for WPI-HMP suspensions.

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Visual observations (data not shown) showed that the deposited material of

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WPI-HMPb suspensions were more fluffy than with HMP as pectin source. It is therefore likely, that different complex structures were built under the impact

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of shear and CaCl2 . Placeholder for Fig. 4

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3.2.2. Intrinsic fluorescence spectroscopy

Intrinsic fluorescence spectroscopy is a non-invasive method to detect conformational changes in complex molecules as well as in globular proteins such

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as β-Lg (Lakowicz, 2006; Simion et al., 2015). It was recently shown, that this method can be applied to detect conformational changes of β-LG in WPIpectin complexes (Qi et al., 2014; Simion et al., 2015). To get more detailed

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information regarding the conformational changes of β-Lg during thermomechanical treatments of the second set-up (SG), the fluorescence spectra were

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further evaluated with special emphasis on the effect of shear rate, biopolymer and CaCl2 concentration on the IR.

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Table 3 shows, that the biopolymer concentration level affected both sus-

pension types similarly. A higher concentration level resulted in an increased IR, which is associated with a stronger unfolding of β-Lg (Daas et al., 1999). Thus, it is likely that with increasing concentration level, the complex structure

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becomes more open, reducing the protective effect of pectin against unfolding of β-Lg (Qi et al., 2014). Similar observations were made for WPI-HMP suspensions without the impact of shear or CaCl2 (Protte et al., 2016). This suggests, that the effect of the biopolymer concentration level on the IR is of basic nature as no interaction with the other two parameters was observed.

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It is well known, that the addition of CaCl2 to pure β-Lg suspensions keeps the IR steady as calcium stabilising the native state of single β-Lg (Petit et al., 2016), preventing an increase in IR during heat treatment. In combination with

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pectin, the scope of this effect seems to depend on the degree of blockiness of the pectin. Tab. 3 shows that the addition of CaCl2 to WPI-HMP suspensions resulted in an increase in IR. In combination with a low DB pectin, the addition

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of CaCl2 reduces stabilising electrostatic interactions between β-Lg and pectin

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by increasing the ionic strength (Sperber et al., 2009; Xu et al., 2015). This might have lead to a looser complex structure, reducing the protective effect of

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pectin on β-Lg and thus increasing IR (Perez et al., 2015).

However, for WPI-HMPb suspensions, an increase in CaCl2 concentration caused a reduction in IR. This contrary effect might have been caused by the

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high DB of the pectin. The patches of negative charge attract the bivalent ions (Sperber et al., 2009; Xu et al., 2015). One could speculate that in this close vicinity, the stabilising effect of calcium on β-Lg can affect better and thus prevent a strong unfolding of the protein.

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The shear rate had no significant effect on the IR and thus the microstructure of the thermomechanically treated WPI-pectin complexes. Similar

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results were reported for pure β-LG (Erabit et al., 2013, 2015), showing no

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changes in residual native fraction due to shear forces or CaCl2 . We deduced that addition of pectin does not affect this relation between shear rate and

Ac ce p

micro-structure of β-Lg.

3.2.3. Evaluation of pressure cell experiments As shown before, the experiments of the second experimental set-up (SG)

revealed that WPI-HMPb complexes had a fragile and fluffy structure due to the

340

calcium sensitivity of HMPb. The sensitivity led to a stronger unfolding of β-Lg making the complexes more prone to environmental changes which also resulted in a distinct variability of the yield of manually separable complexes impeding a robust process design. We deduced that HMPb does not seem suitable as pectin source for the generation of process-stable WPI-pectin complexes and focused

345

the experiments of the third set-up to HMP as pectin source. The experiments showed as well, that high levels of CaCl2 caused an intense unfolding of β-Lg. Moreover, the yield of manually separable complexes of WPI-

14

Page 15 of 37

γ˙

IR

(mM)

(s−1 )

(-)

chigh chigh chigh chigh cmed clow clow clow clow chigh chigh chigh cmed chigh clow clow clow clow

30 30 0 0 15 30 30 0 0 0 0 30 15 30 30 30 0 0

ip t cr

† c

an

CaCl2

(-)

0 500 500 0 150 500 0 500 0 500 0 0 150 500 500 0 500 0

M d

Ac ce p

HMP HMP HMP HMP HMP HMP HMP HMP HMP HMPb HMPb HMPb HMPb HMPb HMPb HMPb HMPb HMPb

Biopolymer conc.

te

Pectin type

us

Table 3: Intensity ratio (IR) of WPI-pectin suspensions of the second experiential set-up after a thermomechanical treatment (90 ◦ C, 250 s) in a pressure cell (SG) as affected by pectin type, biopolymer concentration, CaCl2 concentration and shear rate γ. ˙ Data sorted in descending order of IR.

1.11 1.10 1.09 1.09 1.09 1.09 1.07 1.03 1.03 1.13 1.12 1.12 1.11 1.10 1.09 1.09 1.00 1.00

† c

† a † a

# b * c * c * a * a † a † a † c # b † c * c * c * a * a

∗ # †

significant differences regarding concentration levels (p < 0.05) significant differences regarding CaCl2 concentration (p < 0.05) WPI: whey protein isolate; HMP: statistically deesterified high-methoxylated pectin; HMPb: blockwise deesterified highmethoxylated pectin; chigh : 5.0 % (w/w) WPI + 1.0 % (w/w) pectin; cmed : 2.75 % (w/w) WPI + 0.55 % (w/w) pectin; clow : 0.5 % (w/w) WPI + 0.1 % (w/w) pectin

a b c

15

Page 16 of 37

HMP suspensions got affected by changes in CaCl2 concentrations at high shear rates. We concluded that high concentrations of CaCl2 do not seem suitable for the generation of process-stable WPI-pectin complexes and therefore we limited

ip t

350

CaCl2 concentrations to low and medium levels in the third set-up (SSHE).

cr

3.3. Scraped surface heat-exchanger experiments 3.3.1. Particle size distributions

355

us

Apart from structural characteristics such as composition and charge, the particle size of biopolymer complexes is an essential criterion for possible ap-

an

plications. The microscopic images in Fig. 5 show that the shear rate affected the particle size distributions considerably. At a shear rate of of 150 s−1 , the characteristic values of the particle size distributions (d90,3 , d3,2 and d10,3 ) of all

360

M

systems were ≤ 10 µm. Similar results were also found by Erabit et al. (2013) who demonstrated that high numbers of large particles (≤ 10 µm) occurred upon thermomechanical treatment of pure β-Lg suspensions at moderate shear rates

d

(≤ 150 s−1 ). This can be explained by the collision theory of Smoluchowski

te

(von Smoluchowski, 1916), after which the probability of collisions increases with increasing shear rate, thus the number of large particles. Consistent with 365

the collision theory, Wolz et al. (2016b) reported an increase in d3,2 due to an

Ac ce p

increase in shear rate from 150 to 500 s−1 for pure whey protein concentrates.

However, our experiments revealed a decrease in particle sizes for the same amount of increase in the shear rate, which is also apparent in the microscopic images for d90,3 , d3,2 and d10,3 . Depending on the system, the d90,3 decreased

370

up to 20-fold. It should be noted, that the collision theory assumes an adequate amount of accessible, reactive groups so that bonds between the particles or aggregates can be formed after collisions. The higher the applied shear rates, the more accessible and reactive groups are necessary to form large aggregates despite the defragmenting effects of the shear forces. One can presume that

375

due to the interactions with the pectin molecules, the amount of accessible and reactive groups is smaller than for pure β-Lg or whey protein concentrate suspensions. This reduced amount seemed to be sufficient to form large aggregates 16

Page 17 of 37

at moderate shear rates of 150 s−1 . At high shear rates, however, not enough reactive groups seemed to be present to compensate the defragmenting effects of the shear forces resulting in a defragmentation of the particles and, thus,

ip t

380

reduction of the particle sizes.

cr

Increasing the concentration of calcium ions reduces the repulsive Coulomb forces in pure β-Lg suspensions during a thermal denaturation, whereby aggre-

gation reactions are catalysed (Petit et al., 2011). This mechanism was found valid also under the influence of moderate shear (≤ 150 s−1 ) resulting in a higher

us

385

probability of the formation of stable aggregates after a collision (Erabit et al.,

an

2013). The microscopic images and the particle size confirm this relation for WPI-HMP suspensions. Thus, one can assume, that CaCl2 acted in a similar way in WPI-HMP suspensions permitting the formation of large and stable aggregates, less prone to defragmentation.

M

390

However, the addition of CaCl2 could not supersede the defragmenting effects of shear completely as the d3,2 of the suspensions without CaCl2 at 150 s−1 was

d

larger than for systems at 500 s−1 and 15 mM CaCl2 (Fig. 5; A: 49.0 µm, G:

395

te

16.7 µm; B: 18.7 µm, H: 13.9 µm). This is in good accordance with the statistical evaluation in which the shear rate was of higher significance than CaCl2 .

Ac ce p

At protein concentrations < 5 %, an increase in whey protein concentration accelerates the denaturation kinetics generating larger aggregates due to an increased probability of collisions (Verheul et al., 1998; Fitzsimons et al., 2007; Dissanayake et al., 2013). However, WPI-HMP suspensions at cmed had a larger

400

d3,2 than the corresponding systems at chigh (e.g., A: 49.0 µm, B: 18.7 µm). Studies conducted with higher concentrations (≥ 5-10 %) under the impact of shear showed a decrease in particle size with increasing concentration (Wolz et al., 2016a). Under the assumption that the relation found for concentrations < 5 % protein is also valid under the impact of shear, one can speculate that

405

by increasing the biopolymer concentration from cmed to chigh a threshold was exceeded leading to a reduction in particle size similarly as found by Wolz et al. (2016a).

17

Page 18 of 37

Considering a potential application as fat replacers in dairy systems, a lim410

ited size range similar to milk fat globules (1 - 10 µm) has to be met. The

ip t

experiments on the lab-scale showed, that only the suspensions at high shear rate and without CaCl2 (d3,2 (C): 2.7 µm ; d3,2 (D): 2.4 µm) had particle sizes

cr

within the target range. Apart from the replacement of fat, a common prob-

lem related to the addition of fat replacers is the perception of graininess or grittyness. Several studies showed, that the critical particle size perceived as

us

415

grainy or gritty greatly depends on the surrounding matrix (Engelen & van der Bilt, 2008; Foegeding et al., 2011; Engelen & de Wijk, 2012). Thus, systems not

an

exactly meeting the target range could be applied as fat replacers in an other, suitable matrix. It was shown for pudding that particles up to 20 µm can be 420

added without causing the perception of graininess (Engelen & van der Bilt,

M

2008). Accordingly, suspensions B - D, G and H could be applied there. Hahn et al. (2012) showed that the sensation of graininess in fresh cheese appears for particles ≥ 40 µm. Consequently, also suspensions B and F - H might have the

Placeholder for Fig. 5

te

425

d

potential to be applied as fat replacers in fresh cheese.

3.3.2. Modulation of particle size distributions

Ac ce p

By applying a squares model to the statistically designed data set, we were

able to visualise the impact of varied parameters on the particle size (d3,2 ) of

the thermomechanically treated WPI-HMP complexes (Fig. 6).

430

We found that the shear rate had an negative effect on the particle size

(d3,2 ) resulting in fragmentation of generated WPI-HMP complexes (Fig. 6, left). An increase in CaCl2 concentration can damp this effect to some extend. However, as the orientation of the lines of same diameter illustrates, the main parameter for adjusting the d3,2 was the shear rate. For a potential application

435

as fat-replacers in dairy systems, particle sizes similar to milk fat globules are desirable (green area). By combining shear rates from 330 to 500 s−1 with the corresponding CaCl2 concentrations of 0 - 10 mM, such particles could be achieved.

18

Page 19 of 37

A combination of the effects of biopolymer concentration level and CaCl2 440

concentration (Fig. 6, right) revealed that the increasing biopolymer concentra-

ip t

tion had an increasing effect on the d3,2 . Other than for the combination with

the shear rate (left), the CaCl2 concentration had a supportive effect. Still,

cr

as the orientation of the lines of same d3,2 illustrates, the biopolymer concentration level was the driving force for adjustment of the d3,2 . With respect to

a possible application as fat-replacers in dairy systems, high biopolymer con-

us

445

centrations from 4.01 % WPI + 0.82 % HMP to 5.00 % WPI + 1.00 % HMP should be chosen. Other than for the combination with shear rates, low CaCl2

an

concentrations of 0 - 5 mM are necessary. Combining the previous findings, it turned out that high shear rates (430 - 500 s−1 ), low CaCl2 concentrations (0 450

- 5 mM) and high biopolymer concentrations (4.01 % WPI and 0.82 % HMP to

M

5.00 % WPI and 1.00 % HMP) should be chosen in order to receive WPI-HMP complexes, possessing the potential as fat-replacers in dairy systems. Considering the characteristics of the surrounding matrix (see 3.3.2), other less narrow

20 µ or ≤ 40 µm.

te

455

d

parameter combinations could be chosen resulting in particle sizes such as ≤

Ac ce p

Placeholder for Fig. 6

4. Conclusion

This study demonstrated that process-stable WPI-pectin complexes can be

generated by thermal stabilization in the presence of CaCl2 applying sets of

460

high shear rates and high biopolymer concentrations. A high shear stability during complex generation at 500 s−1 was found for WPIpectin suspensions treated in a high-pressure double gap geometry. Thereby, the degree of blockiness of the high-methoxylated pectins was identified as critical parameter, affecting complex structure and system stability in an interplay with

465

CaCl2 . The micro-structure was also affected by the biopolymer concentration level, leading to a more hydrated structure at high levels. The shear rate was determined as the main parameter for adjusting the macro-structural parameters,

19

Page 20 of 37

yield and particle size. An increase in shear rate lead to a higher yield consisting of smaller particles due to the defragmenting effect of shear. This could be partially compensated by addition of CaCl2 (≤ 10 mM) or by biopolymer

ip t

470

concentration levels at 5.0 % (w/w) WPI + 1.0 % (w/w) pectin. Modelling the

cr

combined parameter effects enabled to tailor WPI-pectin complexes for specific size ranges, that are suitable for an application as fat-replacers (1 - 10 µm).

475

us

With respect to utilisation of whey as protein source, future research will focus on the impact of lactose and shear stress on micro- and macro-structural characteristics. In order to get a detailed understanding of the formed com-

an

plex structure, further studies on the surface characteristics and hardness of the

480

M

complexes are necessary.

5. Acknowledgement

d

This research project was supported by the German Ministry of Economics and Technology (via AiF) and the FEI (Forschungskreis der Ern¨ahrungsindustrie

Ac ce p

te

e. V.), Project AiF 17876N.

20

Page 21 of 37

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Engelen, L., & de Wijk, R. A. (2012). Oral processing and texture perception. In Food Oral Processing (pp. 157–176). Wiley-Blackwell.

Erabit, N., Flick, D., & Alvarez, G. (2013). Effect of calcium chloride and mod-

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Erabit, N., Ndoye, F. T., Alvarez, G., & Flick, D. (2015). A population balance model integrating some specificities of the beta-lactoglobulin thermallyinduced aggregation. Journal of Food Engineering, 144 , 66 – 76.

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Figure captions

exchanger used for the third experimental set-up (SSHE).

• Figure 2: Temperature profile of (left) the second experimental set-up

cr

670

ip t

• Figure 1: Schematic representation of the lab-scale scraped surface heat

(SG) recorded in a TA high pressure cell and of (right) the third experimental set-up (SSHE) recorded in the lab-scale scraped surface heat

us

exchanger.

• Figure 3: Relative viscosity ηrel of pure WPI and HMP suspensions as well as of a WPI-HMP suspension during a thermomechanical treatment

an

675

(γ˙ = 500 s−1 ; 90.0 ± 1.5 ◦ C, 250 s) in a high-pressure double gap cylinder geometry (DG) illustrated as a function of time. Black line: 5.0 % (w/w)

M

WPI + 1.0 % (w/w) HMP, 0 mM CaCl2 ; blue line: 5.0 % (w/w) WPI; green line: 1.0 % (w/w) HMP; dotted line: temperature; m: slope of the viscosities; WPI: whey protein isolate; HMP: statistically deesterified

d

680

te

high-methoxylated pectin

• Figure 4: Weight proportion of manually separable complexes of the total WPI-pectin suspension after a thermomechanical treatment in a

Ac ce p

pressure cell (SG) (90.0 ± 1.5

685

◦

C, 250 s; 5.0 % (w/w) WPI + 1.0 %

(w/w) pectin) as a function of shear rate and calcium concentration. Solid lines: Weight proportion of separable complexes of WPI-HMPb suspensions, dashed lines: Weight proportion of separable complexes of WPI-HMP suspensions; The weight proportions were calculated using a squares model in SigmaPlot. WPI: whey protein isolate; HMP: statisti-

690

cally deesterified high-methoxylated pectin; HMPb: blockwise deesterified high-methoxylated pectin

• Figure 5: Volume based particle size distribution Q3 [x] and structure of WPI-HMP complexes after a thermomechanical treatment at γ˙ = 150 s−1 or 500 s−1 (90.0 ± 1.5

◦

C, 250 s) in a lab-scale scraped surface heat-

28

Page 29 of 37

695

exchanger. chigh : 5.0 % (w/w) WPI + 1.0 % (w/w) HMP, cmed : 2.75 %

ip t

(w/w) WPI + 0.55 % (w/w) HMP. Dashed lines: target range (1 -10 µm) • Figure 6: Particle size (d50,3 ) of WPI-HMP complexes as a function of (left) shear rate and calcium concentration and (right) biopolymer con-

700

cr

centration and CaCl2 concentration, after a thermomechanical treatment in the lab-scale scraped surface heat exchanger (SSHE) (90.0 ± 1.5 C,

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250 s). Areas dyed in green represent systems matching size range similar to milk fat globules (1 - 10 µm). The particle sizes were calculated using

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a squares model in SigmaPlot.

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