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Insignificance of lactose impurities on generation and structural characteristics of thermally stabilised whey protein-pectin complexes
Accepted Manuscript Insignificance of lactose impurities on generation and structural characteristics of thermally stabilised whey protein-pectin complexes Kristin Protte, Jochen Weiss, Jörg Hinrichs PII:
S0958-6946(18)30012-8
DOI:
10.1016/j.idairyj.2018.01.001
Reference:
INDA 4259
To appear in:
International Dairy Journal
Received Date: 16 August 2017 Revised Date:
4 January 2018
Accepted Date: 6 January 2018
Please cite this article as: Protte, K., Weiss, J., Hinrichs, J., Insignificance of lactose impurities on generation and structural characteristics of thermally stabilised whey protein-pectin complexes, International Dairy Journal (2018), doi: 10.1016/j.idairyj.2018.01.001. 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.
ACCEPTED MANUSCRIPT Insignificance of lactose impurities on generation and structural characteristics of thermally stabilised
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Kristin Protte *, Jochen Weiss , Jörg Hinrichs
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whey protein-pectin complexes
University of Hohenheim, Department of Soft Matter Science and Dairy Technology,
Garbenstraße 21, 70599 Stuttgart, Germany
University of Hohenheim, Department of Food Physics and Meat Science, Garbenstraße 23, 70599
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Stuttgart, Germany
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Corresponding author. Tel.: + 49 711 45923616 E-mail address: [email protected] (K. Protte)
ACCEPTED MANUSCRIPT ________________________________________________________________________________ ABSTRACT
We studied the impact of lactose impurities (0, 130, 150 mM) on micro- and macrostructural characteristics of thermally stabilised whey protein-pectin complexes (WPPC) by varying biopolymer
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concentration (chigh = 5.0% WPI + 1.0% pectin; cmed = 2.75% WPI + 0.55% pectin; clow = 0.5% WPI + −1
0.1% pectin), shear rate (0, 150, 500 s ) and scale (lab/pilot plant). We demonstrated that ≤ 150 mM lactose had no significant effect on the microstructure of WPPC, as investigated by fluorescence
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spectroscopy and browning measurements. Unfolding of β-lactoglobulin within WPPC depended on the biopolymer concentration, being strongest at chigh. Measured browning was attributed to reactions between whey proteins and neutral sugars in pectin side chains. Particle size was unaffected by
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lactose, but showed an increase with biopolymer concentration and a decrease with shear rate. Thus, WPPC can likely be generated using whey powders with lactose impurities or other protein sources of lower purity.
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Introduction
In the last decade, the incidence of obesity and related disorders such as hypertension and cardiovascular diseases has increased rapidly (Wharton, 2016). Hence, several national and international institutions recommend frequent consumption of fat-free or low-fat dairy products as part
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of an individual wholesome life (Lichtenstein et al., 2006). However, the consumer acceptance of fatreduced dairy products is still very low, as important textural attributes such as creaminess and
appropriate mouthfeel are often absent (Cayot, Schenker, Houze, Sulmont-Rosse, & Colas 2008;
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Folkenberg & Martens, 2003).
A common approach to overcome these deficiencies is the application of fat replacers, which can substitute or mimic sensory and structural properties of fat in fat-reduced food systems (Lucca &
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Tepper, 1994). Due to their complementary nutritional benefits, protein-based fat-replacers were studied considerably (Patel, 2015). In particular, research focused on whey protein-pectin complexes (WPPC) as they combine functional with high nutritional properties (Dickinson, 2013; Hamaker & Tuncil, 2014; Patel, 2015; Schmitt & Turgeon, 2011) The impacts of environmental parameters were studied thoroughly, including biopolymer ratio (Neirynck et al., 2007), pH (Jones & McClements
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2008), heating temperature (Gentès, St-Gelais, & Turgeon, 2010), ionic strength (Hirt & Jones, 2014) and shear conditions (Thongkaew, Gibis, Hinrichs, & Weiss, 2015). Thus, a tailoring of thermally stabilised WPPC, possessing specific characteristics for fat replacement, is possible. A central
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attribute in fat replacement is creaminess, which can be predicted by the particle size (Krzeminski et al., 2013; Laiho, Williams, Poelman, Appelqvist, & Logan, 2017). In dairy products, particle sizes from
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1 to 10 µm were shown to create the highest creaminess attributes (Krzeminski, Prell, BuschStockfisch, Weiss, & Hinrichs, 2014a). However, when it comes to process transfer, studies on the effects of common impurities in
whey powders such as minerals and lactose are missing. Most of the studies were carried out using ultrapure β-lactoglobulin (β-Lg) or deionised whey protein isolates with low levels of lactose. Still, it was shown that calcium ions as well as lactose have a considerable effect on the denaturation behaviour of β-Lg (Kessler & Beyer, 1991; Petit et al., 2016). As an unfolding of β-Lg is essential for the formation of thermally stabilised WPPC, the impact of those co-solutes is of high importance. In a previous study we showed that low concentrations of calcium ions, as occurring in sweet whey, are
ACCEPTED MANUSCRIPT insignificant for the generation of complexes, if a statistically de-esterified high methoxylated citrus pectin is chosen (Protte et al., 2017). Thus, the applicability of sweet whey as protein source for complex generation seemed feasible. Spiegel (1999) showed that at low lactose concentrations, the aggregation step during denaturation of β-Lg supersedes the unfolding step. As the process of thermally induced complex formation requires an unfolding of β-Lg, the impact of lactose needs to be
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investigated with special emphasis on the micro-structure. To achieve high process yields and
efficiencies, high biopolymer concentrations are required. Still, few studies (Wagoner & Foegeding, 2017) focused on the impact of higher protein concentrations, as most studies were conducted at low
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protein concentrations. In this context, the effect of different shear rates on complex formation is
important, as particle sizes are largely affected by the interplay of biopolymer concentration and shear rates (Wolz, Mersch, & Kulozik, 2016). Moreover, for the generation of thermally stabilised WPPC as
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fat replacers on a large scale, the effect of scale has to be considered, as most studies focused on lab-scale experiments, ignoring problems in up-scaling related to shear rates and heating. The first objective of this study was to investigate the process stability of WPPC focusing on the impact of lactose during shear treatment. Secondly, the effect of different process scales (laboratory and pilot plant) and biopolymer concentrations on micro- and macrostructure of WPPC
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was studied. Based on this, parameter sets and potential protein sources for the generation of process stable WPPC were explored.
Materials and methods
2.1.
Materials
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Whey protein isolate (WPI) was purchased from Fonterra co-operative Group (Auckland, New
Zealand). As stated by the manufacturer, the protein fractions were as follows: 69.2% β-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 protein content was 93.9 ± 0.2% as determined by Dumas method (N × 6.38; IDF 185:2002) and used for further calculations. Ash and lactose concentrations were 1.7% and 0.1%, respectively (as stated by the manufacturer). An unstandardised high-methoxylated citrus pectin (CU 201; HMP) was kindly provided by
ACCEPTED MANUSCRIPT Herbstreith & Fox (Neuenbürg, Germany) and used without further purification. As stated by the manufacturer, the degree of esterification was 71 % and the apparent molecular mass 85 kDa. If not stated elsewhere, all chemicals reagents were of analytical grade and purchased by Carl Roth
2.2.
Preparation of WPI-HMP suspensions
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(Karlsruhe, Germany).
Stock suspensions of WPI and HMP were prepared, resulting in concentrations of 10.0, 5.5
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and 1.0% (w/w) protein and 2.0, 1.1 and 0.2% (w/w) pectin. Lactose (α-lactose monohydrate, Ph. Eur., for biochemistry) was added to the protein suspensions, resulting in concentrations of 0, 260 and 300 mM.
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The weight ratio of protein:pectin was kept at 5:1, which was shown to be optimal for complete saturation of proteins with pectins (Krzeminski, Prell, Weiss, & Hinrichs, 2014b; Stenger, Zeeb, Hinrichs, & Weiss, 2016) Suspensions of protein and pectin were prepared at three different levels [5.0% WPI + 1.0% pectin (chigh), 2.75% WPI + 0.55% pectin (cmed), 0.5% WPI + 0.1% pectin (clow )], covering a wide range of process relevant concentrations. Final suspensions had pH values of
Thermomechanical treatments
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2.3.
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6.10 ± 0.05 and lactose concentrations of 0, 130 and 150 mM, similar to sweet whey.
Experiments were performed as described in Protte et al. (2017). In brief, in the first
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experimental set-up (single-gap: SG), 10.0 mL of unheated WPI-HMP suspensions were placed in a high pressure single gap geometry, equipped with a conical rotor, attached to a AR2000 rheometer (TA-Instruments, New Cas- tle,DE, USA). Samples were sheared at constant rates of 0, 150 and 500 −1
s , while a temperature treatment was applied (90.0 ± 1.5 °C, 250 s). Thereby, a protein denaturation ≥ 90% (Kessler & Beyer, 1991) was achieved. In the second experimental set-up (surface-scraped heat-exchanger: SSHE), 130 mL of unheated WPI-HMP suspensions were placed in a lab-scale scraped surface heat exchanger (Technical Workshop of the University of Hohenheim, Stuttgart, Germany). Samples were sheared at −1
constant rates of 150 or 500 s . A heat treatment similar to the previous experiments (90.0 ± 1.5 °C,
ACCEPTED MANUSCRIPT 250 s) was applied.
2.4.
Determination of the lactose concentration
Lactose concentrations were determined using an enzymatic test kit (Lactose/D-Galactose,
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Megazyme International Ireland Ltd., Wicklow, Ireland). The measuring principle relies on the
enzymatic conversion of galactose and lactose by β-galactosidase, galactose mutarotase and βgalactose dehydrogenase, detected by UV-measurements at 340 nm. All measurements were
Intrinsic fluorescence spectroscopy
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2.5.
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performed in triplicate.
Intrinsic fluorescence measurements were performed as described in Protte et al. (2016, 2017) using a LS 50B fluorescence spectrometer (Perkin Elmer Inc., Waltham, MA, USA). Samples were excited at a wavelength of 295 nm, emission spectra were recorded at 300–500 nm with slit widths of 5 nm. Samples were diluted to a final protein concentration of 0.01% (w/w) with a
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50 mM phosphate buffer (pH 6.1). All measurements were performed at 25.0 °C. SSHE-samples were measured six-fold, all other samples were measured three-fold. The intensity ratio (IR) was calculated from the spectra by dividing the fluorescence intensity I350 nm at λ350 nm by the fluorescence intensity
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I330 nm at λ330 nm. Thereby, predictions on the degree of unfolding of β-lactoglobulin are possible
2.6.
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(Bhattacharjee & Das, 2000; Protte et al., 2016).
Determination of browning
Browning due to the thermomechanical treatment was investigated by measuring the
absorption at 420 nm (Morales & van Boekel, 1998; Yeo & Shibamoto, 1991) using polystryrene cuvettes with a path length of 1 cm. Samples were diluted with phosphate buffer (50 mM, pH 6.1) to a final protein concentration of 0.01% (w/w). Absorbances of untreated and thermomechanically treated samples were recorded with phosphate buffer in a reference cuvette. The absorbance difference ∆A was quantified by subtracting the absorbances of the untreated samples (Auntreated) from that of the
ACCEPTED MANUSCRIPT thermomechanically treated samples (Atreated) using equation (1). Thus, scattering at particles eliminated, as a previous study (Protte et al., 2016) demonstrated the formation of pre-complexes already in unheated samples. ∆A = Atreated − Auntreted
Particle size determination
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2.7.
(1)
Particle size distributions of WPI-HMP complexes were determined using a LS-230 laser scattering particle size analyser (BeckmanCoulter, Brea, CA, USA). The calculations are based on the
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Mie theory allowing for particle detection within a range of 0.01 to 3000 µm. Datasets were evaluated based on a logarithmic density distribution (Sommer, 2001). A refractive index of 1.42 was used for
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the measurements (Krzeminski et al., 2014b). All measurements were performed at room temperature
Optical Microscopy
Light microscopy images were taken with a Canon Power Shot G16 (Canon, Tokyo, Japan)
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mounted on an Axio Scope optical microscope (A1, Carl Zeiss MicroImaging GmbH, Göttingen, Germany). One drop of sample was placed on an objective slide, carefully mixed with a drop of a safranin-O suspension (0.34%, w/v) for contrast enhancement and closed with a cover slip. Images were taken at bright field and 40-fold magnification at room temperature. From each sample, a total
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number of 9 pictures were taken by randomly picking areas for imaging, ensuring a good
2.9.
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representation of the structures.
Experimental design and statistical analysis
To evaluate the effect of biopolymer concentration level c, shear rate γ˙ and lactose
concentration lac on the micro- and macro-structural properties of WPI- HMP suspensions, a 3factorial design was used for the two experimental set-ups (SG and SSHE). The experiments of both set-ups were run in three blocks. All experiments were performed in randomised order (Table 1). The micro- and macrostructural properties were determined at least three-fold, if not stated elsewhere. All measurements were performed on the same day as the corresponding thermomechanical treatment.
ACCEPTED MANUSCRIPT Results were analysed statistically using the GLM and mixed procedures of SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA) including a three-way analysis of variance (ANOVA) as well as linear regression models. Significant differences in micro- (fluorescence spectroscopy and browning measurements) and macrostructural (particle size and light microscopy) properties (p < 0.05) were
Results and discussion
3.1.
Intrinsic fluorescence spectroscopy
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3.
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evaluated.
Table 2 shows the impacts of the varied process parameters on intensity ratio (IR) and
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wavelength at maximum intensity (λmax) with the two different set ups. All thermomechanically treated systems exhibited IR > 1.00 and λmax > 341.00 nm indicating a desired unfolding of β-Lg during complex formation (Lakowicz, 2010; Protte et al., 2016; Ruffin, Schmit, Lafitte, Dollat, & Chambin, 2014). Moreover, the results show that variations of shear rate and set up are not significant for IR and λmax, which is in good accordance with previous studies (Protte et al., 2016, 2017). Variations in
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biopolymer concentration, however, had a significant effect on IR and λmax. At clow, only a slight unfolding occurred (IR 1.03; λmax 341.6 nm for SSHE) compared with the high values of IR and λmax at cmed and chigh (IR 1.08; λmax 343.2 nm for SSHE), between which no difference was observed. This
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indicates that the structure of the complexes at clow is more compact than at the other two levels, which are thought to have a more open and hydrated structures (Protte et al., 2016, 2017).
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Although lactose is known for stabilising the native state of β-Lg during heating (Nicolai et al., 2011; Petit et al., 2016; Spiegel, 1999) addition of lactose had no significant effect on IR and λmax, thus on the state of unfolding of β-Lg within the complexes. One reason for this could be found in the mechanism of lactose stabilisation, which is ascribed to a complexation with β-Lg (Nicolai, Britten, & Schmitt, 2011; Relkin & Mulvihill, 1996). In this case, one could argue that in the presence of pectin, potential binding sites for lactose are no longer accessible, as the long chained pectins electrostatically bind to β-Lg and block the binding sites via steric hindrance. However, it was shown by Morgan et al. (1999), that under chosen conditions, lactose is too slow to undergo a complexation with proteins. Furthermore, Ames (1998) and Petit et al. (2016) showed, that interactions of lactose
ACCEPTED MANUSCRIPT with β-Lg via Maillard-reaction remain limited for temperatures <100 °C, lasting just a few minutes. Hence, a different mechanism might be involved. Kulmyrzaev, Bryant, and McClements (2000) reported that lactose can undergo preferential interactions with proteins such as β-Lg, leading to a stabilising effect against heat-induced denaturation. For this mechanism to come into effect, a certain
were observed.
Browning of thermomechanically treated samples
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3.2.
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lactose concentration is needed, which might not have been the case in this study as no changes
Browning measurements of thermomechanically treated samples (Fig. 1) showed that the
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biopolymer concentration had a significant impact on the formation of coloured products, separating systems at chigh and cmed from systems at clow. The absorbances measured in chigh and cmed systems were up to 4-fold higher than for those at clow. This can be attributed to the different complex structures. As the complexes at chigh and cmed are suggested to have a more open, hydrated structure than those at clow (Protte et al., 2016, 2017), it is probable that primary amines and reducing sugars are in closer proximity than in densely packed, inflexible structures. Thus, a higher degree of
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browning at chigh and cmed is achieved than at clow.
Furthermore, at chigh and cmed, variations in shear rate and set-up caused only slight differences in absorbances, whereas at clow the absorbance using the SG set-up was 2.5-fold higher
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than for the SSHE set-up. The treatment in the SG set-up was dominated by laminar shear profiles leading to a thin layer of deposited material. Here, the heat exposure was higher than in the rest of
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the sample causing an overall increased browning. The treatments in the SSHE set-up involved additional mechanical forces from the blades resulting in an improved mixing. Heated product from the walls was transferred directly into the bulk phase causing a better heat transfer, thus the absorbances were lower. The addition of lactose, however, had no significant effect on browning of the samples, indicating that the added lactose is not involved in ongoing reactions leading to the observed browning. This is in good accordance with Ames (1998) and Petit et al. (2016), who reported for pure β-Lg suspensions that heat treatments <100 °C, lasting just a few minutes, are neglectable as interactions of lactose with β-Lg via Maillard reaction remain limited. Thus, one can assume that the browning is a result of the interactions of reducing sugars in the side chains of the
ACCEPTED MANUSCRIPT pectin with either residual whey proteins or protein impurities in the pectin powder during heat treatment (Flutto, 2003).
3.3.
Particle size distributions
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Fig. 2 shows the d3,2 of thermomechanically treated WPI-pectin suspensions as affected by the varied process parameters at the two different set-ups. A decrease in biopolymer concentration resulted in smaller complexes, especially apparent for SG-systems. This is in good accordance with
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the collision theory of von Smoluchowski (von Smoluchowski, 1916; Wolz et al., 2016), after which the collision probability decreases with decreasing biopolymer concentration resulting in larger aggregates. At lower concentrations, the collision rate decreases and a just few large particles can be
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formed via collision induced aggregation. Thus, just small primary, non-aggregated, particles result. Using the SSHE, the d3,2 of the system at chigh was smaller than at cmed, which seems contradictory at first. However, the treatment in the SSHE is characterised by additional flow forces during mixing and scraping by the blades, increasing the active forces and thus the overall collision probability and fragmentation of large aggregates. Hence, smaller particles result. As no shear forces due to the
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blades occurred in the SG set-ups, it is likely that the d3,2 represents the size without fragmenting forces. Since this relation could be observed only at chigh, one can assume that a certain biopolymer concentration has to be exceeded. Similar findings were already made in a previous study (Protte et
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al., 2017).
The d3,2 of systems at chigh and cmed treated in the pressure cell are in similar size ranges
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despite the different shear rates. One would expect a reduction in particle size due to the higher shear rate. However, as an increase in biopolymer concentration was shown to compensate this effect in comparable set-ups (Wolz et al., 2016; Protte et al., 2017), the similar size ranges at chigh and cmed are consistent.
Fluorescence and browning measurements showed that the addition of lactose to WPI-pectin suspensions had no significant effect on microstructural characteristics. In Fig. 3 exemplary micrographs of thermomechanically treated WPPC with and without lactose addition are shown. There was no apparent difference in size distribution and structure of the complexes. Together with Fig. 2, this supports the hypothesis that lactose is not involved in complex formation and does not
ACCEPTED MANUSCRIPT interact with already formed complexes during the treatment. On the contrary, Spiegel (1999) showed that a high lactose concentration (380 mM) during thermomechanical treatment of whey protein concentrate resulted in larger particle sizes (d3,2 = 25 µm). However, one can presume that due to the complexation with pectin, necessary binding sites for the formation of larger, lactose mediated aggregates are no longer available. Thus, the aggregation facilitating effect of high lactose
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concentrations as described by Spiegel (1999) does not come into effect.
With regard to a potential application of the WPPC as fat-replacers, a size range similar to milk fat globules (1–10 µm) should be met for dairy products. The experiments in the two set-ups −1
in the SSHE-set-up had particle sizes (d3,2 =
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showed that just the systems at chigh, sheared at 500 s
1.03 and 1.08 µm) within the target range. However, the perception of creaminess is closely related to the perception of grittiness, representing a common problem related to the application of fat-replacers.
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As several studies showed that the perception of grittiness largely depends on the surrounding matrix (Engelen & van der Bilt, 2008; Foegeding et al., 2011; Engelen & de Wijk, 2012), the application of further WPPC systems in other matrices seems possible. It was shown for pudding and fresh cheese that particles up to 20 or 40 µm, respectively, can be added without causing the perception of graininess (Engelen & van der Bilt, 2008; Hahn, Sramek, Nöbel, & Hinrichs, 2012). Accordingly, the
Conclusion
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systems sheared at cmed (d3,2 = 7.42 and 18.53 µm) could be applied there.
The effects of lactose on micro- and macro-structural characteristics of WPI- pectin
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complexes were investigated by varying biopolymer concentration, shear rate and experimental setup. On the basis of our data we demonstrated that lactose (≤ 150 mM in final suspensions) had no significant effect on the microstructure of the complexes, as investigated by means of fluorescence spectroscopy and browning measurements. In fact, the unfolding of β-Lg within the complexes was stronger at a higher biopolymer concentration. The measured browning could be attributed to reactions between WPI and neutral sugars in pectin side chains. The macro-structural particle size was not affected by lactose either, showing an increase in size with increasing biopolymer concentration and a decrease with increasing shear rate. Furthermore, statistical evaluation of the experimental data showed no significant interactions among parameters and blocs underlining the
ACCEPTED MANUSCRIPT validity of the findings. Concluding, we showed that lactose impurities in WPI ≤ 150 mM are negligible for the thermomechanical generation of WPI-pectin complexes. Accordingly, other raw materials with lower purity could be used for production at industrial scale. For a pursued utilisation of whey this means that sweet whey has the potential to be used as protein source. Thus, future research will focus on
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investigating the applicability of sweet whey and its concentrates as the protein source for generating process-stable thermally stabilised WPI-pectin complexes. To get a detailed understanding of the underlying mechanisms of the formation of stable complexes, further studies on quantification of the
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stabilising bonds in the complexes are necessary.
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Acknowledgements
The authors thank Mareike Ableitner and Britta Graf for conducting the experiments, Zeynep Atamer for fruitful discussions and Carolin Wedel for proof-reading the manuscript. This research project was supported by the German Ministry of Economics and Technology (via AiF) and the FEI
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Nicolai, T., Britten, M., & Schmitt, C. (2011). β-Lactoglobulin and WPI aggregates: Formation, structure and applications. Food Hydrocolloids, 25, 1945–1962. Patel, S. (2015). Emerging trends in nutraceutical applications of whey protein and its derivatives. Journal of Food Science and Technology, 52, 6847–6858. Petit, J., Moreau, A., Ronse, G., Debreyne, P., Bouvier, L., Blanpain-Avet, P., et al. (2016). Role of whey components in the kinetics and thermodynamics of β-lactoglobulin unfolding and
ACCEPTED MANUSCRIPT aggregation. Food and Bioprocess Technology, 9, 1367–1379. Protte, K., Bollow, C., Sonne, A., Menéndez-Aguirre, O., Weiss, J., & Hinrichs, J. (2016). Impacts on micro- and macro-structure of thermally stabilised whey protein-pectin complexes: A fluorescence approach. Food Biophysics, 11, 226–234. Protte, K., Ruf, T., Atamer, Z., Sonne, A., Weiss, J., & Hinrichs, J. (2017). Influence of shear stress,
pectin complexes. Food Structure, 14, 76–84.
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pectin type and calcium chloride on the process stability of thermally stabilised whey protein-
Relkin, P., & Mulvihill, D. D. M. (1996). Thermal unfolding of β-lactoglobulin, α-lactalbumin, and bovine
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serum albumin. A thermodynamic approach. Critical Reviews in Food Science and Nutrition, 36, 565–601.
Ruffin, E., Schmit, T., Lafitte, G., Dollat, J.-M., & Chambin, O. (2014). The impact of whey protein
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preheating on the properties of emulsion gel bead. Food Chemistry, 151, 324–332. Schmitt, C., & Turgeon, S. L. (2011). Protein/polysaccharide complexes and coacervates in food systems. Advances in Colloid and Interface Science, 167, 63–70. Sommer, K. (2001). 40 years of presentation particle size distribution - yet still incorrect? Particle and Particle Systems Characterization, 18, 22–25.
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Spiegel, T. (1999). Whey protein aggregation under shear conditions: effects of lactose and heating temperature on aggregate size and structure. International Journal of Food Science and Technology, 34, 523–531.
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Stenger, C., Zeeb, B., Hinrichs, J., & Weiss, J. (2016). Formation of concentrated biopolymer particles composed of oppositely charged WPI and pectin for food applications. Journal of Dispersion
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Science and Technology, 38, 1258–1265. Thongkaew, C., Gibis, M., Hinrichs, J., & Weiss, J. (2015). Shear-induced morphological changes in associative and segregative phase-separated biopolymer systems. Food Hydrocolloids, 51, 414–423.
von Smoluchowski, M. (1916). Versuch einer mathematischen theorie der koag- ulationskinetik kolloider lösungen. Zeitschrift fuer Physikalische Chemie, 92, 129–168. Wagoner, T., & Foegeding, E. (2017). Whey protein-pectin soluble complexes for beverage applications. Food Hydrocolloids, 63, 130–138. Wharton, S. (2016). Current perspectives on long-term obesity pharmacotherapy. Canadian Journal
ACCEPTED MANUSCRIPT of Diabetes, 40, 184–191. Wolz, M., Mersch, E., & Kulozik, U. (2016). Thermal aggregation of whey proteins under shear stress. Food Hydrocolloids, 56, 396–404. Yeo, H., & Shibamoto, T. (1991). Effects of moisture content on the Maillard browning model system
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upon microwave irradiation. Journal of Agricultural and Food Chemistry, 39, 1860–1862.
ACCEPTED MANUSCRIPT Figure legends
Fig. 1. Browning of whey protein-pectin suspensions after a thermomechanical treatment (90.0 °C, 250 s) in a pressure cell (SG, black:
and lactose concentration (
) and a scraped surface heat
), affected by biopolymer concentration, shear rate , 0 mM; and , 150 mM). chigh, 5.0% (w/w) whey
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exchanger (SSHE, grey: and
and
and
protein isolate (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; # and *, significant differences regarding
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concentration levels (p < 0.05); a and b, significant differences regarding experimental
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set-up (p < 0.05).
Fig. 2. d3,2 of whey protein-pectin suspensions after a thermomechanical treatment (90.0 °C, 250 s) in a pressure cell (SG, black: exchanger (SSHE, grey: and and lactose concentration (
and
) and a scraped surface heat
), affected by biopolymer concentration, shear rate
and
, 0 mM; and , 150 mM). chigh, 5.0% (w/w) whey
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protein isolate (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; #, * and †, significant differences regarding concentration levels (p < 0.05); a and b, significant differences regarding experimental
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set-up (p < 0.05).
Fig. 3. Structure of whey protein-pectin complexes after a thermomechanical treatment −1
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(90.0 °C, 250 s; 500 s ) in a scraped surface heat exchanger at different biopolymer concentration levels [chigh, 5.0% (w/w) whey protein isolate (WPI) + 1.0% (w/w) pectin; clow, 0.5% (w/w) WPI + 0.1% (w/w) pectin] and lactose concentrations (0 and 150 mM). The scale bar corresponds to 10 µm.
ACCEPTED MANUSCRIPT Table 1
Design of experiments of the three experimental set-ups with the corresponding analysis a
a
SSHE
high pressure single gap geometry scraped surface heat exchanger
Biopolymer conc. (-) clow cmed chigh
Lactose (mM) 150 130 0 150
clow
0 150 130 0 150
cmed chigh
−1
γ˙ (s ) 0 150 x x x x x x x x x
Analysis 500 x
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2
Device
browning particle size
x x
x x
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Set-up No Abbr 1 SG
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methods.
x x
fluorescence browning particle size microscopy
Abbreviations are: SG, high-pressure single gap geometry; SSHE, scraped surface heat exchanger;
chigh, 5.0% (w/w) whey protein isolate (WPI) + 1.0% pectin; cmed, 2.75% (w/w) WPI + 0.55% pectin;
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clow, 0.5% (w/w) WPI + 0.1% pectin.
ACCEPTED MANUSCRIPT Table 2 Intensity ratio (IR) and wavelength at maximum fluorescence intensity (λmax) of WPI-pectin suspensions after a thermomechanical treatment (90 °C, 250 s) in a pressure cell (SG) and a scraped surface heat exchanger (SSHE) as affected by biopolymer concentration, shear rate γ˙ and lactose concentration.
SSHE
a
Lactose (mM) 150 0 130 0 150 0
IR (-) a 1.08 ± 0.02 a 1.09 ± 0.01 a 1.07 ± 0.01 a 1.07 ± 0.01 b 1.03 ± 0.01 b 1.03 ± 0.01
λmax (nm) a 343.7 ± 1.2 a 343.0 ± 1.5 a 342.8 ± 1.5 a 342.1 ± 1.6 b 341.8 ± 1.5 b 341.1 ± 1.2
chigh chigh cmed cmed clow clow
500 500 150 150 500 500
150 0 130 0 150 0
1.08 ± 0.01 a 1.08 ± 0.02 a 1.07 ± 0.01 a 1.08 ± 0.01 b 1.03 ± 0.02 b 1.03 ± 0.01
a
343.2 ± 1.2 a 343.4 ± 1.5 a 342.6 ± 1.1 a 343.7 ± 1.1 b 341.6 ± 1.8 b 341.9 ± 1.7
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γ˙ −1 (s ) 500 500 150 150 500 500
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SG
Biopolymer conc. (-) chigh chigh cmed cmed clow clow
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Set up
a
a
Abbreviations are: chigh, 5.0% (w/w) whey protein isolate (WPI) + 1.0% pectin; cmed, 2.75% (w/w) WPI
+ 0.55% pectin; clow, 0.5% (w/w) WPI + 0.1% pectin. Different superscript letters in a column for IR
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and λmax indicate significant differences regarding concentration levels (p < 0.05), tested by three-way
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ANOVA and linear regression models.
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Figure 1
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chigh
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cmed
clow
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chigh
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Figure 2
cmed
clow
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150 mM
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chigh
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clow
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0 mM
Figure 3
S0958-6946(18)30012-8
DOI:
10.1016/j.idairyj.2018.01.001
Reference:
INDA 4259
To appear in:
International Dairy Journal
Received Date: 16 August 2017 Revised Date:
4 January 2018
Accepted Date: 6 January 2018
Please cite this article as: Protte, K., Weiss, J., Hinrichs, J., Insignificance of lactose impurities on generation and structural characteristics of thermally stabilised whey protein-pectin complexes, International Dairy Journal (2018), doi: 10.1016/j.idairyj.2018.01.001. 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.
ACCEPTED MANUSCRIPT Insignificance of lactose impurities on generation and structural characteristics of thermally stabilised
a
b
a
a
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Kristin Protte *, Jochen Weiss , Jörg Hinrichs
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whey protein-pectin complexes
University of Hohenheim, Department of Soft Matter Science and Dairy Technology,
Garbenstraße 21, 70599 Stuttgart, Germany
University of Hohenheim, Department of Food Physics and Meat Science, Garbenstraße 23, 70599
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Stuttgart, Germany
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b
Corresponding author. Tel.: + 49 711 45923616 E-mail address: [email protected] (K. Protte)
ACCEPTED MANUSCRIPT ________________________________________________________________________________ ABSTRACT
We studied the impact of lactose impurities (0, 130, 150 mM) on micro- and macrostructural characteristics of thermally stabilised whey protein-pectin complexes (WPPC) by varying biopolymer
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concentration (chigh = 5.0% WPI + 1.0% pectin; cmed = 2.75% WPI + 0.55% pectin; clow = 0.5% WPI + −1
0.1% pectin), shear rate (0, 150, 500 s ) and scale (lab/pilot plant). We demonstrated that ≤ 150 mM lactose had no significant effect on the microstructure of WPPC, as investigated by fluorescence
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spectroscopy and browning measurements. Unfolding of β-lactoglobulin within WPPC depended on the biopolymer concentration, being strongest at chigh. Measured browning was attributed to reactions between whey proteins and neutral sugars in pectin side chains. Particle size was unaffected by
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lactose, but showed an increase with biopolymer concentration and a decrease with shear rate. Thus, WPPC can likely be generated using whey powders with lactose impurities or other protein sources of lower purity.
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_________________________________________________________________________________
ACCEPTED MANUSCRIPT 1.
Introduction
In the last decade, the incidence of obesity and related disorders such as hypertension and cardiovascular diseases has increased rapidly (Wharton, 2016). Hence, several national and international institutions recommend frequent consumption of fat-free or low-fat dairy products as part
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of an individual wholesome life (Lichtenstein et al., 2006). However, the consumer acceptance of fatreduced dairy products is still very low, as important textural attributes such as creaminess and
appropriate mouthfeel are often absent (Cayot, Schenker, Houze, Sulmont-Rosse, & Colas 2008;
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Folkenberg & Martens, 2003).
A common approach to overcome these deficiencies is the application of fat replacers, which can substitute or mimic sensory and structural properties of fat in fat-reduced food systems (Lucca &
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Tepper, 1994). Due to their complementary nutritional benefits, protein-based fat-replacers were studied considerably (Patel, 2015). In particular, research focused on whey protein-pectin complexes (WPPC) as they combine functional with high nutritional properties (Dickinson, 2013; Hamaker & Tuncil, 2014; Patel, 2015; Schmitt & Turgeon, 2011) The impacts of environmental parameters were studied thoroughly, including biopolymer ratio (Neirynck et al., 2007), pH (Jones & McClements
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2008), heating temperature (Gentès, St-Gelais, & Turgeon, 2010), ionic strength (Hirt & Jones, 2014) and shear conditions (Thongkaew, Gibis, Hinrichs, & Weiss, 2015). Thus, a tailoring of thermally stabilised WPPC, possessing specific characteristics for fat replacement, is possible. A central
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attribute in fat replacement is creaminess, which can be predicted by the particle size (Krzeminski et al., 2013; Laiho, Williams, Poelman, Appelqvist, & Logan, 2017). In dairy products, particle sizes from
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1 to 10 µm were shown to create the highest creaminess attributes (Krzeminski, Prell, BuschStockfisch, Weiss, & Hinrichs, 2014a). However, when it comes to process transfer, studies on the effects of common impurities in
whey powders such as minerals and lactose are missing. Most of the studies were carried out using ultrapure β-lactoglobulin (β-Lg) or deionised whey protein isolates with low levels of lactose. Still, it was shown that calcium ions as well as lactose have a considerable effect on the denaturation behaviour of β-Lg (Kessler & Beyer, 1991; Petit et al., 2016). As an unfolding of β-Lg is essential for the formation of thermally stabilised WPPC, the impact of those co-solutes is of high importance. In a previous study we showed that low concentrations of calcium ions, as occurring in sweet whey, are
ACCEPTED MANUSCRIPT insignificant for the generation of complexes, if a statistically de-esterified high methoxylated citrus pectin is chosen (Protte et al., 2017). Thus, the applicability of sweet whey as protein source for complex generation seemed feasible. Spiegel (1999) showed that at low lactose concentrations, the aggregation step during denaturation of β-Lg supersedes the unfolding step. As the process of thermally induced complex formation requires an unfolding of β-Lg, the impact of lactose needs to be
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investigated with special emphasis on the micro-structure. To achieve high process yields and
efficiencies, high biopolymer concentrations are required. Still, few studies (Wagoner & Foegeding, 2017) focused on the impact of higher protein concentrations, as most studies were conducted at low
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protein concentrations. In this context, the effect of different shear rates on complex formation is
important, as particle sizes are largely affected by the interplay of biopolymer concentration and shear rates (Wolz, Mersch, & Kulozik, 2016). Moreover, for the generation of thermally stabilised WPPC as
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fat replacers on a large scale, the effect of scale has to be considered, as most studies focused on lab-scale experiments, ignoring problems in up-scaling related to shear rates and heating. The first objective of this study was to investigate the process stability of WPPC focusing on the impact of lactose during shear treatment. Secondly, the effect of different process scales (laboratory and pilot plant) and biopolymer concentrations on micro- and macrostructure of WPPC
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was studied. Based on this, parameter sets and potential protein sources for the generation of process stable WPPC were explored.
Materials and methods
2.1.
Materials
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2.
Whey protein isolate (WPI) was purchased from Fonterra co-operative Group (Auckland, New
Zealand). As stated by the manufacturer, the protein fractions were as follows: 69.2% β-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 protein content was 93.9 ± 0.2% as determined by Dumas method (N × 6.38; IDF 185:2002) and used for further calculations. Ash and lactose concentrations were 1.7% and 0.1%, respectively (as stated by the manufacturer). An unstandardised high-methoxylated citrus pectin (CU 201; HMP) was kindly provided by
ACCEPTED MANUSCRIPT Herbstreith & Fox (Neuenbürg, Germany) and used without further purification. As stated by the manufacturer, the degree of esterification was 71 % and the apparent molecular mass 85 kDa. If not stated elsewhere, all chemicals reagents were of analytical grade and purchased by Carl Roth
2.2.
Preparation of WPI-HMP suspensions
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(Karlsruhe, Germany).
Stock suspensions of WPI and HMP were prepared, resulting in concentrations of 10.0, 5.5
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and 1.0% (w/w) protein and 2.0, 1.1 and 0.2% (w/w) pectin. Lactose (α-lactose monohydrate, Ph. Eur., for biochemistry) was added to the protein suspensions, resulting in concentrations of 0, 260 and 300 mM.
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The weight ratio of protein:pectin was kept at 5:1, which was shown to be optimal for complete saturation of proteins with pectins (Krzeminski, Prell, Weiss, & Hinrichs, 2014b; Stenger, Zeeb, Hinrichs, & Weiss, 2016) Suspensions of protein and pectin were prepared at three different levels [5.0% WPI + 1.0% pectin (chigh), 2.75% WPI + 0.55% pectin (cmed), 0.5% WPI + 0.1% pectin (clow )], covering a wide range of process relevant concentrations. Final suspensions had pH values of
Thermomechanical treatments
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2.3.
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6.10 ± 0.05 and lactose concentrations of 0, 130 and 150 mM, similar to sweet whey.
Experiments were performed as described in Protte et al. (2017). In brief, in the first
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experimental set-up (single-gap: SG), 10.0 mL of unheated WPI-HMP suspensions were placed in a high pressure single gap geometry, equipped with a conical rotor, attached to a AR2000 rheometer (TA-Instruments, New Cas- tle,DE, USA). Samples were sheared at constant rates of 0, 150 and 500 −1
s , while a temperature treatment was applied (90.0 ± 1.5 °C, 250 s). Thereby, a protein denaturation ≥ 90% (Kessler & Beyer, 1991) was achieved. In the second experimental set-up (surface-scraped heat-exchanger: SSHE), 130 mL of unheated WPI-HMP suspensions were placed in a lab-scale scraped surface heat exchanger (Technical Workshop of the University of Hohenheim, Stuttgart, Germany). Samples were sheared at −1
constant rates of 150 or 500 s . A heat treatment similar to the previous experiments (90.0 ± 1.5 °C,
ACCEPTED MANUSCRIPT 250 s) was applied.
2.4.
Determination of the lactose concentration
Lactose concentrations were determined using an enzymatic test kit (Lactose/D-Galactose,
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Megazyme International Ireland Ltd., Wicklow, Ireland). The measuring principle relies on the
enzymatic conversion of galactose and lactose by β-galactosidase, galactose mutarotase and βgalactose dehydrogenase, detected by UV-measurements at 340 nm. All measurements were
Intrinsic fluorescence spectroscopy
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2.5.
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performed in triplicate.
Intrinsic fluorescence measurements were performed as described in Protte et al. (2016, 2017) using a LS 50B fluorescence spectrometer (Perkin Elmer Inc., Waltham, MA, USA). Samples were excited at a wavelength of 295 nm, emission spectra were recorded at 300–500 nm with slit widths of 5 nm. Samples were diluted to a final protein concentration of 0.01% (w/w) with a
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50 mM phosphate buffer (pH 6.1). All measurements were performed at 25.0 °C. SSHE-samples were measured six-fold, all other samples were measured three-fold. The intensity ratio (IR) was calculated from the spectra by dividing the fluorescence intensity I350 nm at λ350 nm by the fluorescence intensity
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I330 nm at λ330 nm. Thereby, predictions on the degree of unfolding of β-lactoglobulin are possible
2.6.
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(Bhattacharjee & Das, 2000; Protte et al., 2016).
Determination of browning
Browning due to the thermomechanical treatment was investigated by measuring the
absorption at 420 nm (Morales & van Boekel, 1998; Yeo & Shibamoto, 1991) using polystryrene cuvettes with a path length of 1 cm. Samples were diluted with phosphate buffer (50 mM, pH 6.1) to a final protein concentration of 0.01% (w/w). Absorbances of untreated and thermomechanically treated samples were recorded with phosphate buffer in a reference cuvette. The absorbance difference ∆A was quantified by subtracting the absorbances of the untreated samples (Auntreated) from that of the
ACCEPTED MANUSCRIPT thermomechanically treated samples (Atreated) using equation (1). Thus, scattering at particles eliminated, as a previous study (Protte et al., 2016) demonstrated the formation of pre-complexes already in unheated samples. ∆A = Atreated − Auntreted
Particle size determination
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2.7.
(1)
Particle size distributions of WPI-HMP complexes were determined using a LS-230 laser scattering particle size analyser (BeckmanCoulter, Brea, CA, USA). The calculations are based on the
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Mie theory allowing for particle detection within a range of 0.01 to 3000 µm. Datasets were evaluated based on a logarithmic density distribution (Sommer, 2001). A refractive index of 1.42 was used for
2.8.
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the measurements (Krzeminski et al., 2014b). All measurements were performed at room temperature
Optical Microscopy
Light microscopy images were taken with a Canon Power Shot G16 (Canon, Tokyo, Japan)
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mounted on an Axio Scope optical microscope (A1, Carl Zeiss MicroImaging GmbH, Göttingen, Germany). One drop of sample was placed on an objective slide, carefully mixed with a drop of a safranin-O suspension (0.34%, w/v) for contrast enhancement and closed with a cover slip. Images were taken at bright field and 40-fold magnification at room temperature. From each sample, a total
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number of 9 pictures were taken by randomly picking areas for imaging, ensuring a good
2.9.
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representation of the structures.
Experimental design and statistical analysis
To evaluate the effect of biopolymer concentration level c, shear rate γ˙ and lactose
concentration lac on the micro- and macro-structural properties of WPI- HMP suspensions, a 3factorial design was used for the two experimental set-ups (SG and SSHE). The experiments of both set-ups were run in three blocks. All experiments were performed in randomised order (Table 1). The micro- and macrostructural properties were determined at least three-fold, if not stated elsewhere. All measurements were performed on the same day as the corresponding thermomechanical treatment.
ACCEPTED MANUSCRIPT Results were analysed statistically using the GLM and mixed procedures of SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA) including a three-way analysis of variance (ANOVA) as well as linear regression models. Significant differences in micro- (fluorescence spectroscopy and browning measurements) and macrostructural (particle size and light microscopy) properties (p < 0.05) were
Results and discussion
3.1.
Intrinsic fluorescence spectroscopy
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3.
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evaluated.
Table 2 shows the impacts of the varied process parameters on intensity ratio (IR) and
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wavelength at maximum intensity (λmax) with the two different set ups. All thermomechanically treated systems exhibited IR > 1.00 and λmax > 341.00 nm indicating a desired unfolding of β-Lg during complex formation (Lakowicz, 2010; Protte et al., 2016; Ruffin, Schmit, Lafitte, Dollat, & Chambin, 2014). Moreover, the results show that variations of shear rate and set up are not significant for IR and λmax, which is in good accordance with previous studies (Protte et al., 2016, 2017). Variations in
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biopolymer concentration, however, had a significant effect on IR and λmax. At clow, only a slight unfolding occurred (IR 1.03; λmax 341.6 nm for SSHE) compared with the high values of IR and λmax at cmed and chigh (IR 1.08; λmax 343.2 nm for SSHE), between which no difference was observed. This
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indicates that the structure of the complexes at clow is more compact than at the other two levels, which are thought to have a more open and hydrated structures (Protte et al., 2016, 2017).
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Although lactose is known for stabilising the native state of β-Lg during heating (Nicolai et al., 2011; Petit et al., 2016; Spiegel, 1999) addition of lactose had no significant effect on IR and λmax, thus on the state of unfolding of β-Lg within the complexes. One reason for this could be found in the mechanism of lactose stabilisation, which is ascribed to a complexation with β-Lg (Nicolai, Britten, & Schmitt, 2011; Relkin & Mulvihill, 1996). In this case, one could argue that in the presence of pectin, potential binding sites for lactose are no longer accessible, as the long chained pectins electrostatically bind to β-Lg and block the binding sites via steric hindrance. However, it was shown by Morgan et al. (1999), that under chosen conditions, lactose is too slow to undergo a complexation with proteins. Furthermore, Ames (1998) and Petit et al. (2016) showed, that interactions of lactose
ACCEPTED MANUSCRIPT with β-Lg via Maillard-reaction remain limited for temperatures <100 °C, lasting just a few minutes. Hence, a different mechanism might be involved. Kulmyrzaev, Bryant, and McClements (2000) reported that lactose can undergo preferential interactions with proteins such as β-Lg, leading to a stabilising effect against heat-induced denaturation. For this mechanism to come into effect, a certain
were observed.
Browning of thermomechanically treated samples
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3.2.
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lactose concentration is needed, which might not have been the case in this study as no changes
Browning measurements of thermomechanically treated samples (Fig. 1) showed that the
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biopolymer concentration had a significant impact on the formation of coloured products, separating systems at chigh and cmed from systems at clow. The absorbances measured in chigh and cmed systems were up to 4-fold higher than for those at clow. This can be attributed to the different complex structures. As the complexes at chigh and cmed are suggested to have a more open, hydrated structure than those at clow (Protte et al., 2016, 2017), it is probable that primary amines and reducing sugars are in closer proximity than in densely packed, inflexible structures. Thus, a higher degree of
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browning at chigh and cmed is achieved than at clow.
Furthermore, at chigh and cmed, variations in shear rate and set-up caused only slight differences in absorbances, whereas at clow the absorbance using the SG set-up was 2.5-fold higher
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than for the SSHE set-up. The treatment in the SG set-up was dominated by laminar shear profiles leading to a thin layer of deposited material. Here, the heat exposure was higher than in the rest of
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the sample causing an overall increased browning. The treatments in the SSHE set-up involved additional mechanical forces from the blades resulting in an improved mixing. Heated product from the walls was transferred directly into the bulk phase causing a better heat transfer, thus the absorbances were lower. The addition of lactose, however, had no significant effect on browning of the samples, indicating that the added lactose is not involved in ongoing reactions leading to the observed browning. This is in good accordance with Ames (1998) and Petit et al. (2016), who reported for pure β-Lg suspensions that heat treatments <100 °C, lasting just a few minutes, are neglectable as interactions of lactose with β-Lg via Maillard reaction remain limited. Thus, one can assume that the browning is a result of the interactions of reducing sugars in the side chains of the
ACCEPTED MANUSCRIPT pectin with either residual whey proteins or protein impurities in the pectin powder during heat treatment (Flutto, 2003).
3.3.
Particle size distributions
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Fig. 2 shows the d3,2 of thermomechanically treated WPI-pectin suspensions as affected by the varied process parameters at the two different set-ups. A decrease in biopolymer concentration resulted in smaller complexes, especially apparent for SG-systems. This is in good accordance with
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the collision theory of von Smoluchowski (von Smoluchowski, 1916; Wolz et al., 2016), after which the collision probability decreases with decreasing biopolymer concentration resulting in larger aggregates. At lower concentrations, the collision rate decreases and a just few large particles can be
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formed via collision induced aggregation. Thus, just small primary, non-aggregated, particles result. Using the SSHE, the d3,2 of the system at chigh was smaller than at cmed, which seems contradictory at first. However, the treatment in the SSHE is characterised by additional flow forces during mixing and scraping by the blades, increasing the active forces and thus the overall collision probability and fragmentation of large aggregates. Hence, smaller particles result. As no shear forces due to the
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blades occurred in the SG set-ups, it is likely that the d3,2 represents the size without fragmenting forces. Since this relation could be observed only at chigh, one can assume that a certain biopolymer concentration has to be exceeded. Similar findings were already made in a previous study (Protte et
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al., 2017).
The d3,2 of systems at chigh and cmed treated in the pressure cell are in similar size ranges
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despite the different shear rates. One would expect a reduction in particle size due to the higher shear rate. However, as an increase in biopolymer concentration was shown to compensate this effect in comparable set-ups (Wolz et al., 2016; Protte et al., 2017), the similar size ranges at chigh and cmed are consistent.
Fluorescence and browning measurements showed that the addition of lactose to WPI-pectin suspensions had no significant effect on microstructural characteristics. In Fig. 3 exemplary micrographs of thermomechanically treated WPPC with and without lactose addition are shown. There was no apparent difference in size distribution and structure of the complexes. Together with Fig. 2, this supports the hypothesis that lactose is not involved in complex formation and does not
ACCEPTED MANUSCRIPT interact with already formed complexes during the treatment. On the contrary, Spiegel (1999) showed that a high lactose concentration (380 mM) during thermomechanical treatment of whey protein concentrate resulted in larger particle sizes (d3,2 = 25 µm). However, one can presume that due to the complexation with pectin, necessary binding sites for the formation of larger, lactose mediated aggregates are no longer available. Thus, the aggregation facilitating effect of high lactose
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concentrations as described by Spiegel (1999) does not come into effect.
With regard to a potential application of the WPPC as fat-replacers, a size range similar to milk fat globules (1–10 µm) should be met for dairy products. The experiments in the two set-ups −1
in the SSHE-set-up had particle sizes (d3,2 =
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showed that just the systems at chigh, sheared at 500 s
1.03 and 1.08 µm) within the target range. However, the perception of creaminess is closely related to the perception of grittiness, representing a common problem related to the application of fat-replacers.
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As several studies showed that the perception of grittiness largely depends on the surrounding matrix (Engelen & van der Bilt, 2008; Foegeding et al., 2011; Engelen & de Wijk, 2012), the application of further WPPC systems in other matrices seems possible. It was shown for pudding and fresh cheese that particles up to 20 or 40 µm, respectively, can be added without causing the perception of graininess (Engelen & van der Bilt, 2008; Hahn, Sramek, Nöbel, & Hinrichs, 2012). Accordingly, the
Conclusion
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4.
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systems sheared at cmed (d3,2 = 7.42 and 18.53 µm) could be applied there.
The effects of lactose on micro- and macro-structural characteristics of WPI- pectin
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complexes were investigated by varying biopolymer concentration, shear rate and experimental setup. On the basis of our data we demonstrated that lactose (≤ 150 mM in final suspensions) had no significant effect on the microstructure of the complexes, as investigated by means of fluorescence spectroscopy and browning measurements. In fact, the unfolding of β-Lg within the complexes was stronger at a higher biopolymer concentration. The measured browning could be attributed to reactions between WPI and neutral sugars in pectin side chains. The macro-structural particle size was not affected by lactose either, showing an increase in size with increasing biopolymer concentration and a decrease with increasing shear rate. Furthermore, statistical evaluation of the experimental data showed no significant interactions among parameters and blocs underlining the
ACCEPTED MANUSCRIPT validity of the findings. Concluding, we showed that lactose impurities in WPI ≤ 150 mM are negligible for the thermomechanical generation of WPI-pectin complexes. Accordingly, other raw materials with lower purity could be used for production at industrial scale. For a pursued utilisation of whey this means that sweet whey has the potential to be used as protein source. Thus, future research will focus on
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investigating the applicability of sweet whey and its concentrates as the protein source for generating process-stable thermally stabilised WPI-pectin complexes. To get a detailed understanding of the underlying mechanisms of the formation of stable complexes, further studies on quantification of the
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stabilising bonds in the complexes are necessary.
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Acknowledgements
The authors thank Mareike Ableitner and Britta Graf for conducting the experiments, Zeynep Atamer for fruitful discussions and Carolin Wedel for proof-reading the manuscript. This research project was supported by the German Ministry of Economics and Technology (via AiF) and the FEI
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ACCEPTED MANUSCRIPT Figure legends
Fig. 1. Browning of whey protein-pectin suspensions after a thermomechanical treatment (90.0 °C, 250 s) in a pressure cell (SG, black:
and lactose concentration (
) and a scraped surface heat
), affected by biopolymer concentration, shear rate , 0 mM; and , 150 mM). chigh, 5.0% (w/w) whey
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exchanger (SSHE, grey: and
and
and
protein isolate (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; # and *, significant differences regarding
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concentration levels (p < 0.05); a and b, significant differences regarding experimental
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set-up (p < 0.05).
Fig. 2. d3,2 of whey protein-pectin suspensions after a thermomechanical treatment (90.0 °C, 250 s) in a pressure cell (SG, black: exchanger (SSHE, grey: and and lactose concentration (
and
) and a scraped surface heat
), affected by biopolymer concentration, shear rate
and
, 0 mM; and , 150 mM). chigh, 5.0% (w/w) whey
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protein isolate (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; #, * and †, significant differences regarding concentration levels (p < 0.05); a and b, significant differences regarding experimental
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set-up (p < 0.05).
Fig. 3. Structure of whey protein-pectin complexes after a thermomechanical treatment −1
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(90.0 °C, 250 s; 500 s ) in a scraped surface heat exchanger at different biopolymer concentration levels [chigh, 5.0% (w/w) whey protein isolate (WPI) + 1.0% (w/w) pectin; clow, 0.5% (w/w) WPI + 0.1% (w/w) pectin] and lactose concentrations (0 and 150 mM). The scale bar corresponds to 10 µm.
ACCEPTED MANUSCRIPT Table 1
Design of experiments of the three experimental set-ups with the corresponding analysis a
a
SSHE
high pressure single gap geometry scraped surface heat exchanger
Biopolymer conc. (-) clow cmed chigh
Lactose (mM) 150 130 0 150
clow
0 150 130 0 150
cmed chigh
−1
γ˙ (s ) 0 150 x x x x x x x x x
Analysis 500 x
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2
Device
browning particle size
x x
x x
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Set-up No Abbr 1 SG
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methods.
x x
fluorescence browning particle size microscopy
Abbreviations are: SG, high-pressure single gap geometry; SSHE, scraped surface heat exchanger;
chigh, 5.0% (w/w) whey protein isolate (WPI) + 1.0% pectin; cmed, 2.75% (w/w) WPI + 0.55% pectin;
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clow, 0.5% (w/w) WPI + 0.1% pectin.
ACCEPTED MANUSCRIPT Table 2 Intensity ratio (IR) and wavelength at maximum fluorescence intensity (λmax) of WPI-pectin suspensions after a thermomechanical treatment (90 °C, 250 s) in a pressure cell (SG) and a scraped surface heat exchanger (SSHE) as affected by biopolymer concentration, shear rate γ˙ and lactose concentration.
SSHE
a
Lactose (mM) 150 0 130 0 150 0
IR (-) a 1.08 ± 0.02 a 1.09 ± 0.01 a 1.07 ± 0.01 a 1.07 ± 0.01 b 1.03 ± 0.01 b 1.03 ± 0.01
λmax (nm) a 343.7 ± 1.2 a 343.0 ± 1.5 a 342.8 ± 1.5 a 342.1 ± 1.6 b 341.8 ± 1.5 b 341.1 ± 1.2
chigh chigh cmed cmed clow clow
500 500 150 150 500 500
150 0 130 0 150 0
1.08 ± 0.01 a 1.08 ± 0.02 a 1.07 ± 0.01 a 1.08 ± 0.01 b 1.03 ± 0.02 b 1.03 ± 0.01
a
343.2 ± 1.2 a 343.4 ± 1.5 a 342.6 ± 1.1 a 343.7 ± 1.1 b 341.6 ± 1.8 b 341.9 ± 1.7
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γ˙ −1 (s ) 500 500 150 150 500 500
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SG
Biopolymer conc. (-) chigh chigh cmed cmed clow clow
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Set up
a
a
Abbreviations are: chigh, 5.0% (w/w) whey protein isolate (WPI) + 1.0% pectin; cmed, 2.75% (w/w) WPI
+ 0.55% pectin; clow, 0.5% (w/w) WPI + 0.1% pectin. Different superscript letters in a column for IR
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and λmax indicate significant differences regarding concentration levels (p < 0.05), tested by three-way
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ANOVA and linear regression models.
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Figure 1
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chigh
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cmed
clow
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chigh
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Figure 2
cmed
clow
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150 mM
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chigh
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clow
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0 mM
Figure 3